Targeting GD2 antibody coupling drug based on NPV linker sequence, and preparation method and application thereof

Abstract

The invention provides an NPV linker sequence-based GD2 targeting antibody coupling drug, a preparation method and an application, and belongs to the technical field of biological medicines. According to the invention, an Asn-Pro-Val (NPV) sequence sensitive to neutrophil elastase is taken as a linker, and MMAE and Hu 3F8 are coupled by using a cysteine coupling technology, so that the antibody conjugate Hu 3F8-NPV-MMAE is obtained. The antibody conjugate shows good tumor cell killing ability, can significantly reduce the tumor volume, and is high in safety. And after passing through intercellular, the elastase of the antibody conjugate is split to release MMAE to play a role, so that the bystander effect can be enhanced, the curative effect of ADC can be improved, the drug-resistant mechanism related to ADC endocytosis and intracellular processing can be avoided, the enzyme specific splitting 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 an NPV linker sequence, a preparation method and application. BACKGROUND

[0002] An antibody drug conjugate (ADC) is a new type of anti-tumor drug, and its principle is to connect a cytotoxin to an antibody, so as to transport the cytotoxin to a target point through the recognition of the antibody to a specific antigen on the surface of a cancer cell, thereby achieving the purpose of targeted treatment of malignant tumors. Compared with traditional small-molecule anti-tumor drugs, the ADC can realize the targeted recognition of the antibody, and has high toxin activity, so as to have higher specificity and effectiveness.

[0003] The ADC includes three different components, an antibody, a linker and a cytotoxin. The antibody realizes targeting, the linker ensures the stability of the ADC in the blood transport process, and after reaching the target point, the toxin plays a role in killing cancer cells. According to different mechanisms, the toxins suitable for the ADC include microtubule inhibitors, DNA damaging agents, RNA polymerase inhibitors and the like. At present, the toxins used in the ADCs on the market and in clinical trials mainly include microtubule inhibitors, including compounds based on dolastatin, such as MMAE, MMAE and MMAD, and compounds based on maytansine, such as DM1 and DM4. In terms of linkers, the main application is cleavable, such as valine-citriline and cyclohexyl formic acid (MCC).

[0004] Traditional ADCs usually need to be internalized into cancer cells to release toxins, but this brings strict restrictions to cancer treatment, and requires that the cancer target has high expression of internalization antigens and effective intracellular processing.

[0005] Therefore, how to eliminate the dependence on high antigen expression and improve the tumor cell killing effect is crucial. SUMMARY

[0006] The present application aims to provide an antibody conjugate drug targeting GD2 based on an NPV linker sequence, a preparation method and application, which can significantly reduce the tumor volume and has good tumor cell killing ability.

[0007] In order to achieve the above-mentioned purposes, the present application provides the following technical solutions: The present application provides an antibody conjugate Hu 3F8-NPV-MMAE, which is obtained by coupling MMAE with Hu 3F8 using a cysteine coupling technology with a neutrophil elastase-sensitive Asn-Pro-Val sequence as a linker.

[0008] The present application also provides a preparation method of the antibody conjugate Hu 3F8-NPV-MMAE, which comprises the following steps: (1) Preparation of Fmoc-Val-PAB: dissolve Fmoc-Val-OH in DCM, add PAB and EEDQ successively, react, filter, wash, and dry to obtain Fmoc-Val-PAB; (2) Preparation of Val-PAB: dissolve Fmoc-Val-PAB in DCM, add Piperidine, react, concentrate, purify and elute, collect the eluate, and concentrate to obtain Val-PAB; (3) Preparation of Fmoc-Asn-Pro-Val-PAB: dissolve Fmoc-Asn-Pro-OH and Val-PAB in DMF, add HATU and DIPEA successively, react, continue to add an aqueous citric acid solution, extract and combine the organic phases, dry, concentrate, mix with silica gel, purify and elute, and collect the eluate to obtain Fmoc-Asn-Pro-Val-PAB; (4) Preparation of Asn-Pro-Val-PAB: dissolve Fmoc-Asn-Pro-Val-PAB in DCM, add DEA, react, concentrate, dissolve in acetonitrile, purify and elute, collect the eluate, and freeze-dry to obtain Asn-Pro-Val-PAB; (5) Preparation of MC-Asn-Pro-Val-PAB: dissolve Asn-Pro-Val-PAB in DMF, then add MC-OSU and DIPEA successively, react, add silica gel for concentration and mixing, purify and elute, collect the eluate, and concentrate to obtain MC-Asn-Pro-Val-PAB; (6) Preparation of MC-Asn-Pro-Val-PAB-PNP: dissolve MC-Asn-Pro-Val-PAB in DMF, add DNPC and DIPEA successively, react, extract and combine the organic phases, dry and concentrate, mix with silica gel, purify and elute, collect the eluate, and concentrate to obtain MC-Asn-Pro-Val-PAB-PNP; (7) Preparation of MC-NPV-PAB-PNP-MMAE: MC-Asn-Pro-Val-PAB-PNP-MMAE was prepared by dissolving MC-Asn-Pro-Val-PAB-PNP in DMF, adding MMAE, HOBt and DIPEA in sequence, reacting, purifying and eluting, collecting the eluate and freeze-drying to obtain MC-Asn-Pro-Val-PAB-PNP-MMAE. (8) Preparation of antibody conjugate Hu 3F8-NPV-MMAE: The antibody Hu 3F8 was replaced into a buffer, TCEP was added into the antibody solution and stirred, MC-NPV-PAB-PNP-MMAE was added, and stirring was continued, and Hu 3F8-NPV-MMAE was obtained after washing.

[0009] Preferably, in step (1), the mass-volume ratio of Fmoc-Val-OH, DCM, PAB and EEDQ is 5-10 g:50-100 mL:3-7 g:5-10 g; and the reaction time is 16-20 h; in step (2), the mass-volume ratio of Fmoc-Val-PAB, DCM and Piperidine is 5-15 g:50-150 mL:5-15 mL; and the reaction time is 1-2 h.

[0010] Preferably, in step (3), the mass-volume ratio of Fmoc-Asn-Pro-OH, Val-PAB, DMF, HATU and DIPEA is 1-2 g:1-2 g:4-10 mL:1-3:1-2 g; the reaction time is 1-2 h; and the concentration of the aqueous citric acid solution is 5-15%.

[0011] Preferably, in step (4), the mass-volume ratio of Fmoc-Asn-Pro-Val-PAB, DCM and DEA is 1-2 g:10-20 mL:1-2 mL; and the reaction time is 1-2 h.

[0012] Preferably, in step (5), the mass-volume ratio of Asn-Pro-Val-PAB, DMF, MC-OSU and DIPEA is 300-700 mg:3-7 mL:300-700 mg:300-700 mg; and the reaction time is 2-3 h.

[0013] Preferably, in step (6), the mass-volume ratio of MC-Asn-Pro-Val-PAB, DMF, DNPC and DIPEA is 500-1000 mg:3-10 mL:900-1500 mg:500-1000 mg; and the reaction time is 2-3 h.

[0014] Preferably, in step (7), the mass-volume ratio of MC-Asn-Pro-Val-PAB-PNP, DMF, MMAE, HOBt, DIPEA is 100-300 mg:1-3 mL:100-200 mg:50-100 mg:100-200 mg; the reaction time is 2-3 h.

[0015] Preferably, in step (8), the mass-volume ratio of Hu 3F8, TCEP, MC-NPV-PAB-PNP-MMAE is 40-50 mg:75-100 µL:400-500 µL; the stirring time is 2-3 h; the continued stirring time is 0.5-1 h.

[0016] The application further provides a preparation method of the antibody conjugate Hu 3F8-NPV-MMAE and application of the antibody conjugate Hu 3F8-NPV-MMAE in preparation of a cancer treatment drug.

[0017] Compared with the prior art, the application has the following beneficial effects: Hu 3F8 is a monoclonal humanized antibody (molecular formula is C 3207 H 4969 N 859 O 997 S 22 ) targeting GD2, and the application uses a neutrophil elastase-sensitive Asn-Pro-Val (NPV) sequence as a linker to couple MMAE and Hu 3F8 by using a cysteine coupling technology to obtain an antibody conjugate Hu 3F8-NPV-MMAE. The antibody conjugate exhibits good tumor cell killing ability, can significantly reduce the tumor volume, and has high safety. And through intercellular post, the elastase of the antibody conjugate is cleaved to release MMAE to play a role, can enhance the bystander effect, improve the ADC efficacy, avoid the drug resistance mechanism related to ADC endocytosis and intracellular processing, improve the enzyme-specific 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 It is a synthesis flow chart of MC-Asn-Pro-Val-PAB-PNP-MMAE in the embodiment 1 of the application. Figure 2 Structure of Hu3F8-NPV-MMAE prepared in Example 1 of the present application; Figure 3 HIC-HPLC profile of Hu3F8-NPV-MMAE prepared in Example 1 of the present application; Figure 4 SEC-HPLC profile of Hu3F8-NPV-MMAE prepared in Example 1 of the present application; Figure 5 Plot of average tumor volume in mice in Test Example 1 of the present application; Figure 6 Plot of average body weight in mice in Test Example 1 of the present application; Figure 7-1 Statistical plot of apoptotic effect in Test Example 2 of the present application, where A is the -NE group and B is the +NE group; Figure 7-2 Plot of apoptotic effect in Test Example 2 of the present application; Figure 8-1 Plot of cycle arrest in Test Example 2 of the present application; Figure 8-2 Statistical plot of cycle arrest in Test Example 2 of the present application, where A is the -NE group and B is the +NE group; Figure 9 Plot of cytotoxicity at the cellular level in Test Example 3 of the present application. DETAILED DESCRIPTION

[0020] Various illustrative embodiments of the present application are now described in detail below. The detailed description is made with reference to the accompanying drawings, wherein the same or like components have the same or like reference numbers. The present application should not be considered limited to the particular examples described herein, but rather should be understood to cover any and all alternatives, modifications, permutations, or equivalents falling within the scope of the application. Numerous specific details of the present application are described below to provide a thorough understanding of the application. However, it will be clear to one skilled in the art that the present application can be practiced without many of the specific details. In some instances, well-known structures and components are shown in block diagram form in order to avoid obscuring the concepts of the present application.

[0021] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, it is to be understood that the use of any of the following terms, or terms of similar import, in the present application is intended to be interpreted in the broadest possible manner consistent with the context of the present application. Additionally, for numerical ranges expressed in the format "from X to Y," "X to Y," or "X or lower," or "Y or higher," each numerical limit of the range is a separate and independent numerical limitation, and thus each numerical limit can be combined with any of the other numerical limits to form a new range. For example, a range of "1 to 10" can be combined with a range of "1 to 12" to form a new range of "1 to 12" or "1 to 10" or "1 to 10" or "1 to 12." In addition, it is to be understood that the use of the terms "about" and "substantially" are used to describe approximations which are near the point of comparison, but do not have to be exact.

[0022] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains unless clearly indicated otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application. All literature and similar materials cited in this application, including but not limited to, patents, genetic sequences, and other documents, are hereby expressly incorporated by reference.

[0023] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in scope by the

[0024] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0025] The application also provides a preparation method of the antibody conjugate Hu 3F8-NPV-MMAE, comprising the following steps: (1) Preparation of Fmoc-Val-PAB: Fmoc-Val-OH is dissolved in DCM, PAB and EEDQ are sequentially added, and then the reaction is carried out. After filtration, washing and drying, Fmoc-Val-PAB is obtained. The mass-volume ratio of Fmoc-Val-OH, DCM, PAB and EEDQ is 5-10 g: 50-100 mL: 3-7 g: 5-10 g, and more preferably 6 g: 60 mL: 4.3 g: 8.4 g. The reaction time is 16-20 h. (2) Preparation of Val-PAB: Fmoc-Val-PAB is dissolved in DCM, Piperidine is added, and then the reaction is carried out. After concentration, purification elution, collection of eluent and concentration, Val-PAB is obtained. The mass-volume ratio of Fmoc-Val-PAB, DMF and Piperidine is 5-15 g: 50-150 mL: 5-15 mL, and more preferably 6 g: 60 mL: 9 mL. The reaction time is 1-2 h. The purification elution is carried out by using a medium-pressure normal-phase column, and the eluents are A: DCM and eluent B: (DCM / MeOH=4 / 1). When the product peak appears, the proportion of eluent B is 35-40%.

[0026] (3) Preparation of Fmoc-Asn-Pro-Val-PAB: Fmoc-Asn-Pro-OH and Val-PAB were dissolved in DMF, HATU and DIPEA were added in sequence, and the reaction was carried out. Citric acid aqueous solution was added, the combined organic phase was extracted, dried, concentrated, silica gel was added for sample mixing, purified elution was performed, and the eluate was collected to obtain Fmoc-Asn-Pro-Val-PAB; wherein the mass-volume ratio of Fmoc-Asn-Pro-OH, Val-PAB, DMF, HATU, and DIPEA was 1-2 g:1-2 g:4-10 mL:1-3 :1-2 g, and more preferably 1.5 g:1.1 g:5 mL:1.9 :1.3 g; the reaction time was 1-2 h, the concentration of the citric acid aqueous solution was 5-15%, and more preferably 10%; the purified elution was performed by using a medium-pressure normal-phase column, and the eluents were A: DCM and eluent B: (DCM / MeOH=4 / 1) system elution, and the product peak was obtained at a B ratio of 20-25%.

[0027] (4) Preparation of Asn-Pro-Val-PAB: Fmoc-Asn-Pro-Val-PAB was dissolved in DCM, DEA was added, and the reaction was carried out. After concentration, acetonitrile was added for dissolution, purified elution was performed, the eluate was collected, and freeze-drying was performed to obtain Asn-Pro-Val-PAB; wherein the mass-volume ratio of Fmoc-Asn-Pro-Val-PAB, DCM, and DEA was 1-2 g:10-20 mL:1-2 mL, and more preferably 1.5 g:15 mL:1.5 mL; the reaction time was 1-2 h; the purified elution was performed by using a medium-pressure reversed-phase column, the column was Santai 120 g, 40-60 um, elution was performed with an ACN / H2O system, and the product peak was obtained at an ACN ratio of 10%.

[0028] (5) Preparation of MC-Asn-Pro-Val-PAB: Asn-Pro-Val-PAB was dissolved in DMF, and then MC-OSU and DIPEA were added in sequence, and the reaction was carried out. Silica gel was added for concentration and sample mixing, purified elution was performed, the eluate was collected, and concentration was performed to obtain MC-Asn-Pro-Val-PAB; wherein the mass-volume ratio of Asn-Pro-Val-PAB, DMF, MC-OSU, and DIPEA was 300-700 mg:3-7 mL:300-700 mg:300-700 mg, and more preferably 500 mg:5 mL:532 mg:446 mg; the reaction time was 2-3 h, the purified elution was performed by using a medium-pressure normal-phase column, the eluents were A: DCM and eluent B: (DCM / MeOH=4 / 1) system elution, and the product peak was obtained at a B ratio of 30-35% (6) Preparation of MC-Asn-Pro-Val-PAB-PNP: MC-Asn-Pro-Val-PAB was dissolved in DMF, DNPC, DIPEA were added in turn, and the reaction was carried out. The combined organic phase was extracted and dried and concentrated. The sample was mixed with silica gel, and purified and eluted. The eluate was collected and concentrated to obtain MC-Asn-Pro-Val-PAB-PNP. The mass-volume ratio of MC-Asn-Pro-Val-PAB, DMF, DNPC, and DIPEA was 500-1000 mg:3-10 mL:900-1500 mg:500-1000 mg, and more preferably 680 mg:5 mL:990 mg:700 mg. The reaction time was 2-3 h. The purified elution was purified by a medium-pressure normal-phase column, and the eluate was A: DCM and eluate B: (DCM / MeOH=4 / 1) system elution. The product was eluted at a B ratio of 20-25%.

[0029] (7) Preparation of MC-NPV-PAB-PNP-MMAE: MC-Asn-Pro-Val-PAB-PNP was dissolved in DMF, MMAE, HOBt, and DIPEA were added in turn, and the reaction was carried out. The purified elution was collected, and the eluate was freeze-dried to obtain MC-NPV-PAB-PNP-MMAE. The mass-volume ratio of MC-Asn-Pro-Val-PAB-PNP, DMF, MMAE, HOBt, and DIPEA was 100-300 mg:1-3 mL:100-200 mg:50-100 mg:100-200 mg, and more preferably 200 mg:2 mL:152 mg:67 mg:129 mg. The reaction time was 2-3 h. The purified elution was purified by a medium-pressure reversed-phase column, and the column was Santai 120 g, 40-60 um. Elution was performed with an ACN / H2O system. The product was eluted at an ACN ratio of 40%.

[0030] (8) Preparation of antibody conjugate Hu 3F8-NPV-MMAE: The antibody Hu 3F8 was replaced in the buffer, TCEP was added to the antibody solution and stirred, and MC-NPV-PAB-PNP-MMAE was added. The stirring was continued, and washing was performed to obtain Hu 3F8-NPV-MMAE. The mass-volume ratio of Hu 3F8, TCEP, and MC-NPV-PAB-PNP-MMAE was 40-50 mg:75-100 µL:400-500 µL, and more preferably 44.46 mg:89.6 µL:494 µL. The stirring time was 2-3 h, and the continued stirring time was 0.5-1 h.

[0031] Example 1 Example 1 of the present application provides a preparation method of antibody conjugate Hu3F8-NPV-MMAE, the flow chart is shown as Figure 1 The specific steps are as follows: (1) Preparation of Fmoc-Val-PAB: 6.0 g of Fmoc-Val-OH (compound 1) was dissolved in 60 mL of DCM, 4.3 g of PAB (compound 2) and 8.4 g of EEDQ were added in sequence, and the reaction was carried out at room temperature for 20 h. LC-MS monitoring, Fmoc-Val-OH disappeared, the reaction liquid was filtered, the filter cake was washed with DCM, and the filter cake was dried to obtain the product, Fmoc-Val-PAB (compound 3) 6.0 g, yield 76%.

[0032] (2) Preparation of Val-PAB: 6 g of Fmoc-Val-PAB (compound 3) was dissolved in 60 mL of DCM, 9 mL of Piperidine was added, and the reaction was carried out at room temperature for 1 h. LC-MS monitoring, Fmoc-Val-PAB disappeared, the reaction liquid was directly concentrated, and the sample was mixed with silica gel. After mixing, the product was purified by medium pressure normal phase column, eluent A: DCM, eluent B: (DCM / MeOH=4 / 1), the proportion of eluent B was 35-40% when the product peak appeared, and the eluent was collected and concentrated to obtain Val-PAB (compound 4) 3.0 g.

[0033] (3) Preparation of Fmoc-Asn-Pro-Val-PAB: 1.5 g of Fmoc-Asn-Pro-OH (compound 5) and 1.1 g of Val-PAB (compound 4) were dissolved in 5 mL of DMF, 1.9 g of HATU and 1.3 g of DIPEA were added in sequence, and the reaction was carried out at room temperature for 2 h. LC-MS monitoring, Fmoc-Asn-Pro-OH disappeared, 10% citric acid aqueous solution was added to the reaction liquid, extracted with DCM three times, combined organic phase, dried with sodium sulfate, concentrated, and mixed with silica gel. The residue was purified by medium pressure normal phase column, eluent A: DCM, eluent B: (DCM / MeOH=4 / 1) system elution, the proportion of B was 20-25% when the product peak appeared, and the eluent was collected to obtain Fmoc-Asn-Pro-Val-PAB (compound 6) 1.5 g, yield 60%.

[0034] (4) Preparation of Asn-Pro-Val-PAB: 1.5 g of Fmoc-Asn-Pro-Val-PAB (compound 6) was dissolved in 15 mL of DCM, 1.5 mL of DEA was added, and the reaction was carried out at room temperature for 1 h, and LC-MS was used for monitoring. Fmoc-Asn-Pro-Val-PAB disappeared, the reaction solution was concentrated to dryness, a small amount of acetonitrile was added for dissolution, and then a medium-pressure reverse-phase column was used for purification. The column was Santai 120 g, 40-60 um. Elution was carried out using an ACN / H2O system, and the product was eluted at an ACN ratio of 10%. The eluate was collected and freeze-dried to obtain Asn-Pro-Val-PAB (compound 7) 790 mg, with a yield of 80%.

[0035] (5) Preparation of MC-Asn-Pro-Val-PAB: 500 mg of Asn-Pro-Val-PAB (compound 7) was dissolved in 5 mL of DMF at room temperature, and then 532 mg of MC-OSU (compound 8) and 446 mg of DIPEA were added in sequence. The reaction was carried out for 2-3 h, and LC-MS was used for monitoring. Asn-Pro-Val-PAB disappeared, a large amount of silica gel was added to the reaction solution, and then concentrated and mixed. After mixing, a medium-pressure normal-phase column was used for purification. The eluent was A: DCM, and the eluent B: (DCM / MeOH=4 / 1) system elution. The product was eluted at a B ratio of 30-35%. The eluate was collected and concentrated to obtain MC-Asn-Pro-Val-PAB (compound 9) 680 mg, with a yield of 95%.

[0036] (6) Preparation of MC-Asn-Pro-Val-PAB-PNP: 680 mg of MC-Asn-Pro-Val-PAB (compound 9) was dissolved in 5 mL of DMF, and then 990 mg of DNPC (compound 10) and 700 mg of DIPEA were added in sequence. The reaction was carried out at room temperature for 2 h, and LC-MS was used for monitoring. MC-Asn-Pro-Val-PAB disappeared, and then an appropriate amount of water was added to the reaction solution, and then extracted with DCM three times. The organic phase was combined and dried with sodium sulfate. The concentrated and mixed silica gel was used for purification. The residue was purified by a medium-pressure normal-phase column. The eluent was A: DCM, and the eluent B: (DCM / MeOH=4 / 1) system elution. The product was eluted at a B ratio of 20-25%. The eluate was collected and concentrated to obtain MC-Asn-Pro-Val-PAB-PNP (compound 11) 500 mg, with a yield of 58%.

[0037] (7) Preparation of MC-NPV-PAB-PNP-MMAE: The 200 mg MC-Asn-Pro-Val-PAB-PNP (compound 11) was dissolved in 2 mL DMF, and 152 mg MMAE (compound 12), 67 mg HOBt, 129 mg DIPEA were added in turn, and the reaction was carried out at room temperature for 2 h, and LC-MS was used for monitoring. When MC-Asn-Pro-Val-PAB-PNP disappeared, the reaction solution was directly purified by medium pressure reverse phase column. The column was Santai 120 g, 40-60 um. The product was eluted with ACN / H2O system, and the ACN ratio was 40% when the product peak appeared. The eluent was collected and freeze-dried to obtain MC-NPV-PAB-PNP-MMAE (compound 13) 205 mg, with a yield of 60%.

[0038] (8) Preparation of antibody conjugate Hu3F8-NPV-MMAE: The antibody XM001AB01 (Hu3F8) was replaced into a buffer containing (5 mM PB, 5 mM EDTA, pH 7.2). The antibody (9.88 mg / mL, 4.5 mL) was taken 44.46 mg, TCEP (10 mM, 2.9 eq) 89.6 μL was added, and the mixture was stirred at room temperature for 2 h. MC-NPV-PAB-PNP-MMAE (5 mM, DMSO, 8 eq) 494 μL was added, and the mixture was stirred at room temperature for 30 min. HIC-HPLC detection showed that the conjugation was completed. The reaction solution was washed with PBS to remove excess small molecules and organic solvents, and Hu3F8-NPV-MMAE (structure as shown in Figure 2 ) 35 mg (9.22 mg / mL, 3.8 mL) was obtained.

[0039] Hu3F8-NPV-MMAE was characterized by HIC-HPLC and SEC-HPLC, and the results are shown in Figure 3 、 4 .

[0040] Figure 3 As shown in Figure 4 , Hu3F8-NPV-MMAE was analyzed by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), and the results showed that Hu3F8-NPV-MMAE accounted for 98% of the total product, indicating that it had high purity.

[0041] Test Example 1 The anti-tumor activity of the Hu3F8-NPV-MMAE prepared in Example 1 on GD2-positive tumor-bearing mice was detected in Test Example 1 of the present application, and the specific steps are as follows: A M21 cell suspension (5 x 10 6 / 0.2 mL / mouse) was inoculated into the right armpit of nude mice, and when the tumor volume reached 80 mm 3 , the mice were grouped, with 6 mice in each group, and a total of two groups. The antibody conjugate Hu3F8-NPV-MMAE prepared in Example 1 was administered to the administration group by tail vein injection at a concentration of 10 mg / kg of body weight, and the control group was administered with the same amount of normal saline by the same method. The administration was performed once a week for four weeks, and during this period, the body weight and tumor volume of the nude mice were recorded every 3-4 days. After the fourth administration, the mice were observed for one week, and then sacrificed and photographed, and the results are shown in Figure 5 、 6 .

[0042] Figure 5 、 6 It is shown that the antibody conjugate Hu3F8-NPV-MMAE can significantly reduce the tumor volume compared with the control group, has good tumor cell killing ability, and is safe.

[0043] Test Example 2 The apoptosis and cycle arrest effects of the Hu3F8-NPV-MMAE prepared in Example 1 on M21 cells were detected in Test Example 2 of the present application.

[0044] 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 5.0 x 10 5 cells per well, and incubated overnight. The next day, Hu3F8-NPV-MMAE was added to the cells at the following concentration gradient: 0, 1, 10, and 100 μg / ml, and enzyme-added and non-enzyme-added control groups were set up. After 48 h of administration and uptake, the cells were collected and stained, and the data was detected and counted on the machine. The results are shown in Figure 7-1 、 Figure 7-2 、 Figure 8-1 、 Figure 8-2 .

[0045] Figure 7-1 、 Figure 7-2 、 Figure 8-1 、 Figure 8-2 It is shown that with the increase of the dose, the apoptosis and cycle arrest effects also increase, which has a dose-dependent effect. At the same time, the cycle effect and cell arrest effect of the enzyme-added experimental group are slightly increased compared with the non-enzyme-added experimental group.

[0046] Test Example 3 Experimental Example 3 of the present invention tested the cytotoxicity of Hu3F8-NPV-MMAE prepared in Example 1 to cells with different GD2 expression levels. GD2-positive cells were M21 cells, and GD2-negative cells were BE2M17 cells.

[0047] The specific steps are as follows: M21 cells and BE2M17 cells were cultured in 200 μL per well, with 5.0×10 3 The cells were evenly seeded into 96-well cell culture plates at a density of 100 cells / well and cultured overnight. The next day, Hu3F8-NPV-MMAE was added to the cells at the following concentration gradient: 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μg / ml. Each concentration was repeated in triplicate. 48 hours after administration, 20 μL of 5 mg / mL MTT solution was added to each well in the dark and cultured at 37°C for 4 hours. After the incubation period, the drug-containing medium was discarded and 150 μL of DMSO was added to each well. After shaking the plate for 5 minutes, the absorbance of each well at 570 nm was measured and the cytotoxicity was calculated. The results are shown in Figure 2. Figure 9 shown.

[0048] Figure 9 It showed that GD2-positive cells had higher cytotoxicity than GD2-negative cells.

[0049] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An antibody conjugate Hu3F8-NPV-MMAE, characterized in that The antibody conjugate Hu3F8-NPV-MMAE uses the neutrophil elastase-sensitive Asn-Pro-Val sequence as a linker, and uses cysteine ​​coupling technology to couple MMAE to Hu 3F8.

2. A method for preparing the antibody conjugate Hu3F8-NPV-MMAE according to claim 1, characterized in that: The steps include: (1) Preparation of Fmoc-Val-PAB: Dissolve Fmoc-Val-OH in DCM, add PAB and EEDQ in sequence, react, filter, wash, and dry to obtain Fmoc-Val-PAB; (2) Preparation of Val-PAB: Dissolve Fmoc-Val-PAB in DMF, add Piperidine, react, concentrate, purify and elute, collect the eluate, and concentrate to obtain Val-PAB; (3) Preparation of Fmoc-Asn-Pro-Val-PAB: Fmoc-Asn-Pro-OH and Val-PAB were dissolved in DMF, HATU and DIPEA were added in sequence, and the reaction was continued. A citric acid aqueous solution was added, and the organic phases were extracted and combined. The organic phases were dried, concentrated, mixed with silica gel, purified and eluted, and the eluate was collected to obtain Fmoc-Asn-Pro-Val-PAB. (4) Preparation of Asn-Pro-Val-PAB: Fmoc-Asn-Pro-Val-PAB was dissolved in DCM, DEA was added, the reaction was carried out, the reaction was concentrated, acetonitrile was added to dissolve the mixture, the eluate was purified and eluted, and the eluate was collected and lyophilized to obtain Asn-Pro-Val-PAB; (5) Preparation of MC-Asn-Pro-Val-PAB: Asn-Pro-Val-PAB was dissolved in DMF, and then MC-OSU and DIPEA were added in sequence. The mixture was reacted, and silica gel was added to concentrate the sample. The mixture was purified and eluted. The eluate was collected and concentrated to obtain MC-Asn-Pro-Val-PAB. (6) Preparation of MC-Asn-Pro-Val-PAB-PNP: MC-Asn-Pro-Val-PAB was dissolved in DMF, and DNPC and DIPEA were added in sequence. The reaction was carried out, and the organic phases were extracted and combined. The organic phases were dried and concentrated. The sample was mixed with silica gel, purified and eluted, and the eluate was collected and concentrated to obtain MC-Asn-Pro-Val-PAB-PNP. (7) Preparation of MC-NPV-PAB-PNP-MMAE: MC-Asn-Pro-Val-PAB-PNP was dissolved in DMF, and MMAE, HOBt, and DIPEA were added in sequence, reacted, purified and eluted, and the eluate was collected and lyophilized to obtain MC-NPV-PAB-PNP-MMAE; (8) Preparation of antibody conjugate Hu3F8-NPV-MMAE: The antibody Hu3F8 was replaced in a buffer solution, TCEP was added to the antibody solution and stirred, MC-NPV-PAB-PNP-MMAE was added, stirring was continued, and washing was performed to obtain Hu3F8-NPV-MMAE.

3. The method for preparing the antibody conjugate Hu3F8-NPV-MMAE according to claim 2, wherein: In step (1), the mass volume ratio of Fmoc-Val-OH, DCM, PAB, and EEDQ is: 5-10 g: 50-100 mL: 3-7 g: 5-10 g; and the reaction time is 16-20 h. In step (2), the mass volume ratio of Fmoc-Val-PAB, DCM, and Piperidine is: 5-15 g: 50-150 mL: 5-15 mL; and the reaction time is 1-2 h.

4. The method for preparing the antibody conjugate Hu3F8-NPV-MMAE according to claim 2, wherein In step (3), the mass volume ratio of Fmoc-Asn-Pro-OH, Val-PAB, DMF, HATU, and DIPEA is: 1~2 g:1~2 g:4~10 mL:1~3:1~2 g; the reaction time is 1-2 h, and the concentration of the citric acid aqueous solution is 5~15%.

5. The method for preparing the antibody conjugate Hu3F8-NPV-MMAE according to claim 2, wherein: In step (4), the mass volume ratio of the Fmoc-Asn-Pro-Val-PAB, DCM, and DEA is: 1-2 g: 10-20 mL: 1-2 mL; and the reaction time is 1-2 h.

6. The method for preparing the antibody conjugate Hu3F8-NPV-MMAE according to claim 2, wherein: In step (5), the mass volume ratio of Asn-Pro-Val-PAB, DMF, MC-OSU, and DIPEA is: 300-700 mg: 3-7 mL: 300-700 mg: 300-700 mg; and the reaction time is 2-3 h.

7. The method for preparing the antibody conjugate Hu3F8-NPV-MMAE according to claim 2, wherein: In step (6), the mass volume ratio of MC-Asn-Pro-Val-PAB, DMF, DNPC, and DIPEA is: 500-1000 mg: 3-10 mL: 900-1500 mg: 500-1000 mg; and the reaction time is 2-3 h.

8. The method for preparing the antibody conjugate Hu3F8-NPV-MMAE according to claim 2, wherein: In step (7), the mass volume ratio of MC-Asn-Pro-Val-PAB-PNP, DMF, MMAE, HOBt, and DIPEA is: 100-300 mg: 1-3 mL: 100-200 mg: 50-100 mg: 100-200 mg; and the reaction time is 2-3 h.

9. The method for preparing the antibody conjugate Hu3F8-NPV-MMAE according to claim 2, wherein: In step (8), the mass volume ratio of Hu 3F8, TCEP, and MC-NPV-PAB-PNP-MMAE is: 40-50 mg: 75-100 µL: 400-500 µL; the stirring time is 2-3 h; and the continued stirring time is 0.5-1 h.

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

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