Trop2-targeted antibody-drug conjugate, and preparation method therefor and use thereof

Abstract

Disclosed are a TROP2-targeted antibody-drug conjugate (ADC), and a preparation method therefor and a use thereof. The TROP2-targeted ADC has a structure as shown in formula I. In the present invention, glycosyl site-directed technology is used to specifically link the cell toxins MMAE and Eribulin, and the like to an antibody glycosylation site, and an obtained ADC compound has good in-vitro uniformity and toxicity for TROP2-positive cells, and can be used for preparing anti-tumor drugs for TROP2-positive tumors.

Description

Antibody-drug conjugate targeting TROP2 and preparation method and use thereof Technical Field

[0001] The present invention belongs to the fields of medicinal chemistry and biotechnology drugs, and specifically relates to sugar-targeted technology, and more specifically to a class of antibody-drug conjugates targeting TROP2, and a preparation method and use thereof. Background Art

[0002] Trop2 (Tumor-associated calcium signal transducer 2, also known as TACSTD2, EGP-1, GA733-1) is a type of cell surface single-pass transmembrane glycoprotein encoded by the TACSTD2 gene. It is highly expressed in lung adenocarcinoma and triple-negative breast cancer, and is associated with tumor growth, cancer metastasis and poor prognosis of patients. It is a target with clinical translational value.

[0003] Antibody-Drug Conjugates (ADCs) are complexes formed by chemically coupling antibodies with highly cytotoxic small molecules (payloads), which can deliver cytotoxic small molecules to cancer cells in a targeted manner. Currently, more than ten ADC drugs have been approved for marketing. Among them, Sacituzumab govitecan (SG, IMMU-132) is a new ADC drug targeting Trop2 developed by Immunomedics, Inc. It consists of a humanized antibody hRS7, a maleimide-polyethylene glycol-acid-sensitive cleavable carbonate linker and an irinotecan metabolite (SN-38), and is approved for the treatment of metastatic triple-negative breast cancer and urothelial carcinoma. In addition, there are several Trop2-targeted ADCs drugs in clinical research, such as Datopotamab deruxtecan (Dato-Dxd, DS-1062a), a Trop2-targeted ADC drug developed by Daiichi Sankyo Co., Ltd. The I inhibitor Dxd was linked to the Datopotamab antibody by random conjugation; RN927C achieved site-specific modification of the C-terminus of the antibody heavy chain of the humanized anti-Trop2 IgG1 antibody (PF-06478924, RN926) with AcLys-VC-PF-06380101 through an enzymatic reaction to produce a highly uniform conjugate. This ADC compound was terminated in a Phase I trial due to excessive toxicity and limited objective tumor response; SKB264 is a Trop2-targeted ADC drug under development by Kelun Biotech, currently in Phase III clinical trials. It has the same monoclonal antibody as IMMU-132 and uses a novel drug linker intermediate TL033 to couple the payload KL610023 (T030, a topoisomerase I inhibitor belotecan derivative that arrests the cell cycle in the G2 / S phase after internalization, leading to cell death) through a cysteine ​​site, with a drug-antibody ratio of 7 to 8.

[0004] Most of the Trop2-targeted ADCs listed or under development adopt a random conjugation strategy, which involves partially reducing the interchain disulfide bonds of the antibody to expose reactive sulfhydryl groups for drug-linker modification. Although this method is simple to operate, it still has the following shortcomings:

[0005] 1) Reduction of interchain disulfide bonds in antibodies may affect ADC product stability; 2) ADC product homogeneity is poor; 3) The structure formed by maleimide and sulfhydryl groups may undergo anti-Markovnikov addition, resulting in poor plasma stability and potential off-target toxicity; 4) The strong hydrophobicity of small toxin molecules may lead to insufficient aggregation and stability of ADCs. To improve ADC homogeneity and enhance its stability, site-specific conjugation technology has become the preferred choice for next-generation ADCs.

[0006] In addition, the Trop2 target is widely distributed throughout the body. Most Trop2-targeted ADC drugs currently on the market or under clinical development use DNA topoisomerase inhibitors as cytotoxins. Microtubule inhibitors such as MMAE have strong toxicity and can cause on-target toxicity. Most ADC compounds using MMAE as a toxin have been terminated in the preclinical and clinical stages due to safety issues. Summary of the Invention

[0007] Based on the above problems in the prior art, the purpose of the present invention is to provide an antibody-drug conjugate targeting TROP2 with high stability, high homogeneity, high tumor inhibitory activity and high safety, as well as a preparation method and use thereof.

[0008] In order to achieve the above objectives, the technical solutions adopted by this application are as follows:

[0009] In a first aspect, the present invention provides an antibody-drug conjugate targeting TROP2, which has a structure shown in the following formula I:

[0010] wherein m is 1 or 2, preferably 2;

[0011] Ab represents an antibody targeting TROP2, which is an IgG with a conserved N-glycosylation site at N297 in the Fc region;

[0012] Each X is independently -(CH=NO) p0 -(CH2) p1 ; wherein p0 is 0 or 1, p1 is an integer between 1 and 20, preferably an integer between 1 and 6, for example 2;

[0013] Y represents -W-L3-, wherein W represents a structure formed by the reaction of orthogonal groups to form a stable covalent bond, and the orthogonal groups are selected from azide (N3) group, dibenzocyclooctyl (DBCO), bicyclo[6.1.0]nonyne (BCN) and dibenzooctylcycloalkyne (DIBO); L3 represents a linking group, the structure of which is selected from -(CH2) p2 -CO-, -(CH2CH2O) p3 -CO-, -(CH2) p4 -CONH-(CH2CH2O) p5 -(CH2) p6 -CO-; wherein p2, p3, p4, p5, and p6 are each independently selected from an integer between 1 and 20, preferably an integer between 2 and 6, such as 2 or 4;

[0014] n1 is 0 or 1;

[0015] L1 represents a linking group having three or more linking sites, for example, a linking group derived from glutamic acid (Glu), β-glutamic acid, lysine (Lys), a Lys-Glu dipeptide or a lysine-β-glutamic acid dipeptide, or a linking group derived from diethanolamine;

[0016] L2 represents a short peptide linear linker with two or more linking sites that may or may not be linked to p-aminobenzyl carboxylate, for example, a tumor microenvironment cleavable or non-cleavable structural unit such as Val-Cit-PAB, Val-Ala-PAB, or Gly-Gly-Phe-Gly;

[0017] Furthermore, L1 and L2 are optionally connected to a PEG chain at the end of the group they contain. For example, when L1 is a linker group derived from Lys, a PEG chain is further connected to the amino group of lysine via an amide bond. The PEG chain is, for example, -(CH2CH2O) p7 -CH3, wherein p7 is an integer from 2 to 20, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14;

[0018] D represents a group derived from a toxin small molecule drug;

[0019] n2 is 0 and n3 is 1; or n2 is 1 and n3 is an integer from 2 to 4, for example, n2 is 1, and n3 is 2, 3 or 4, preferably n3 is 2;

[0020] When n2 is 1, n3 L2-Ds are covalently connected to L1, and n3≤the number of connection sites of L1-1.

[0021] In a specific embodiment, the Ab is an antibody selected from IgG1, IgG2 or IgG4, preferably IgG1;

[0022] Specifically, the antibody is a monoclonal antibody, a polyclonal antibody, a bifunctional antibody, a trifunctional antibody, a nanobody fused with an Fc domain, a therapeutic antibody or a functional antibody of different species (mouse, rat, sheep, rabbit, human, etc.).

[0023] Specifically, the antibodies include certolizumab and datopotamab, preferably certolizumab.

[0024] In a specific embodiment, the W structure is selected from

[0025] In a specific embodiment, L1 represents a linking group derived from glutamic acid (Glu), lysine (Lys), diethanolamine, preferably, L1 is selected from and

[0026] In a specific embodiment, L2 is

[0027] In a specific embodiment, the small molecule toxin includes MMAE, MMAF, Eribulin or Dxd, Exatecan, DM1, DM4, etc.; preferably, the small molecule toxin is a microtubule inhibitor MMAE or Eribulin.

[0028] Specifically, D is selected from the structures shown below:

[0029] In a specific embodiment, the anti-TROP2 antibody-drug conjugate has the structure shown in Formula I-1 or Formula I-2 below:

[0030] Where W, L1, L2, L3, D, n2, n3, and the definition of the antibody are as described above.

[0031] In a specific embodiment, the DAR value of the anti-TROP2 antibody-drug conjugate is 0 < DAR ≤ 4, preferably 0.5 ≤ DAR ≤ 4, more preferably, the DAR value is 1-4, still more preferably, the DAR value is 2 or 4.

[0032] In a specific embodiment, the anti-TROP2 antibody-drug conjugate has the structure shown below:

[0033] Where in the above ADC-1 to 6, the antibody is cetuximab;

[0034] Specifically, the anti-TROP2 antibody-drug conjugate has the structure shown in ADC-5.

[0035] In the second aspect of the present invention, a method for preparing the anti-TROP2 antibody-drug conjugate I-1 or I-2 described in the first aspect is provided:

[0036] Where Z represents an orthogonal group that can react with an azide group (N3) to form the W structure, such as dibenzocyclooctyl (DBCO), bicyclo[6.1.0]nonyne (BCN);

[0037] Ab, W, L1, L2, L3, D, n2, n3 are defined as described above;

[0038] Method 1: Preparation of Antibody-Drug Conjugate I-1

[0039] Step a: The toxin small molecule drug-disaccharide oxazoline complex D-ox reacts with the wild-type antibody Ab under the action of the wild-type endoglycosidase Endo-S2 to obtain the antibody-drug conjugate I-1 (ADC-5);

[0040] Method 2: Preparation of Antibody-Drug Conjugate I-2

[0041] Step b: Azide disaccharide oxazoline G1 reacts with wild-type antibody under the action of endoglycosidase Endo-S2 to obtain non-natural glycoengineered antibody Ab-1;

[0042] Step c: Non-natural glycoengineered antibody Ab-1 and toxin small molecule drug containing an orthogonal group capable of reacting with the azide group (N3) - linker Z-L3-(L1) n2 -(L2-D) n3 (i.e. compounds D1-D4, D6) were reacted to obtain antibody-drug conjugate I-2 (ADC-1 to ADC-4 and ADC-6).

[0043] In a specific embodiment, in step a of the above-mentioned preparation method 1, the concentrations of the toxin small molecule drug-disaccharide oxazoline complex D-ox, the wild-type antibody, and the wild-type endoglycosidase Endo-S2 are 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively; the reaction conditions are: pH 6.5, and incubation at 30°C for 1 hour.

[0044] In a specific embodiment, in method 1, the toxin small molecule drug-disaccharide oxazoline complex D-ox has the structure shown below:

[0045] In step b of the above preparation method 2, the concentrations of azidodisaccharide oxazoline G1, wild-type antibody, and endoglycosidase Endo-S2 are 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively. The reaction conditions are: pH 6.5, and incubation at 30°C for 1 hour.

[0046] In step c of the above preparation method 2, the concentrations of the non-natural glycoengineered antibody Ab-1 and the toxin small molecule drug-linker were 5 mg / mL and 0.3 mM respectively; the reaction was carried out in a 50 mM PB, pH 7.4 buffer system at 37°C.

[0047] In a specific embodiment, in method 2, the toxin small molecule drug-linker Z-L3-(L1) n2 -(L2-D) n3 A compound selected from the following structures:

[0048] The second aspect of the present invention provides use of the antibody-drug conjugate targeting TROP2 described in the first aspect and the antibody-drug conjugate prepared by the method described in the second aspect in the preparation of anti-tumor drugs.

[0049] In a specific embodiment, the tumor is a tumor with high expression of TROP2. Preferably, the tumor includes: breast cancer (especially breast squamous cell carcinoma), gastric cancer, pancreatic cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, ovarian cancer, urothelial carcinoma, lung cancer, especially breast cancer (especially breast squamous cell carcinoma), gastric cancer. Beneficial effects

[0050] This invention utilizes novel sugar-targeted technology to specifically link cytotoxins such as MMAE and Eribulin to antibody glycosylation sites. The resulting ADC compounds exhibit excellent in vitro homogeneity, stability, and toxicity against cells with high TROP2 expression. In particular, ADCs 1-2, 5-6 exhibit little toxicity against cells with low TROP2 expression, and even more particularly, ADC-5 exhibits little toxicity against cells with moderate TROP2 expression. This selectivity improves drug safety.

[0051] In the NCI-N87 tumor model, ADC-5 exhibited better in vivo tumor inhibitory activity than other sugar chain-directed ADC compounds (e.g., DS1062, ADC-7). BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG1 shows the hydrophobic interaction chromatography (HIC) analysis data of the antibody-drug conjugate ADC in Test Example 1.

[0053] FIG2 shows the size exclusion chromatography (SEC) analysis data of the antibody-drug conjugate ADC in Test Example 2.

[0054] FIG3 shows the reverse phase (RP) analysis data of the antibody-drug conjugate ADC in Test Example 3.

[0055] FIG4 shows the size exclusion chromatography (SEC) analysis data of the antibody-drug conjugate ADC aggregation stability test in Test Example 4.

[0056] FIG5 shows the in vitro activity experimental data results of the antibody-drug conjugate ADC in Pharmacological Example 1.

[0057] FIG6 shows the in vivo activity experimental data of the antibody-drug conjugates ADC-5 and ADC-7 in Pharmacological Example 2 in NCI-N87 tumor-bearing mice. DETAILED DESCRIPTION

[0058] The endoglycosidase used in the present invention is expressed in the Escherichia coli system and is laboratory expressed. The small molecule cytotoxic drugs Eribulin and monomethyl auristatin E (MMAE) used were purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.; DBCO compounds (such as compound 5) and BCN compounds (such as compound 7 and compound 25) were purchased from Chengdu Baierkang Biotechnology Co., Ltd.; 3-azidopropylamine was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. K2HPO4, (NH4)2SO4, KH2PO4 were purchased from Bid Pharmaceutical; 20×PBS was purchased from Sinopharm Group. Other compounds and reagents, unless further explained, were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0059] The instruments used in the present invention include: high-resolution mass spectrometer (Waters Xevo G2-XS QTOF), analytical high-performance liquid chromatograph (Thermo ultimate 3000), high-performance liquid chromatograph (WATERS W2690), high-performance liquid chromatograph (Angilent 1260), analytical high-performance liquid chromatograph (Beijing Innovation Tongheng LC3000), preparative high-performance liquid chromatograph (Beijing Innovation Tongheng LC3000). Chromatographic columns include: Thermo C18 (Acclaim TM 120, 5μm, 4.6 x 250mm), Agilent SB-C18 (5μm, 4.6 x 150mm), Waters C18 column (ACQUITY UPLC BEH C18, 1.7μm, 2.1 x 50mm). Waters C4 column (ACQUITY UPLC Protein BEH C4, 1.7μm, 2.1mm x 50mm). Thermo, MAbPac HIC-Butyl column (4.6 x 100mm, 3.5μm). Agilent PLRP-S Chromatographic column (4.6 x 250mm, 8μm). BioCore SEC-300 chromatography column (5μm, 7.8*300mm).

[0060] I: Synthesis of Azidodisaccharide Oxazoline G1

[0061] Step 1: CHO-LacNAc 1 (18 mg, 47.2 μmoL) was weighed and dissolved in 200 μL of 50 mM PB, pH 7.4 buffer. O-(2-azidoethyl)hydroxylamine hydrochloride (5.3 mg, 51.9 μmoL) was added to the above reaction system and reacted at 37°C for 2 h. LC-MS monitored the reaction completion. Compound 2 was isolated and purified using a semi-preparative C18 column to obtain compound 2 (20 mg, 91% yield). HRMS, calculated value: C16H 27 N5O 11 [M+H] + 466.1785, measured value 466.1732.

[0062] Step 2: Weigh compound 2 (10 mg, 21.4 μmoL) and dissolve it in 428 μL D2O. Add 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride (CDMBI, 23.1 mg, 107 μmoL) to the above system, mix well and place on ice to cool to 0°C. Add potassium phosphate (68.5 mg, 321 μmoL) and react at 0°C for 12 hours. LC-MS monitoring of the reaction system showed that the reaction was complete. A semi-preparative C18 column was used for separation and purification. The target product was collected and lyophilized to obtain compound G1. HRMS, calculated value: C16H 25 N5O 10 [M+H] + 448.1679, measured value 448.1662.

[0063] II: Synthesis of non-natural glycoengineered antibodies

[0064] The non-natural glycoengineered antibody Ab-1 is obtained by compound G1 and wild-type Sacituzumab antibody through the following step b method of the general operation 2 for ADC preparation.

[0065] The preparation method of the non-natural glycoengineered antibody Ab-2 is as follows:

[0066] Wild-type sacituzumab antibody, endoglycosidase Endo-S, phosphatase (AP), UDP-6-N3-GalNAc3, and TnGalNAcT (purchased from Wuhan Tangzhi Pharmaceutical Co., Ltd.) were sequentially added to a buffer solution (20 mM histidine, 150 mM NaCl, pH 6.5) to adjust the reaction concentrations to 7 mg / mL, 0.08 mg / mL, 0.75 μg / mL, 0.5 mM, and 0.2 mg / mL, respectively. The pH of the reaction system was adjusted to 7.5, and 10% 10 mM MnCl2 was added to the above system. The mixture was incubated at 30°C for 16 hours. After LC-MS confirmed the conversion to a product with two azides, the desired glycoengineered antibody Ab-2 was purified using Protein A.

[0067] III: Preparation of small molecule drug-linkers

[0068] Example 1: Synthesis of Compound D1

[0069] Steps: Compound 5 (8.06 mg, 0.0264 mM) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 16.73 mg, 0.044 mM) were weighed and dissolved in 650 μL N,N-dimethylformamide (DMF). Compound 4 (41 mg, 0.022 mM) and N,N-diisopropylethylamine (DIEA, 11.5 μL, 0.066 mM) were added to the above system in sequence. After the reaction system was mixed, it was reacted at room temperature for 2 hours. LC-MS monitoring of the reaction system showed that the reaction was complete. Semi-preparative C18 column was used for separation and purification. The target product was collected and lyophilized to obtain compound D1 (21.7 mg, 45.8% yield). HRMS: [M+H] + 2153.283.

[0070] Example 2: Synthesis of Compound D2

[0071] Step 1: Compound 7 (31 mg, 86 μM) was weighed and dissolved in 0.4 mL of N,N-dimethylformamide (DMF). Compound 8 (36 mg, 18 μM) and triethylamine (20 μL, 14.2 μM) were added to the above system in sequence. The reaction was carried out at room temperature for 20 minutes. The reaction system was directly carried out to the next step without purification.

[0072] Step 2: Compound 9 (75 μL, 20.5 μM) and triethylamine (20 μL, 14.2 μM) were added to the crude product (31 mg, 32.4 μM) in sequence. The reaction was continued for 1 hour and monitored by LC-MS to obtain compound 10. The reaction system was directly carried out to the next step without purification.

[0073] Step 3: Compound 11 (62.4 mg, 32.4 μM) and triethylamine (20 μL, 14.2 μM) were added to the crude product and reacted for 1 hour. After the reaction was completed as monitored by LC-MS, compound 12 was separated and purified using a semi-preparative C18 column and then lyophilized to obtain compound 12 in a yield of 33.3%.

[0074] Step 4: Compound 12 (16 mg, 19.48 μM) was weighed and dissolved in 1 mL of N,N-dimethylformamide (DMF). Compound 13 (54.7 mg, 48.7 μM) and triethylamine (10.8 μL, 7.92 μM) were added to the above system in sequence. The reaction was incubated at 37°C for 2 hours and monitored by LC-MS. After the reaction was complete, the target fraction was separated and purified by reverse phase chromatography and lyophilized to obtain compound D2 as a white solid. HRMS: [M+H] + 2788.5628.

[0075] Example 3: Synthesis of Compound D3

[0076] Step 1: Compound 14 (11.48 mg, 0.0205 mM) and 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU, 11.67 mg, 0.0307 mM) were weighed and dissolved in 2 mL of N, N-dimethylformamide (DMF). Compound 15 (15 mg, 0.0205 mM) and N, N-diisopropylethylamine (DIEA, 7.94 mg, 0.0614 mM) were added to the above system in sequence. After the reaction system was mixed, the reaction was allowed to react at room temperature for 2 hours. LC-MS showed that there was basically no starting material remaining, and the main peak was the product. The reaction solution was directly used for the next chemical reaction. HRMS: [M+H-18] + 1271.8.

[0077] Step 2: Diethylamine (DEA, 14.99 mg, 0.205 mM) was added dropwise to the reaction mixture from the previous step and stirred at room temperature for 1 hour. LC-MS showed that the reaction of the raw materials was complete and product was generated. The reaction mixture was centrifuged and semi-preparative separation and purification was performed. The target product was collected and lyophilized to obtain compound 16 (14 mg, yield 65.15%). HRMS: [MH] + 1048.4.

[0078] Step 3: Weigh compound 17 (4.12 mg, 0.0067 mM) and 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU, 7.64 mg, 0.0201 mM) and dissolve them in 2 mL of N, N-dimethylformamide (DMF). Add compound 16 (14 mg, 0.0134 mM) and N, N-diisopropylethylamine (DIEA, 5.20 mg, 0.0402 mM) to the above system in sequence. After the reaction system is mixed, react at room temperature for 2 hours. LC-MS shows that there is basically no starting material remaining, the main peak is the product, and the reaction solution is directly used for the next chemical reaction. HRMS: [M / 2+H] + 1339.2.

[0079] Step 4: Diethylamine (DEA, 9.93 mg, 0.134 mM) was added dropwise to the reaction mixture from the previous step and stirred at 25°C for 1 hour. LCMS indicated that the reaction of the starting materials was complete and product was generated. The reaction mixture was centrifuged and semi-preparative purification was performed. The target product was collected and lyophilized to obtain compound 18 (8 mg, 24.32% yield). HRMS: [M / 2+H] + 1228.2.

[0080] Step 5: Compound 5 (2.0 mg, 0.0066 mM) and 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU, 1.88 mg, 0.0049 mM) were dissolved in 2 mL of N,N-dimethylformamide (DMF). Compound 18 (8.0 mg, 0.0033 mM) and N,N-diisopropylethylamine (DIEA, 1.28 mg, 0.0099 mM) were added sequentially to the above system. The reaction system was mixed and reacted at room temperature for 2 hours. LC-MS showed that the starting materials had reacted completely and product was produced. The reaction solution was centrifuged and semi-preparative separation and purification were performed. The target product was collected and lyophilized to obtain compound D3 (5.2 mg, 40.32% yield). HRMS: [M+H] + 2742.19.

[0081] Example 4: Synthesis of Compound D4

[0082] Step 1: Weigh compound 19 (15.72 mg, 0.0205 mM) and 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU, 11.67 mg, 0.0307 mM) and dissolve them in 2 mL of N, N-dimethylformamide (DMF). Add compound 15 (15 mg, 0.0205 mM) and N, N-diisopropylethylamine (DIEA, 7.95 mg, 0.0615 mM) to the above system in sequence. After the reaction system is mixed, react at room temperature for 2 hours. LC-MS shows that there is basically no starting material remaining, the main peak is the product, and the reaction solution is directly used for the next chemical reaction. HRMS: [M+H] + 1357.8.

[0083] Step 2: Diethylamine (DEA, 14.99 mg, 0.205 mM) was added dropwise to the reaction mixture from the previous step and stirred at 25°C for 1 hour. LC-MS showed that the reaction of the starting materials was complete and product was generated. The reaction mixture was centrifuged and semi-preparative separation and purification were performed. The target product was collected and lyophilized to obtain compound 20 (18 mg, 77.34% yield). HRMS: [MH] + 1135.8.

[0084] Step 3: Weigh compound 17 (3.26 mg, 0.00528 mM) and 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU, 6.02 mg, 0.0158 mM) and dissolve them in 2 mL of N, N-dimethylformamide (DMF). Add compound 20 (12 mg, 0.0105 mM) and N, N-diisopropylethylamine (DIEA, 4.09 mg, 0.0317 mM) to the above system in sequence. After the reaction system is mixed, react at room temperature for 2 hours. LC-MS shows that there is basically no starting material remaining, the main peak is the product, and the reaction solution is directly used for the next chemical reaction. HRMS: [M+H] + 1426.3.8.

[0085] Step 4: Diethylamine (DEA, 7.78 mg, 0.105 mM) was added dropwise to the reaction mixture from the previous step and stirred at 25°C for 1 hour. LC-MS showed that the reaction of the starting materials was complete and product was generated. The reaction mixture was centrifuged and semi-preparative separation and purification were performed. The target product was collected and lyophilized to obtain compound 21 (5.5 mg, 19.92%). HRMS: [M / 2+H] + 1315.1.

[0086] Step 5: Compound 5 (1.28 mg, 0.0042 mM) and 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU, 1.20 mg, 0.00315 mM) were dissolved in 2 mL of N,N-dimethylformamide (DMF). Compound 21 (5.5 mg, 0.0021 mM) and N,N-diisopropylethylamine (DIEA, 0.81 mg, 0.0063 mM) were added sequentially to the solution. The reaction system was mixed and allowed to react at room temperature for 2 hours. LC-MS showed that the starting materials had reacted completely and product was produced. The reaction solution was centrifuged and semi-preparative purification was performed. The target product was collected and lyophilized to obtain compound D4 (1.7 mg, 27.76% yield). HRMS: [M+H] + 1459.7.

[0087] Example 5: Synthesis of Compound D5

[0088] Step 1: Compound 22 (20 mg, 0.0178 mM) was weighed and dissolved in 200 μL N,N dimethylformamide (DMF). Compound 1 (27 mg, 0.0712 mM) and sodium cyanoborohydride (NaCNBH3, 22.4 mg, 0.356 mM) were added to the above system in sequence. After the reaction system was mixed evenly, it was reacted at 37°C overnight. LC-MS monitoring of the reaction system showed that most of the product was generated. A semi-preparative C18 column was used for separation and purification. The target product was collected and lyophilized to obtain product 24 (15 mg, 56.6% yield). HRMS: [M+H] + 1488.8453.

[0089] Step 2: Compound 24 was weighed and dissolved in deionized water (ddH2O). 2-1,3-Dimethylimidazolium chloride (DMC) and triethylamine were added sequentially to final concentrations of 10 mM, 200 mM, and 600 mM, respectively. The mixture was mixed and reacted at 4°C for 4 h. LC-MS monitoring of the reaction system showed that the reaction was almost complete. The target product was isolated and purified using a basic semi-preparative C18 column and lyophilized to obtain D5. HRMS: [M+2H] 2+ 735.9212.

[0090] Example 6: Synthesis of Compound D6

[0091] Step 1: Compound 25 (50 mg, 33 μM) was weighed and dissolved in 4 mL of dichloromethane. Compound 26 (29 μL, 20.4 μM) and triethylamine (40 μL, 27.2 μM) were added to the reaction system in sequence. After reacting at room temperature for 20 minutes, compound 27 (94 μL, 71 μM) was added and the reaction continued for 15 minutes, monitored by LC-MS. After the reaction was complete, 150 μL of dichloromethane was added for extraction, and the mixture was dried to obtain crude compound 7.

[0092] Step 2: Weigh the crude compound 7 (31 mg, 86 μM) from the previous step and dissolve it in 1 mL of N,N-dimethylformamide (DMF). Add compound 8 (36 mg, 18 μM) and triethylamine (20 μL, 14.2 μM). React at room temperature for 40 minutes. After monitoring the reaction completion by LC-MS, proceed directly to the next step.

[0093] Step 3: Compound 13 (54.7 mg, 48.7 μM) was added to the crude compound from the previous step (25.2 mg, 48 μM), followed by triethylamine (62.5 μL, 44.5 μM), and the mixture was allowed to react at room temperature for 2 hours. LC-MS monitored the reaction to completion. Compound D6 was isolated and purified using a semi-preparative C18 column and lyophilized to obtain compound D6 (yield 61.3%). HRMS: [M+H] + 1508.82.

[0094] IV: Synthesis of Glycan-Directed Antibody-Drug Conjugates ADC-1 to ADC-7

[0095] The following reaction scheme summarizes the preparation route of the sugar chain-directed antibody-drug conjugate (ADC) of the present invention.

[0096] General operation for ADC preparation:

[0097] Step a: The prepared toxin-disaccharide oxazoline complex D-ox (e.g., compound D5), wild-type sacituzumab antibody, and wild-type endoglycosidase Endo-S2 are added to 50 mM PB at concentrations of 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively. The pH of the reaction system is adjusted to 6.5, and the mixture is incubated at 30°C for 1 hour. After LC-MS confirms the conversion to a product with two disaccharide-toxin complexes, the target antibody-drug conjugate I-1 (e.g., ADC-5) is obtained by protein A purification.

[0098] General operation 2 for ADC preparation:

[0099] Step b: The prepared azidodisaccharide oxazoline G1, wild-type sacituzumab antibody, and endoglycosidase Endo-S2 were added to 50 mM PB to adjust the concentrations to 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 6.5, and the mixture was incubated at 30°C for 1 hour. Protein A was then used for purification to obtain the desired non-natural glycoengineered antibody Ab-1.

[0100] Step c: Add the non-natural glycoengineered antibody Ab-1 and drug-linker Z-L3-(L1) prepared above to a 50mM PB, pH 7.4 buffer system. n2 -(L2-D) n3 Compounds (e.g., compounds D1-D4, D6) were added to 50 mM PB containing no more than 20% DMF to a concentration of 5 mg / mL and 0.3 mM, respectively. After confirming the final pH of the reaction system was 7.4, the mixture was incubated at 37°C for 2 hours. After LC-MS confirmed complete conversion to product, protein A purification was performed to obtain the desired antibody-drug conjugate I-2 (e.g., ADC-1 to ADC-4 and ADC-6).

[0101] Note: When choosing general operation 1 or 2 to prepare ADC compounds, several factors need to be considered: (1) The enzyme recognition ability of endoglycosidase to different substrates is different. The enzyme recognition ability of molecules with large structures is weak, so general operation 2 should be used;

[0102] (2) The enzyme specifically recognizes the sugar oxazoline structure, which needs to be prepared under alkaline conditions. Drug-linkers that are intolerant to alkaline conditions need to use general operation 2.

[0103] Example 7: Synthesis of ADC-1 to ADC-4 and ADC-6

[0104] Antibody-drug conjugates ADC-1 to ADC-4 and ADC-6 were prepared by reacting compounds D1 to D4 and D6 with the non-natural glycoengineered antibody Ab-1 via step c of the general ADC preparation procedure 2. Their structures are as follows:

[0105] in,

[0106] Example 8: Synthesis of ADC-5

[0107] Antibody-drug conjugate ADC-5 is prepared by combining compound D5 and wild-type sacituzumab antibody using the ADC preparation general procedure 1. Its structure is as follows:

[0108] Comparative Example 1: Synthesis of ADC-7

[0109] The non-natural glycoengineered antibody Ab-2 prepared above and the drug-linker (i.e., compound D6) were added to a 50 mM PB, pH 7.4 buffer system to adjust the concentrations to 5 mg / mL and 0.3 mM, respectively. After confirming that the final pH of the reaction system was 7.4, the reaction was incubated at 37°C. After LC-MS confirmed complete conversion to product, protein A purification was performed to obtain the desired antibody-drug conjugate ADC-7.

[0110] Test Example 1: Hydrophobic Interaction Chromatography Detection of ADC Compounds

[0111] This test example performs hydrophobic interaction chromatography detection of ADC compounds according to the following conditions:

[0112] Sample preparation: Take ADC sample and dilute it to 1 mg / mL with 1 x PBS.

[0113] Mobile phase preparation: Take 88.3 g of ammonium sulfate and 7.878 g of sodium phosphate, dilute to 0.95 L with purified water, adjust the pH to 7.0, and add 50 mL of isopropanol to prepare phase A. Take 6.56 g of sodium phosphate, dilute to 0.8 L with purified water, adjust the pH to 7.0, and add 200 mL of isopropanol to prepare phase B. Sterilize by filtration through a 0.22 μm PES membrane, sonicate for 20 min, and set aside.

[0114] Chromatographic conditions:

[0115] Set the mobile phase gradient as shown in the following table:

[0116] The hydrophobic interaction chromatograms of the ADCs are shown in FIG1 , respectively, indicating that the ADCs prepared by the present invention have a good drug to antibody ratio (DAR) and most ADCs have good homogeneity.

[0117] Test Example 2: Size Exclusion Chromatography (SEC) Analysis of ADC

[0118] This test example performed SEC analysis of the ADC compound according to the following conditions:

[0119] Sample preparation: dilute the ADC to be tested to a concentration of 1 mg / mL with 1×PBS.

[0120] Chromatographic conditions:

[0121] The size exclusion chromatography (SEC) analysis of ADC is shown in FIG2 , indicating that the ADC prepared in the present invention has good purity.

[0122] Test Example 3: Reverse Phase (RP) Analysis of ADC

[0123] This test example performs RP analysis of ADC compounds according to the following conditions:

[0124] Sample preparation: dilute the ADC to be tested to a concentration of 1 mg / mL with 1×PBS.

[0125] Chromatographic conditions:

[0126] Set up the mobile phase gradient as follows:

[0127] Table 1. DAR values ​​of ADCs calculated based on RP results

[0128] The RP analysis data of the ADC are shown in FIG3 and Table 1, indicating that the antibody-drug ratio (DAR) of the ADC prepared by the present invention is good and is comparable to the theoretical DAR value.

[0129] Test Example 4: Aggregation stability analysis of ADC

[0130] The prepared ADC compounds ADC-1-2 and ADC5-6 were heated at 60° C., and samples were taken after 6 hours and 24 hours to observe their aggregation. The samples were then tested according to the size exclusion chromatography (SEC) analysis method for ADC in Test Example 2 above.

[0131] The observation results showed that ADC-1~2 and ADC-5~6 did not produce aggregation phenomenon. The SEC chromatograms are shown in Table 4, indicating that ADC-1~2 and ADC-5~6 have good aggregation stability.

[0132] Pharmacological Example 1: In vitro activity detection of ADC

[0133] TROP2 high-expressing cell lines BXPC-3, NCI-N87, MDA-MB-468, and SK-BR-3, TROP2 medium-expressing cell line COLO205, and TROP2 low-expressing cell line MDA-MB-231 were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The cell activity and cytotoxicity of the ADC molecules (ADC-1 to ADC-7) prepared above were tested by the MTT assay.

[0134] The specific procedure is as follows: 6000 cells / well of the cells to be tested are seeded into a 96-well cell culture plate and cultured overnight. The drug is diluted 5-fold starting from a maximum concentration of 100 nM, with nine dilution steps. Drug is added sequentially, with three replicates set up for each gradient. The outermost circle contains PBS; wells B11, C11, and D11 are negative controls (wells without drug); wells E11, F11, and G11 are blank controls (wells without either cells or drug). ADC-7 is used as a positive control.

[0135] After 144 hours of drug exposure, the original culture medium was discarded, and 10 μL of premixed culture medium containing MTT was added to each well. Incubation continued in a cell culture incubator for 3-4 hours. Subsequently, 100 μL of SDS lysis buffer was added to each well, and the cells were incubated overnight at 37°C to fully lyse the cells. Absorbance was measured at 570 nm. Data were processed using GraphPad Prism 8. The results are shown in Figure 5 and Table 2.

[0136] Analysis of results: Since the Trop2 target is widely distributed in normal tissues throughout the body, the selectivity of ADC drugs can be improved when the ADC drugs have high toxicity to Trop2-high-expressing cell lines but no toxicity or low toxicity to Trop2-medium-low-expressing cell lines. As shown in Figure 5 and Table 2, ADC-1, ADC-2, ADC-5, and ADC-7 showed high cytotoxicity against TROP2-high-expressing cells (BxPC-3, NCI-N87, MDA-MB-468, and SK-Br-3), and decreased toxicity against TROP2-moderate or low-to-medium-expressing cell lines (COLO205 and MDA-MB-231). In particular, ADC-5 and ADC-7 showed little toxicity against TROP2-moderate or low-to-medium-expressing cell lines, which may contribute to the improved safety profile of ADC-5. ADC-3, ADC-4, and ADC-6 showed high cytotoxicity against high-expressing (BxPC-3, NCI-N87, MDA-MB-468, and SK-Br-3) or medium-expressing (COLO205) cell lines, and good safety against the low-to-medium-expressing cell line MDA-MB-231, making them suitable for the treatment of certain indications with moderate or higher TROP2 expression.

[0137] Table 2. ADC-1 to ADC-7 ICs50 value

[0138] Pharmacological Example 2: In vivo antitumor activity experiment of ADC-5

[0139] A BALB / c nude mouse xenograft tumor model was constructed using gastric cancer cells NCI-N87 and human breast squamous cell carcinoma cells HCC1806. The mice were grouped into large, medium and small groups using ear hole marking, with six mice in each group.

[0140] ADC-5 and ADC-7 were selected for animal activity evaluation, DS-1062 was used as a positive control, and PBS was used as a negative control. All samples were sterilized using a 0.22 mm filter membrane before administration.

[0141] All samples were administered intravenously at concentrations of 1 mg / kg and 3 mg / kg, respectively, and administered once. Starting from the first dose, tumor size and mouse body weight were measured twice a week using a vernier caliper. The experimental procedures complied with animal ethics requirements. The measured data were analyzed using GraphPad Prism 8 software. The results are shown in Figure 6: In the NCI-N87 tumor model, ADC-5 had significantly better tumor inhibitory activity than ADC-7 and DS1062 at doses of 3 mg / kg and 1 mg / kg, demonstrating good in vivo tumor inhibitory activity.

Claims

1. An antibody-drug conjugate targeting TROP2, having a structure shown in the following formula I: in, m is 1 or 2, preferably 2; Ab represents an antibody targeting TROP2, which is an IgG having a conserved N-glycosylation site at N297 in the Fc region; Each X is independently -(CH=NO) p0 -(CH2) p1 ; wherein p0 is 0 or 1, p1 is an integer between 1 and 20, preferably an integer between 1 and 6; Y represents -W-L3-; where W represents a structure formed by an orthogonal group reaction to form a stable covalent bond, and the orthogonal group is selected from an azide (N3) group, dibenzocyclooctyl (DBCO), bicyclo[6.1.0]nonyne (BCN), and dibenzocyclooctyne (DIBO); L3 represents a linking group, the structure of which is selected from -(CH2) p2 -CO-, -(CH2CH2O) p3 -CO-, -(CH2) p4 -CONH-(CH2CH2O) p5 -(CH2) p6 -CO-; wherein p2, p3, p4, p5, and p6 are each independently selected from an integer between 1 and 20, preferably an integer between 2 and 6; n1 is 0 or 1; L1 represents a linking group having more than 3 linking sites, such as a linking group derived from glutamic acid (Glu), β-glutamic acid, lysine (Lys), Lys-Glu dipeptide or Lys-β-glutamic acid dipeptide, or a linking group derived from diethanolamine; L z It represents a short peptide linear linker with two or more linking sites, which may or may not be linked to p-aminobenzyl carboxylate, for example, a tumor microenvironment cleavable or non-cleavable structural unit such as Val-Cit-PAB, Val-Ala-PAB, Gly-Gly-Phe-Gly; Furthermore, L1 and L2 are optionally connected to a PEG chain at the end of the group they contain. For example, when L1 is a linker group derived from Lys, a PEG chain is further connected to the amino group of lysine via an amide bond. The PEG chain is, for example, -(CH2CH2O) p7 -CH3, wherein p7 is an integer from 2 to 20; D represents a group derived from a small molecule toxin drug; n2 is 0 and n3 is 1; or n2 is 1 and n3 is an integer of 2-4, for example, n2 is 1 and n3 is 2, 3 or 4, preferably n3 is 2; where, when n2 is 1, n3 L2-Ds are covalently linked to L1 respectively, and n3 ≤ the number of linking sites of L1 - 1.

2. The antibody-drug conjugate targeting TROP2 according to claim 1, characterized in that The Ab is an antibody selected from IgG1, IgG2 or IgG4, preferably IgG1; Specifically, the antibody is a monoclonal antibody, polyclonal antibody, bispecific antibody, trispecific antibody, nanobody fused with an Fc domain, therapeutic antibody or functional antibody from different species, and the species is selected from mouse, rat, sheep, rabbit and human; Specifically, the antibody includes sacituzumab, Datopotamab, preferably sacituzumab.

3. The antibody-drug conjugate targeting TROP2 according to claim 1, characterized in that The W structure is selected from and / or L1 represents a linking group derived from glutamic acid (Glu), lysine (Lys), or diethanolamine. Preferably, L1 is selected from and / or L2 is and / or The small molecule toxin includes MMAE, MMAF, Eribulin, Dxd, Exatecan, DM1 or DM4; preferably, the small molecule toxin is a microtubule inhibitor MMAE or Eribulin; Specifically, D is selected from the structures shown below:

4. The antibody-drug conjugate targeting TROP2 according to claim 1, characterized in that The antibody-drug conjugate targeting TROP2 has a structure as shown in the following Formula I-1 or Formula I-2: where W, L1, L2, L3, D, n2, n3, and the antibody definition are as described in claim 1 respectively.

5. The antibody-drug conjugate targeting TROP2 according to claim 1, characterized in that The DAR value of the antibody-drug conjugate targeting TROP2 is 0 < DAR ≤ 4, preferably 0.5 ≤ DAR ≤ 4, more preferably, the DAR value is 1-4, still more preferably, the DAR value is 2 or 4.

6. The antibody-drug conjugate targeting TROP2 according to any one of claims 1 to 5, characterized in that: The antibody-drug conjugate targeting TROP2 has the following structure: Among them, in the above-mentioned ADC-1 to 6, the antibody is certolizumab; Particularly, the antibody-drug conjugate targeting TROP2 has the structure shown in ADC-5.

7. A method for preparing the TROP2-targeting antibody-drug conjugate I-1 or I-2 according to claim 4, wherein the preparation method is as follows: in, Z represents an orthogonal group that can react with an azide group to form the W structure, such as dibenzocyclooctyl (DBCO), bicyclo[G.1.0]nonyne (BCN); The definitions of Ab, W, L1, L2, L3, D, n2, n3 are as described in claim 1 respectively; Method 1: Preparation of antibody-drug conjugate I-1 Step a: The small molecule toxin drug-disaccharide oxazoline complex D-ox reacts with the wild-type antibody Ab under the action of the wild-type glycoside endonuclease Endo-S2 to obtain the antibody-drug conjugate I-1; Method 2: Preparation of antibody-drug conjugate I-2 Step b: Azidodisaccharide oxazoline G1 reacts with the wild-type antibody under the action of the glycoside endonuclease Endo-S2 to obtain the unnatural sugar engineered antibody Ab-1; Step c: Non-natural glycoengineered antibody Ab-1 and toxin small molecule drug containing an orthogonal group capable of reacting with an azide group - linker Z-L3-(L1) n2 -(L2-D) n3 The reaction yielded antibody-drug conjugate I-2.

8. The preparation method according to claim 7, characterized in that In step a of method 1, the concentrations of the toxin small molecule drug-disaccharide oxazoline complex D-ox, the wild-type antibody, and the wild-type endoglycosidase Endo-S2 are 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively; the reaction conditions are: pH 6.5, incubation at 30°C for 1 hour; and / or In step b of method 2, the concentrations of azidodisaccharide oxazoline G1, wild-type antibody, and endoglycosidase Endo-S2 are 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively, and the reaction conditions are: pH 6.5, incubation at 30°C for 1 hour; and / or In step c of method 2, the non-natural glycoengineered antibody Ab-1 and the toxin small molecule drug-linker Z-L3-(L1) n2 -(L2-D) n3 The concentrations were 5 mg / mL and 0.3 mM; the reaction was carried out in 50 mM PB, pH 7.4 buffer system at 37°C.

9. The preparation method according to claim 7, characterized in that In step a of method 1, the toxin small molecule drug-disaccharide oxazoline complex D-ox has the following structure: and / or In step c of method 2, the toxin small molecule drug-linker Z-L3-(L1) n2 -(L2-D) n3 A compound selected from the following structures:

10. Use of the antibody-drug conjugate targeting TROP2 according to any one of claims 1 to 6 and the antibody-drug conjugate prepared by the method according to any one of claims 7 to 9 in the preparation of anti-tumor drugs; Preferably, the tumor is a tumor with high expression of TROP2. Preferably, the tumor comprises: Breast cancer (especially squamous cell carcinoma of the breast), gastric cancer, pancreatic cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, ovarian cancer, urothelial carcinoma, lung cancer, especially breast cancer (especially squamous cell carcinoma of the breast), gastric cancer.

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