WO2018036403A1 - 一种寡糖连接子以及利用该寡糖连接子制备的定点连接的抗体-药物偶联物 - Google Patents
一种寡糖连接子以及利用该寡糖连接子制备的定点连接的抗体-药物偶联物 Download PDFInfo
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- WO2018036403A1 WO2018036403A1 PCT/CN2017/097439 CN2017097439W WO2018036403A1 WO 2018036403 A1 WO2018036403 A1 WO 2018036403A1 CN 2017097439 W CN2017097439 W CN 2017097439W WO 2018036403 A1 WO2018036403 A1 WO 2018036403A1
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Definitions
- the present invention relates to a novel oligosaccharide linker, a site-directed antibody-drug conjugate prepared using the oligosaccharide linker, and a process for the preparation and use thereof.
- Antibody-based biomacromolecules occupy an important position in the field of cancer therapy.
- IgG-type monoclonal antibodies are an important class of therapeutic proteins for the treatment of cancer, autoimmune diseases and infectious diseases.
- ADCs Antibody Drug Conjugates
- the antibody-drug conjugate is mainly composed of an antibody, a small molecule cytotoxic drug, and a linker fragment, and the small molecule cytotoxic component is specifically transported to the tumor tissue by the antigen-targeting antibody, thereby exerting the function of killing the cancer cell.
- An IgG antibody consists of two heavy chains and two light chains that combine to form three different proteins joined by a flexible hinge region, including two variable Fab domains and a constant (crystallizable) ) Fc domain.
- Fab domains are involved in the recognition and binding of antibody-antigens, while Fc domains are involved in Fc receptor-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
- the Fc domain is a homodimer with two N-glycans at a conserved N-glycosylation site (Asn297), and the attached oligosaccharide is a biantennary complex with considerable Structural heterogeneity.
- the glycoform of the wild type monoclonal antibody Fc domain mainly includes galactose and its deleted N-oligosaccharide chain.
- the main feature of the present invention is that a non-homogeneous N-glycan chain of the Asn297 site in the Fc region is transformed into a single-structure N-glycan chain with a reactive group by a sugar chain modification technique, and the reactive group carried by the sugar chain is utilized.
- the group realizes site-directed coupling of small molecule drugs with antibodies to obtain site-directed antibody-drug conjugates.
- Another object of the present invention is to provide a process for the preparation of the oligosaccharide linker.
- a further object of the present invention is to provide a site-directed antibody-drug conjugate prepared from the oligosaccharide linker, which is catalyzed by a glycosidase ligase to the Fc region glycosylation site of the antibody Asn297, a small molecule drug is coupled at the Y position of the non-natural sialic acid structure at the end of the oligosaccharide linker by a suitable ligation fragment to form a fixed point.
- Linked antibody-drug conjugate prepared from the oligosaccharide linker, which is catalyzed by a glycosidase ligase to the Fc region glycosylation site of the antibody Asn297
- Still another object of the invention is to provide a process for the preparation of said antibody-drug conjugate.
- a dual drug conjugated antibody-drug conjugate is provided.
- It is still another object of the present invention to provide a pharmaceutical composition comprising the antibody-drug conjugate and/or the bis-conjugated antibody-drug conjugate.
- the advantage of the site-directed coupling of the present invention is that the problems of drug heterogeneity, decreased antigen binding force, and drug inhomogeneity caused by random coupling of the conventional antibody-drug conjugate on the antibody amino acid residues are avoided.
- the novelty of the present invention resides in that the novel modified oligosaccharide linker is specifically ligated to the Fc region glycosylation site Asn297 under the enzymatic catalysis, while the modifying group on the oligosaccharide linker provides a small molecule drug site The reaction sites are coupled to form a novel structure-directed linked antibody-drug conjugate molecule.
- the present inventors utilized the conserved N-glycosyl group of the Fc domain as a site for site-directed linkage, and designed and prepared an oligosaccharide linker having a reactive group fragment by using an antibody glycosylation modification method of the antibody Fc domain.
- the enzyme-catalyzed reaction is site-directed to the N-glycosylation site Asn297 of the Fc region of the antibody, and the introduced reactive group is further coupled with a small molecule drug to achieve antibody-drug conjugates site-directed to the Fc glycosylation site Asn297. .
- the present inventors utilized the hydrolysis and transglycosylation reactivity of a class of endo- ⁇ -N-acetylglucosaminidase to perform glycosylation modification and modification of the antibody Fc domain.
- the glycosidase can specifically hydrolyze the N-glycan of the Fc region of the IgG antibody by cleaving the ⁇ -1,4-glycosidic bond in the chitosan core of the N-glycan to obtain a glycoform structure of acetylglucosamine.
- An antibody modified with sugar or fucose ⁇ 1,6 acetylglucosamine disaccharide An antibody modified with sugar or fucose ⁇ 1,6 acetylglucosamine disaccharide.
- the oligosaccharide linker of formula (I) with a reactive group acts as an enzyme-catalyzed transglycosylation reaction substrate, and is efficiently and specifically linked to the antibody Fc region N- A glycosylation site (Asn297), a reactive group is introduced into the antibody, and the subsequent reactive group is coupled to a small molecule drug to effect a site-linked antibody-drug conjugate.
- Endo-S Endo-S
- Endo-F3 Endo-F3
- a small molecule drug can be controlled to control the position and number of small molecule coupling, and a uniform antibody-drug conjugate can be prepared, avoiding the traditional random coupling antibody-drug
- the heterogeneous structure of the conjugate has obvious advantages in drug quality control, drug stability, and consistency of metabolic behavior in vivo.
- n may be an integer selected from 0-6;
- Y can be selected from the following groups:
- Z may be selected from the group consisting of:
- R 1 and R 2 may be independently selected from H or CH 3 , respectively.
- L may be selected from the following groups:
- a, c and d may each independently be an integer between 2 and 6; b may be an integer selected from 3 to 20,
- R 3 and R 4 may be independently selected from CH 3 -, CH 3 CH(CH 3 )-, PhCH 2 -, NH 2 (CH 2 ) 4 -, NH 2 CONH(CH 2 ) 3 -;
- D may be a small molecule drug or a small molecule active compound, preferably selected from but not limited to maytansin, DM-1, MMAE, MMAF, MMAP, auristatin E, vincristine, vinblastine, vinorelbine, VP- 16. Camptothecin, SN-38, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicine, estramustine, simultane, and eleugen Loss or the like; or more preferably selected from the group consisting of:
- the oligosaccharide linker represented by the formula (I) is selected from the following compounds:
- the method comprises the steps of: oxidizing a sialic acid glycopeptide obtained by extracting egg yolk to obtain a sialic acid glycopeptide having an aldehyde group reactive group, and the aldolated sialic acid glycopeptide is subjected to a reductive amination reaction to introduce another living organism Orthogonal reaction value functional groups (such as azide, alkynyl, etc.), and under the action of endoglycosidase, release oligosaccharide chains with special labeling groups, and obtain oligosaccharide linkers through a cyclization process;
- Orthogonal reaction value functional groups such as azide, alkynyl, etc.
- the method comprises the steps of: preparing a oligosaccharide linker by preparing a sialic acid glycopeptide obtained by extracting egg yolk through a glycosidase hydrolysate terminal sialic acid oligosaccharide chain through oxidation, reductive amination reaction, or cyclization process;
- the method comprises the steps of: oxidizing a sialic acid glycopeptide obtained by egg yolk extraction to obtain a sialic acid glycopeptide having an aldehyde group reactive group, which is hydrolyzed by a glycosidase and subjected to a reduced amine The reaction, introducing other functional groups with bioorthogonal reaction value (such as azide, alkynyl, etc.), and releasing the oligosaccharide chain with special labeling group under the action of endoglycosidase, and obtained through the cyclization process Oligosaccharide linker.
- bioorthogonal reaction value such as azide, alkynyl, etc.
- the extraction of the sialic acid glycopeptide may comprise the steps of: lyophilizing the egg yolk powder, which is sequentially washed by diethyl ether (for example, 2 times) and 70% aqueous acetone solution (for example, 2 times). Most of the fat-soluble impurities and the like are removed, and the obtained solid is further extracted (for example, twice) using a 40% aqueous acetone solution, and the resulting solution is concentrated and adsorbed using activated carbon, and pure water, 5% acetonitrile aqueous solution, and 20% acetonitrile aqueous solution are sequentially used.
- the oxidation of the sialic acid glycopeptide may comprise the following steps:
- sialic acid glycopeptide was oxidized in a phosphate buffer using sodium periodate in an ice bath for 15 minutes.
- the egg yolk sialogopeptide is oxidized by sodium periodate to obtain a sialic acid glycopeptide having two aldehyde groups, and then the aldehyde group is subjected to reductive amination, coupling reaction, oxime reaction, The Wittig reaction or the like introduces a specific reactive functional group or other small molecule to obtain a structurally modified non-natural sialic acid glycopeptide.
- the non-native glycopeptides are enzymatically hydrolyzed to remove the polypeptide
- the acetylglucosamine at the reducing end of the sugar chain is cyclized to form the corresponding oligosaccharide linker.
- the oligosaccharide linker can also be prepared in one step from the aldehyde sialic acid glycopeptide by a one-pot method in combination with the above steps.
- an antibody-drug conjugate based on a site-directed N-glycosylation site (Asn297) of an antibody Fc region represented by the following formula (II):
- n may be an integer selected from 0 to 6; m, p and q may each independently be selected from 0 or 1;
- Ab is an antibody, which may preferably be selected from, but not limited to, the following antibodies: trastuzumab, pertuzumab, rituximab, cetuximab, morozumab, gemtuzumab, Abciximab, daclizumab, adalimumab, palivizumab, basiliximab, bevacizumab, panitumumab, nimotuzumab, denisizumab, etc.;
- the Y group can be D-L-Z-
- Z may be selected from the group consisting of:
- R 1 and R 2 may be independently selected from H or CH 3 , respectively.
- L can be selected from the group consisting of:
- a, c and d may each independently be an integer selected from 2 to 6; b may be an integer selected from 3 to 20,
- R 3 and R 4 may be independently selected from the group consisting of CH 3 -, CH 3 CH(CH 3 )-, PhCH 2 -, NH 2 (CH 2 ) 4 -, NH 2 CONH(CH 2 ) 3 -,
- D may be a small molecule drug or a small molecule active compound, preferably selected from but not limited to maytansin, DM-1, MMAE, MMAF, MMAP, auristatin E, vincristine, vinblastine, vinorelbine, VP- 16. Camptothecin, SN-38, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicine, estramustine, simultane, and eleugen Loss or the like; or more preferably selected from the group consisting of:
- An antibody for example, an antibody prepared in a eukaryotic expression system
- a glycosidase is cleaved by a glycosidase to obtain an N-glycosylation site (Asn297) of the Fc region as an acetylglucosamine monosaccharide or a fucoid a sugar ⁇ 1,6 acetylglucosamine disaccharide modified antibody;
- the glycosidase or a mutant thereof may be fucose hydrolyzed
- the small molecule drug having a corresponding group modification capable of performing a specific coupling reaction with the specific reactive group on the sugar chain of the antibody is selected from, but not limited to, The following structural compounds:
- a double drug-conjugated antibody-drug conjugate is provided, that is, at a point of formula (II) Based on the conjugated antibody-drug conjugate, the amino group of the lysine contained on the antibody is linked to another different small molecule drug to form a two drug-conjugated antibody-drug conjugate.
- the double drug-conjugated antibody-drug conjugate is preferably a structure represented by the following formula (III):
- the lysine-linked small molecule drug is maytansine DM1; the Asn297 sugar chain-linked small molecule drug has the same definition as the antibody (II) antibody drug conjugate.
- a site-directed antibody-drug conjugate of the formula (II) prepared from the oligosaccharide linker of the formula (I), the chemical structure of which is a site-specific quantitative homogeneous structure, and a non-homogeneous structure of a marketed antibody-drug Compared with the conjugate, it has the advantages of clear structure, controllable quality and so on. Meanwhile, the antibody is preferably connected to the point - drug conjugates exhibit good anti-tumor activity, IC 50 can be achieved 0.12nM (0.018ug / mL).
- 1 is a schematic diagram showing the structure of a non-native glycosylated antibody 7a according to an embodiment of the present invention.
- FIG. 2A shows a mass spectrum data map (m/z) of antibody 7a
- FIG. 2B shows a SDS-PAGE pattern of antibody 7a prepared in one pot
- FIG. 2C shows a full-length molecular weight mass spectrum data of antibody 7a.
- Fig. 3 is a schematic diagram showing the structure of a non-naturally glycosylated antibody 7b according to an embodiment of the present invention.
- FIG. 4A shows a mass spectrum data chart (m/z) of 7b
- FIG. 4B shows a SDS-PAGE pattern of antibody 7b prepared in one pot
- FIG. 4C shows a full-length molecular weight mass spectrum data of antibody 7b.
- Fig. 5 is a schematic diagram showing the structure of a non-naturally glycosylated antibody 7c according to an embodiment of the present invention.
- Fig. 6A shows a mass spectrum data chart (m/z) of 7c
- Fig. 6B shows a SDS-PAGE pattern of one-pot preparation of antibody 7c
- Fig. 6C shows a full-length molecular weight mass spectrum data of antibody 7c.
- Figure 7 is a schematic diagram showing the structure of a non-native glycosylated antibody 7d according to an embodiment of the present invention.
- Fig. 8A shows a mass spectrum data chart (m/z) of 7d
- Fig. 8B shows a SDS-PAGE pattern of a one-pot preparation antibody 7d
- Fig. 8C shows a full-length molecular weight mass spectrum data of antibody 7d.
- Figure 9 is a schematic diagram showing the structure of a non-naturally glycosylated antibody 7e according to an embodiment of the present invention.
- Fig. 10A shows a mass spectrum data chart (m/z) of 7e
- Fig. 10B shows a SDS-PAGE pattern of one-pot preparation of antibody 7e
- Fig. 10C shows a full-length molecular weight mass spectrum data of antibody 7e.
- Figure 11 shows the structure of the compound of ADC-9a, the mass spectrum data (m/z) and the full-length mass spectrum data (inline).
- Figure 12 shows the structure of the compound of the ADC-9b, the mass spectrum data (m/z) and the full-length mass spectrum data (inline).
- Figure 13 shows the compound structure, mass spectrometry data (m/z) and full-length mass spectrometry data (inline) of ADC-9c.
- Figure 14 shows the structure of the compound of the ADC-9d, the mass spectrum data (m/z) and the full-length mass spectrum data (inline).
- Figure 15 shows the structure of the compound of ADC-9e, mass spectrogram data (m/z) and full-length mass spectrometry data (inline).
- Figure 16 shows the structure of the compound of the ADC-9f, the mass spectrum data (m/z) and the full-length mass spectrum data (inline).
- Figure 17 shows the structure of the compound of the ADC-9g, the mass spectrum data (m/z) and the full-length mass spectrum data (inline).
- Figure 18 is a graph showing the structure of the compound of Compound 10 (ADC-10) and the full-length mass spectrum data, and the numbers in the figure are the number of randomly linked small molecules DM1.
- Figure 19 is a graph showing the structure of the compound of Compound 11 (ADC-11) and the full-length mass spectrum data, and the numbers in the figure are the number of randomly linked small molecules DM1.
- Figure 20 is a graph showing the anti-tumor activity of the antibody-drug conjugate examples.
- the glycoside hydrolase and its mutant enzyme used in the present invention are both expressed in an Escherichia coli system.
- the 3-azidopropylamine, propargylamine and acetonitrile used in the invention were purchased from Shanghai BEHRINGER Chemical Technology Co., Ltd.; the small molecule cytotoxic drugs DM1 and MMAE were purchased from Nanjing Lianning Biopharmaceutical Company; click on the chemical linker DBCO-amine and DBCO-PEG4-acid was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; amino acid compounds were purchased from Jill Biochemical (Shanghai) Co., Ltd.; other compounds, reagents and solvents were purchased from Sinopharm Chemical Reagent Co., Ltd. without further explanation. .
- the instruments and columns used in the present invention include: analytical high performance liquid chromatography (Beijing Innovation Tongheng LC3000), preparative high performance liquid chromatography (Beijing Innovation Tongheng LC3000); Agilent SB-C18 (5 ⁇ m, 4.6 x 150mm), Waters OBD-C18 (5 ⁇ m, 19 x 250 mm);
- Antibody molecular weight measuring instrument liquid chromatography-mass spectrometry (LC-MS), Agilent 6230LC-TOF-MS, with Agilent C-18 column (3.5 ⁇ m, 50 x 2.1 mm) at 55 °C.
- LC-MS liquid chromatography-mass spectrometry
- Agilent 6230LC-TOF-MS Agilent 6230LC-TOF-MS
- Agilent C-18 column 3.5 ⁇ m, 50 x 2.1 mm
- Sialyl glycopeptide 1 (100 mg) was dissolved in 5 mL of phosphate buffer (0.2 M, pH 7.1) while 5 mL of sodium periodate solution (30 mM) was added and stirred in an ice bath. After stirring for 15 minutes, purification was carried out with dextran G25, and the product fractions were collected and lyophilized to give the product compound 2 (90 mg, yield 94%).
- N-hydroxyphthalimide (1.14 g, 7.0 mmol) was added to a 25 mL eggplant flask and dissolved in 8 mL DMF, followed by dropwise addition of 1,2-dibromoethane (3 mL, 34.8 mmol).
- Triethylamine (2 mL, 14 mmol) was added and stirred at room temperature in the dark. The reaction was stirred overnight and was monitored by TLC. After the reaction was completed, the precipitate was filtered, and the precipitate was washed twice with DMF. A large amount of DMF was removed by a rotary evaporator, and the remaining solution was precipitated with a large amount of water, and the precipitate was collected and washed with water.
- the obtained compound S2a (500 mg, 1.85 mmol, 1.0 eq) was dissolved in 10 mL of acetone, and iced, and sodium azide (NaN 3 , 360 mg, 5.55 mmol) previously dissolved in 2 mL of water was added dropwise to the solution dropwise. 3.0eq). After the incubation was completed for 30 minutes in an ice bath, the reaction solution was placed at 60 ° C and the progress of the reaction was monitored by TLC. When S2a was completely consumed, it was diluted with 30 mL of dichloromethane and extracted with water and saturated brine, respectively. Finally, anhydrous sodium sulfate was dried and concentrated to give the product S3 (184 mg, yield: 79%).
- N-Boc-aminooxyacetic acid (1.0 g, 5.23 mmol, 1.0 eq) was dissolved in 20 mL of dichloromethane and stirred in an ice bath, and N-hydroxysuccinimide (NHS, 662.5 mg, 5.75 mmol, 1.1 eq) and dicyclohexylcarbodiimide (DCC, 1.3 g, 6.28 mmol, 1.2 eq) were placed and allowed to react at room temperature for 2 hours after stirring for 10 minutes. Subsequently, the reaction mixture was extracted with EtOAc EtOAc EtOAc.
- the compound S6a was dissolved in 8 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added to obtain a 20% trifluoroacetic acid reaction mixture. After stirring at room temperature for 1 hour, the reaction was completed, and concentrated in vacuo to give a brown oil, which was dissolved in 2 mL of water and used. 2 mL of dichloromethane was extracted twice, and the aqueous portion was collected and concentrated to give product M3 as colorless oil. HRMS calcd.
- the first step S7 (320 mg, 2 mmol, 1.2 eq), triethylamine (278 mL, 1.2 eq) was dissolved in 30 mL of absolute ethanol, stirred in an ice bath, and gradually added to Malay previously dissolved in 10 mL of absolute ethanol.
- the anhydride (163 mg, 1.66 mmol, 1.0 eq) was stirred in an ice bath for 4 hours after completion of the dropwise addition.
- Second step After the first step was completed, the organic solvent in the reaction mixture was evaporated under vacuum and re-dissolved with 8 mL of acetic anhydride while sodium carbonate (193 mg, 1.1 eq) was added. The reaction solution was heated to 65 ° C to continue the reaction, and TLC was detected. However, after the reaction was completed, the temperature was lowered to room temperature, and a certain volume of water was added thereto, followed by extraction with ethyl acetate. The organic phase was collected and extracted with saturated aqueous sodium hydrogen sulfate and brine.
- the resulting derivatized SGP (ie, compound 3a-e) (10 mg), wild type glycosidase EndoM (50) ⁇ g) co-dissolved in phosphate buffer (100 ⁇ L, 50 mM, pH 6.25), incubated at 30 ° C for 3-4 hours, high-phase liquid chromatography. After the hydrolysis was completed, it was diluted with water to a concentration of 50 mM of the non-natural oligosaccharide, while 2-chloro-1,3-dimethylimidazolium (DMC, 15 eq) and sodium hydroxide (40 eq) were added.
- DMC 2-chloro-1,3-dimethylimidazolium
- glycosyltransfer substrate oxazoline and deglycosylation Herceptin 6 were sequentially added to Tris-Cl, 50 mM, pH 7.4.
- the glucosidase EndoS-D233Q was incubated at a concentration of 1.5 mM, 5.0 mg/mL, and 0.15 mg/mL, respectively, and incubated at 30 ° C for about 2 hours to obtain Examples 9-13 (ie, antibodies 7a-7e). ).
- Antibody 7a (Example 9) or 7b (Example 10) (10.0 mg) with azide labeling group, together with DBCO-derived cytotoxic small molecule 8a-e (130 ⁇ M, 20 eq.) in 10 mL of 10% DMSO Incubate overnight in phosphate buffer (50 mM, pH 7.5) and monitor by polyacrylamide gel electrophoresis or LC-MS. Immediately after completion of the reaction, purification was carried out using Protein A resin. Examples 14-20 (i.e., compounds 9a-9f) were obtained, respectively.
- Example 1 Derivatization of SGP-Compound 3a, which was prepared by the general procedure 1 for Compound 2 and M1.
- Example 2 Derivatization of SGP-Compound 3b, which was prepared by the general procedure 2 for Compound 2 and 3-azidopropylamine.
- HSQC ((1H, 500MHz) / (13C, 126MHz), yttrium oxide) 5.05/99.53 (H1c/C1c), 4.96/78.22 ( H1a'/C1a'), 4.85/96.87 (H1c'/C1c'), 4.69/100.38 (H1b/C1b), 4.59/49.84 (H ⁇ Asn/C ⁇ Asn), 4.53/99.58 (H1a/C1a, H1d/C1d, H1d'/ C1d'), 4.38/58.04 (H ⁇ Thr/C ⁇ Thr), 4.37/103.51 (H1e/C1e, H1e'/C1e'), 4.32/53.51 (H ⁇ Lys/C ⁇ Lys), 4.31/67.02 (H ⁇ Thr/C ⁇ Thr ), 4.23/49.46 (H ⁇ Ala/C ⁇ Ala), 4.17/70.16 (H2b/C2b), 4.11/76
- Example 3 Derivatization of SGP-Compound 3c, which was prepared by the general procedure 1 for Compounds 2 and M3.
- Example 4 Derivatization of SGP-Compound 3d, which was prepared by the general procedure 1 for Compounds 2 and M4.
- HSQC ((1H, 500MHz) / (13C, 126MHz), yttrium oxide) 7.49/150.71 (H1g/C1g), 6.90/150.28 (H1g /C1g), 5.06/99.53 (H1c/C1c), 5.03/67.24 (H6f/C6f, H6f'/C6f'), 4.96/78.25 (H1a'/C1a'), 4.88/96.92 (H1c'/C1c'), 4.70/100.20 (H1b/ C1b), 4.60/49.87 (H ⁇ Asn/C ⁇ Asn), 4.53/99.93 (H1a/C1a, H1d/C1d, H1d'/C1d'), 4.51/72.14 (H2g/C2g), 4.39/58.01 (H ⁇ Thr/C ⁇ ) Thr), 4.37/103.63 (H1e/C1e, H1e'/C1e'
- Example 5 Derivatization of SGP-3e, which was prepared by the general procedure 1 for compounds 2 and M2.
- Example 6 Derivatization of SGP-Compound 3f, which was prepared by the general procedure 2 for Compound 2 and propargylamine.
- HSQC ((1H, 500MHz) / (13C, 126MHz), yttrium oxide) 5.06/100.47 (H1c/C1c), 4.96/79.04 ( H1a'/C1a'), 4.86/97.82 (H1c'/C1c'), 4.70/01.42 (H1b/C1b), 4.59/50.87 (H ⁇ Asn/C ⁇ Asn), 4.53/100.62 (H1a/C1a, H1d/C1d, H1d'/C1d'), 4.37/103.51 (H1e/C1e, H1e'/C1e'), 4.32/54.42 (H ⁇ Lys/C ⁇ Lys), 4.31/67.02 (H ⁇ Thr/C ⁇ Thr), 4.22/50.36 (H ⁇ Ala/C ⁇ Ala), 4.17/71.01 (H2b/C2b), 4.12/77.38 (H2c/C2c), 4.06/60.90 (
- 2-Azideethyl sialic acid glycopeptide (compound SGP-compound 3a, 8.7 mg) was dissolved in phosphate buffer (50 mM, pH 6.5, 87 mL), and 43.5 mg of glycosidase was added and incubated at 30 ° C. overnight. After completion of the detection reaction, high performance liquid chromatography was carried out to obtain white powder compound 4 (5.5 mg, yield 88.87%).
- HSQC ((1H, 500MHz) / (13C, 126MHz), yttrium oxide) 7.39 / 149.30 (H1g / C1g), 6.83 / 149.30 (H1g / C1g), 5.11/90.40 (H1aa/C1aa), 5.05/99.56 (H1c/C1c), 4.95/67.23 (H6f/C6f, H6f'/C6f'), 4.86/96.91 (H1c'/C1c'), 4.68/100.36 (H1b/C1b), 4.62/94.73 (H1ab/C1ab), 4.50/99.20 (H1d/C1d, H1d'/C1d'), 4.34/103.64 (H1e/C1e, H1e'/C1e'), 4.16/72.65 (H2b) /C2b, H2g/C2g, H6f/C6f, H6
- HSQC ((1H, 500MHz) / (13C, 126MHz), yttrium oxide) 7.39/150.81 ( H1g/C1g), 6.82/150.84 (H1g/C1g), 6.02/100.66 (H1a/C1a oxa), 5.07/100.38 (H1c/C1c), 4.90/97.41 (H1c'/C1c'), 4.67/102.13 (H1b/ C1b), 4.54/100.31 (H1d/C1d, H1d'/C1d'), 4.36/104.59 (H1e/C1e, H1e'/C1e'), 4.32/70.15 (H3a/C3a, oxa), 4.23/73.66 (H6f/ C6f, H6f'/C6f'), 4.17/73.32 (H2g/C2g), 4.13/66.06 (H2a/C2a, oxa),
- the non-native glycosylated antibody 7a is prepared by the deglycosylation antibody 6 and the compound SGP-3a by the general procedure 3; or the compound 5 and the deglycosylated antibody 6 are prepared by the action of the EndoS mutant enzyme, and refer to the general operation. 3 completed.
- the non-native glycosylated antibody 7b is prepared by the deglycosylation antibody 6 and the compound SGP-compound 3b by the general procedure 3.
- the non-native glycosylated antibody 7c is prepared by the deglycosylated antibody 6 and the compound SGP-compound 3c by the general procedure 3.
- the non-native glycosylated antibody 7d is prepared by deglycosylating antibody 6 and compound SGP-compound 3d by general procedure 3.
- the non-native glycosylated antibody 7e is prepared by the deglycosylation antibody 6 and the compound SGP-compound 3e by the general procedure 3.
- ADC-9a was prepared by a general procedure 4 of azido antibody 7a and small molecule cytotoxic compound 8a.
- ADC-9b was prepared by a general procedure 4 of azido antibody 7a and small molecule cytotoxic compound 8b.
- ADC-9c was prepared by a general procedure 4 of azido antibody 7b and small molecule cytotoxic compound 8b.
- ADC-9d was prepared by a general procedure 4 of azido antibody 7a and small molecule cytotoxic compound 8c.
- ADC-9e was prepared by a general procedure 4 of azido antibody 7b and small molecule cytotoxic compound 8c.
- ADC-9f was prepared by a general procedure 4 of azido antibody 7b and small molecule cytotoxic compound 8e.
- ADC-9g was prepared by a general procedure 4 of azido antibody 7b and small molecule cytotoxic compound 8d.
- Non-natural oligosaccharide Herceptin 7a (1.0 mg/mL, 5 mg, 1.0 eq), DM1-SMCC (100 ⁇ M, 15.0 eq) was co-dissolved in 10% DMSO in phosphate buffer (50 mM, pH 7.5) and at 37 Incubate for 1 hour at °C.
- LC-MS showed that the number of DM1s connected immediately after one hour was mainly between 2-3, and the DAR value was about 2.6.
- the mixture was immediately subjected to purification of Protein A resin to obtain an ADC compound N 3 -T-DM1 (Compound 10) having an azide group.
- the structure and mass spectrum data of Compound 10 (Example 21) are shown in Fig. 18.
- adherent cells were decanted, and 10% (w/v) trichloroacetic acid (100 ⁇ L/well) was added and fixed at 4 ° C for 1 h, then washed five times with distilled water, dried at room temperature, and added to each well. 100 ⁇ L of SRB solution (Sigma, St.
- Example 17, Example 18, Example 19, and Example 22 are shown in FIG. As shown in Figure 20, the activity of the good ADCs of Example 17, Example 18, Example 19, and Example 22 is shown.
Abstract
一种新型寡糖连接子,由该寡糖连接子制备的定点连接的抗体-药物偶联物,双药偶联的抗体-药物偶联物。该寡糖连接子包含双天线N-糖链结构、末端唾液酸带有修饰基团Y-(CH 2) n-X、末端乙酰葡萄糖胺包含噁唑啉环。前述抗体-药物偶联物、双药偶联的抗体-药物偶联物可用于制备抗肿瘤、抗炎症、抗病毒、抗感染或其他免疫治疗药物。
Description
本发明涉及一种新型寡糖连接子、利用该寡糖连接子制备的定点连接的抗体-药物偶联物以及它们的制备方法和用途。
抗体类生物大分子药物在肿瘤治疗领域占据重要位置,IgG类型的单克隆抗体(mAb)是治疗癌症、自身免疫病和传染病的一类重要的治疗性蛋白。伴随着单克隆抗体的不断发展,新一代抗体药物——抗体-药物偶联物(Antibody Drug Conjugates,ADCs)显示出了显著的肿瘤治疗效果。抗体-药物偶联物主要由抗体、小分子细胞毒药物、链接片段组成,通过抗原靶向性的抗体将小分子细胞毒成分特异性的运输到肿瘤组织,从而发挥杀伤癌细胞的作用。在ADCs药物的设计中,抗体的选择、小分子细胞毒成分的筛选以及链接片段的设计与优化都至关重要。其中,将小分子细胞毒成分特异性定点定量偶联到抗体上从而得到均一性的抗体药物偶联物是本发明的一大基础与特点。
IgG抗体是由两条重链和两条轻链构成,它们相结合形成由柔韧性铰链区相连的三个不同的蛋白质构成,包括两个可变的Fab结构域和一个恒定的(可结晶的)Fc结构域。Fab结构域涉及抗体-抗原的识别与结合,而Fc结构域参与Fc受体介导的效应子功能,例如抗体依赖性细胞毒性(ADCC)和补体依赖性细胞毒性(CDC)。Fc结构域是在保守的N-糖基化位点(Asn297)处带有两个N-聚糖的同源二聚体,其所附连的寡糖为双触角复杂型,具有相当大的结构非均质性。研究证实,野生型单克隆抗体Fc结构域的糖型主要是包括半乳糖及其缺失的N-寡糖链。本发明的主要特点在于,通过糖链改造技术将Fc区域Asn297位点的非均一结构N-糖链改造成带反应性基团的单一结构N-糖链,利用该糖链携带的反应性基团实现小分子药物与抗体的定点偶联,得到定点连接抗体-药物偶联物。
发明内容
本发明的一个目的是提供一种新型寡糖连接子,包含双天线N-糖链结构、末端唾液酸带有修饰基团Y-(CH2)n-X、末端乙酰葡萄糖胺包含噁唑啉环。
本发明的另一个目的是提供所述寡糖连接子的制备方法。
本发明的再一个目的是提供由该寡糖连接子制备的定点连接的抗体-药物偶联物,双天线N-糖链连接子在糖苷酶催化下连接在抗体的Fc区域糖基化位点Asn297,小分子药物在寡糖连接子末端非天然唾液酸结构的Y位置处通过合适的连接片段偶联,构成定点
连接的抗体-药物偶联物。
本发明的又一个目的是提供所述抗体-药物偶联物的制备方法。
本发明的还一个方面,提供了一种双药偶联的抗体-药物偶联物。
本发明的还一个目的是提供一种药物组合物,其包含所述抗体-药物偶联物和/或所述双药偶联的抗体-药物偶联物。
本发明的还一个目的是提供所述抗体-药物偶联物、双药偶联的抗体-药物偶联物或所述药物组合物在制备抗肿瘤、抗炎症、抗病毒、抗感染性疾病或其他免疫治疗药物中的用途。
本发明的定点偶联的优势在于避免了传统抗体-药物偶联物在抗体氨基酸残基上随机偶联造成的药物不均一性、抗原结合力下降、药效不均一性等问题。本发明的新颖性在于新颖的带修饰基团寡糖连接子在酶催化下特异性连接至抗体Fc区域糖基化位点Asn297,同时寡糖连接子上的修饰基团提供了小分子药物定点偶联反应位点,构成结构新颖的定点链接抗体-药物偶联物分子。
本发明人利用Fc结构域的保守N-糖基作为定点链接的位点,利用抗体Fc结构域的抗体糖基化修饰方法,设计和制备带有反应性基团片段的寡糖连接子,通过酶催化反应定点连接在抗体Fc区域N-糖基化位点Asn297,引入的反应性基团进一步与小分子药物偶联,实现定点连接于Fc糖基化位点Asn297的抗体-药物偶联物。本发明人利用一类内切-β-N-乙酰氨基葡萄糖苷酶的水解和转糖基化反应活性,来进行抗体Fc结构域的糖基化修饰和改造。该类糖苷酶能够通过切开N-聚糖的壳二糖核心中的β-1,4-糖苷键,特异性水解IgG抗体的Fc区域N-聚糖,得到糖型结构为乙酰葡萄糖胺单糖或岩藻糖α1,6乙酰葡萄糖胺二糖修饰的抗体。同时,利用此类糖苷酶或其突变酶的转糖基化反应活性,可将不同单一结构的N-糖链噁唑啉底物连接至上述水解得到的去糖基化抗体上,完成Fc结构域的糖基化改造,如以下流程图所示。
在以上流程图所示的抗体糖基化改造中,带有反应性基团的式(I)寡糖连接子作为酶催化转糖基化反应底物,高效特异的连接到抗体Fc区域N-糖基化位点(Asn297),将反应性基团定点引入抗体,随后的反应性基团与小分子药物偶联实现定点链接的抗体-药物偶联物。天然糖基化抗体的去糖基化应用野生型酿脓链球菌内切糖苷酶-S(Endo-S),该糖苷酶特异性切除抗体Fc区域N-糖链;转糖基化反应所用糖苷酶为Endo-F3(Elizabethkingia miricola内切糖苷酶-F3)或突变型Endo-S。
基于抗体Fc区域N-糖基化位点定点连接小分子药物,可以控制小分子偶联的位置和数量,制备结构均一的抗体-药物偶联物,避免了传统的随机偶联的抗体-药物偶联物的非均一结构,在药物质量控制、药效稳定性、体内代谢行为一致性等方面具有明显的优势。
本发明的一个方面,提供了一种如下式(I)所示的寡糖连接子:
其中:
n可为选自0-6之间的整数;
X可以选自-NHCH2-、-ONH=CH-、-CONHCH2-、-NHCO-CH=CH-;
Y可以选自以下基团:
其中,Z可以选自以下基团:
其中,R1和R2可以分别独立选自H或CH3,
L可以选自以下基团:
其中,a、c和d可以分别独立地为2-6之间的整数;b可为选自3-20之间的整数,
R3和R4可以分别独立选自CH3-、CH3CH(CH3)-、PhCH2-、NH2(CH2)4-、NH2CONH(CH2)3-;
D可为小分子药物或小分子活性化合物,可优选选自但不局限于美登素、DM-1、MMAE、MMAF、MMAP、auristatin E、长春新碱、长春碱、长春瑞滨、VP-16、喜树碱、SN-38、紫杉醇、多烯紫杉醇、埃博霉素A、埃博霉素B、诺考达唑、秋水仙碱、雌莫司汀、西马多丁、艾榴塞洛素等;或可更优选选自以下基团:
本发明中,优选地,所述式(I)所示的寡糖连接子选自以下化合物:
本发明的另一个方面,提供了式(I)所示的寡糖连接子的制备方法,如以下流程图所示:
其中,n、X和Y的定义与上述式(I)中的定义相同,
所述方法包括以下步骤:经蛋黄提取得到的唾液酸糖肽通过氧化得到具有醛基活性基团的唾液酸糖肽,该醛基化的唾液酸糖肽经过还原胺化反应,引入其他具有生物正交反应价值的官能团(如叠氮、炔基等),并在糖苷内切酶的作用下释放还有特殊标记基团的寡糖链,并经过环合过程获得寡糖连接子;
或者,所述方法包括以下步骤:经蛋黄提取得到的唾液酸糖肽通过糖苷酶水解产物末端唾液酸寡糖链,经过氧化、还原胺化反应、环合过程制备获得寡糖连接子;
或者,所述方法包括以下步骤:经蛋黄提取得到的唾液酸糖肽通过氧化得到具有醛基活性基团的唾液酸糖肽,该醛基化的唾液酸糖肽经过糖苷酶水解、经过还原胺化反应,引入其他具有生物正交反应价值的官能团(如叠氮、炔基等),并在糖苷内切酶的作用下释放还有特殊标记基团的寡糖链,并经过环合过程获得寡糖连接子。
本发明上述方法中,优选地,唾液酸糖肽的提取可以包括以下步骤:将冻干得到的蛋黄粉,依次经过乙醚(例如,2次)、70%丙酮水溶液(例如,2次)洗涤除掉大部分脂溶性杂质等,得到的固体进一步使用40%丙酮水溶液进行萃取(例如,2次),所得溶液浓缩并使用活性炭进行吸附,并依次使用纯水、5%乙腈水溶液、20%乙腈水溶液进行洗脱,收集20%乙腈洗脱液并进行浓缩,所得溶液使用葡聚糖凝胶G25以及半制备等进行进一步纯化,得到天然底物唾液酸糖肽;
本发明上述方法中,优选地,唾液酸糖肽的氧化可以包括以下步骤:
将所得到的唾液酸糖肽在磷酸缓冲液中使用高碘酸钠在冰浴进行氧化,时间为15分钟。
在上述制备方法中,首先,蛋黄唾液酸糖肽通过高碘酸钠氧化得到了具有两个醛基基团的唾液酸糖肽,然后醛基通过还原胺化、偶联反应、成肟反应、Wittig反应等,引入特殊的反应性官能团或其他小分子,获得结构修饰的非天然唾液酸糖肽。最后,这些非天然糖肽在酶催化水解去除多肽后,糖链还原末端的乙酰葡萄糖胺经环合成噁唑啉得到相应寡糖连接子。该寡糖连接子也可以采取一锅法合并上述步骤,从醛基唾液酸糖肽一步制备得到。
本发明的另一方面,提供了一种如下式(II)所示的基于抗体Fc区域N-糖基化位点(Asn297)的定点连接的抗体-药物偶联物:
其中:
n可以为选自0-6之间的整数;m、p和q可以分别独立地选自0或1;
Ab为抗体,可优选选自但不局限于以下抗体:曲妥珠单抗、帕妥珠单抗、利妥昔单抗、西妥昔单抗、莫罗单抗、吉妥珠单抗、阿昔单抗、达利珠单抗、阿达木单抗、帕利珠单抗、巴利昔单抗、贝伐单抗、帕尼单抗、尼妥珠单抗、德尼单抗等;
X可以选自-NHCH2-、-ONH=CH-、-CONHCH2-、-NHCO-CH=CH-;
Y基团可为D-L-Z-,
其中,Z可以选自以下基团:
其中,R1和R2可以分别独立选自H或CH3,
其中,L可以选自以下基团:
其中,a、c和d可以分别独立地为选自2-6之间的整数;b可为选自3-20之间的整数,
其中,R3和R4可以分别独立选自CH3-、CH3CH(CH3)-、PhCH2-、NH2(CH2)4-、
NH2CONH(CH2)3-,
D可为小分子药物或小分子活性化合物,可优选选自但不局限于美登素、DM-1、MMAE、MMAF、MMAP、auristatin E、长春新碱、长春碱、长春瑞滨、VP-16、喜树碱、SN-38、紫杉醇、多烯紫杉醇、埃博霉素A、埃博霉素B、诺考达唑、秋水仙碱、雌莫司汀、西马多丁、艾榴塞洛素等;或可更优选选自以下基团:
本发明的又一个方面,提供了式(II)所示的抗体-药物偶联物的制备方法,包括以下步骤:
a)将抗体(例如,真核表达体系中制备的抗体)通过糖苷酶切除其N-糖链,得到其Fc区域N-糖基化位点(Asn297)为乙酰葡萄糖胺单糖或岩藻糖α1,6乙酰葡萄糖胺二糖修饰的抗体;
b)将式(I)所示的寡糖连接子通过糖苷酶或其突变体催化,连接到步骤a)所得到的乙酰葡萄糖胺单糖或岩藻糖α1,6乙酰葡萄糖胺二糖修饰的抗体上,制备包含特殊反应性基团的式(I)所示的寡糖连接子修饰的抗体;
c)将步骤b)所得到的包含特殊反应性基团的式(I)所示的寡糖连接子修饰的抗体,与带有能与抗体糖链上该特殊反应性基团进行特异性偶联反应的对应基团修饰的小分子药物进行偶联,制备得到式(II)所示的抗体-药物偶联物。
上述制备方法中,优选地,步骤a)和b)中,所述糖苷酶或其突变体可为岩藻糖水解
酶、N-乙酰葡萄糖胺内切水解酶或它们的突变体,更优选地,所述N-乙酰葡萄糖胺内切水解酶包括Endo-S(酿脓链球菌内切糖苷酶-S)、Endo-F3(Elizabethkingia miricola内切糖苷酶-F3)、Endo-S2(Endoglycosidase-S2,酿脓链球菌内切糖苷酶-S2)、Endo-Sd(Endoglycosidase-Sd,酿脓链球菌内切糖苷酶-Sd)以及Endo-CC(Endoglycosidase-CC,酿脓链球菌内切糖苷酶-CC);优选地,步骤b)和c)所述的特殊反应性基团和能与该基团进行特异性偶联反应的对应基团可分别为叠氮与炔基、巯基与马来酰亚胺、巯基与巯基或巯基活化形式、醛基与氨基、醛基与羟胺或肼基等两两对应组合。
上述制备方法中,优选地,步骤c)中,所述带有能与抗体糖链上该特殊反应性基团进行特异性偶联反应的对应基团修饰的小分子药物选自但不局限于以下结构化合物:
本发明的还一个方面,提供了一种双药偶联的抗体-药物偶联物,即在式(II)定点
连接的抗体-药物偶联物基础上,在其抗体上所含赖氨酸的氨基与另一不同小分子药物连接,形成一个两个药物偶联的抗体-药物偶联物。
本发明中,优选地,所述双药偶联的抗体-药物偶联物优选自如下式(III)所示的结构:
其中,n、m、p、q、X和Y的定义与上述式(II)中的定义相同,
其中,赖氨酸所连接小分子药物为美登素DM1;Asn297糖链连接小分子药物与式(II)抗体药物偶联物中定义相同。
由式(I)所示的寡糖连接子制备的式(II)所示的定点连接的抗体-药物偶联物,其化学结构为定点定量的均一结构,与非均一结构的上市抗体-药物偶联物相比,具有结构明确单一、质量可控等优势。同时,优选的定点连接的抗体-药物偶联物表现出良好的抗肿瘤活性,IC50可达到0.12nM(0.018ug/mL)。
图1为显示根据本发明的一个实施方式的非天然糖基化抗体7a的结构的示意图。
图2A显示抗体7a的质谱数据图(m/z);图2B显示一锅法制备抗体7a的SDS-PAGE图谱;图2C显示抗体7a的全长分子量质谱数据图。
图3为显示根据本发明的一个实施方式的非天然糖基化抗体7b的结构的示意图。
图4A显示7b的质谱数据图(m/z);图4B显示一锅法制备抗体7b的SDS-PAGE图谱;图4C显示抗体7b的全长分子量质谱数据图。
图5为显示根据本明发的一个实施方式的非天然糖基化抗体7c的结构的示意图。
图6A显示7c的质谱数据图(m/z);图6B显示一锅法制备抗体7c的SDS-PAGE图谱;图6C显示抗体7c的全长分子量质谱数据图。
图7为显示根据本发明的一个实施方式的非天然糖基化抗体7d的结构的示意图。
图8A显示7d的质谱数据图(m/z);图8B显示一锅法制备抗体7d的SDS-PAGE图谱;图8C显示抗体7d的全长分子量质谱数据图。
图9为显示根据本发明的一个实施方式的非天然糖基化抗体7e的结构的示意图。
图10A显示7e的质谱数据图(m/z);图10B显示一锅法制备抗体7e的SDS-PAGE图谱;图10C显示抗体7e的全长分子量质谱数据图。
图11显示ADC-9a的化合物结构、质谱数据图(m/z)和全长质谱数据图(内嵌)。
图12显示ADC-9b的化合物结构、质谱数据图(m/z)和全长质谱数据图(内嵌)。
图13显示ADC-9c的化合物结构、质谱数据图(m/z)和全长质谱数据图(内嵌)。
图14显示ADC-9d的化合物结构、质谱数据图(m/z)和全长质谱数据图(内嵌)。
图15显示ADC-9e的化合物结构、质谱数据图(m/z)和全长质谱数据图(内嵌)。
图16显示ADC-9f的化合物结构、质谱数据图(m/z)和全长质谱数据图(内嵌)。
图17显示ADC-9g的化合物结构、质谱数据图(m/z)和全长质谱数据图(内嵌)。
图18为化合物10(ADC-10)的化合物结构和全长质谱数据图,图中所标数字为随机连接的小分子DM1的数量。
图19为化合物11(ADC-11)的化合物结构和全长质谱数据图,图中所标数字为随机连接的小分子DM1的数量。
图20为显示抗体-药物偶联物实施例的抗肿瘤活性的图。
本发明中所使用的糖苷水解酶及其突变酶,均表达与大肠杆菌系统。发明中所使用的3-叠氮基丙胺、炔丙胺和乙腈购买自上海百灵威化学技术有限公司;小分子细胞毒药物DM1以及MMAE购买自南京联宁生物制药公司;点击化学连接子DBCO-amine以及DBCO-PEG4-acid购买自西格玛奥德里奇(上海)贸易有限公司;氨基酸化合物购买自吉尔生化(上海)有限公司;其他化合物、试剂和溶剂如未做进一步解释均购买自国药集团化学试剂有限公司。
本发明中所使用的仪器、色谱柱包括:分析型高效液相色谱仪(北京创新通恒LC3000),制备型高效液相色谱仪(北京创新通恒LC3000);安捷伦SB-C18(5μm,4.6x 150mm),Waters OBD-C18(5μm,19x 250mm);
抗体分子量测定仪器:液相色谱质谱联用(LC-MS),安捷伦6230LC-TOF-MS,配用Agilent C-18column(3.5μm,50x 2.1mm)at 55℃。
制备实施例
唾液酸糖肽(SGP,化合物1)的分离纯化
取200个新鲜的鸡蛋,取其蛋黄,并加入等量的水进行冷冻干燥,得到干燥的蛋黄粉约1.2Kg。分别用甲基叔丁基醚(2x 3L)、70%丙酮溶液(2x 1.8L)和40%丙酮溶液(2x 1.8L)进行洗涤,收集40%丙酮溶液组分并进行浓缩。将获得的浓缩液吸附于活性炭/硅藻土(1:1,150g),并分别用2体积纯水、2体积5%乙腈水溶液、2体积20%乙腈水溶液以及2体积30%乙腈水溶液洗脱。将20%的洗脱液进行浓缩并使用葡聚糖G25纯化,将得到的产品再经过制备高效液相色谱纯化得到纯SGP约1.1g。
醛基化唾液酸糖肽(CHO-SGP,化合物2)的制备
将唾液酸糖肽1(100mg)溶于5mL磷酸缓冲液中(0.2M,pH 7.1),同时加入5mL高碘酸钠溶液(30mM)并于冰浴中搅拌。待搅拌15分钟后用葡聚糖G25进行纯化,收集产物组分并进行冷冻干燥,得到产物化合物2(90mg,产率94%)。
1H NMR(500MHz,氧化氘)δ5.05(1H,s,H1c),4.96(1H,d,J=9.7Hz,H1a’),4.88(3H,m,H1c’,H of CHO–C(H)-(OH)2),4.70(1H,s,H1b),4.60(1H,t,J=6.7Hz,Asn Hα),4.53(3H,d,
J=7.4Hz,H1a,H1d,H1d’),4.39(1H,d,J=3.5Hz,Thr Hα),4.37(1H,d,J=2.5Hz,H1e),4.35-4.27(3H,m,H1e’,Thr Hβ,Lys Hα),4.24(1H,q,J=7.1Hz,Ala Hα),4.17(1H,s,H2b),4.12(1H,d,J=3.4Hz,H2c),4.08(1H,d,J=7.5Hz,Val Hα),4.03(1H,d,H2c’),3.99(3H,t,J=6.4Hz,Lys Hα,2H),2.92(m,4H,Lys Hε),2.82-2.59(2H,m,Asn Hβ),2.51(2H,dd,J=12.7,4.2Hz,H3feq,H3f’eq),2.09-1.88(19H,m,6x 3Ac,Val Hβ),1.82(6H,m,Lys Hβ,H3fax,H3f’ax),1.75-1.56(4H,m,J=7.5Hz,Lys Hδ),1.36(4H,m,Lys Hγ),1.30(3H,d,J=7.5Hz,Ala Hβ),1.12(3H,J=6.5Hz,Thr Hγ),0.89(6H,d,7.0Hz,Val Hγ).HSQC((1H,500MHz)/(13C,126MHz),氧化氘)5.05/100.10(H1c/C1c),4.96/78.91(H1a’/C1a’),4.88/89.17(H1g/C1g),4.87/97.65(H1c’/C1c’),4.70/101.14(H1b/C1b),4.59/50.77(Hα Asn/Cα Asn),4.53/100.41(H1a/C1a,H1d/C1d,H1d’/C1d’),4.38/58.86(Hα Thr/CαThr),4.37/104.22(H1e/C1e,H1e’/C1e’),4.33/54.21(Hα Lys/Cα Lys),4.31/67.76(Hβ Thr/Cβ Thr),4.23/50.13(Hα Ala/Cα Ala),4.17/71.26(H2b/C2b),4.11/77.18(H2c/C2c),4.07/60.15(Hα Val/Cα Val),4.03/76.82(H2c’/C2c’),3.99/53.60(Hα Lys/Cα Lys),2.92/39.73(Hε Lys/CεLys),2.66/37.45(HβAsn/CβAsn),2.51/40.00(H3f/C3f,H3f’/C3f’),2.00/31.01(Hβ Val/Cβ Val),1.83/31.01(Hβ Lys/Cβ Lys),1.69/39.96(H3f/C3f,H3f’/C3f’),1.61/26.93(Hδ Lys/Cδ Lys),1.37/22.00(Hγ Lys/Cγ Lys),1.30/17.37(Hβ Ala/Cβ Ala),1.12/19.63(Hγ Thr/Cγ Thr),0.89/19.33(Hγ Val/Cγ Val).HRMS,计算值C108H177N15O66[M+3H]3+914.3730,测量值914.3734;计算值[M+H2O+3H]3+920.3765,测量值920.3787.
羟胺类化合物M1-M4的制备
N-(2-溴乙基氧基)邻苯二甲酰亚胺(S2a)的合成
在25mL茄形瓶中加入N-羟基邻苯二甲酰亚胺(1.14g,7.0mmol)并用8mL DMF溶解,随后逐滴滴加1,2-二溴乙烷(3mL,34.8mmol),最终加入三乙胺(2mL,14mmol)并在室温下避光搅拌。将反应液搅拌过夜并用TLC进行监测。待反应结束后,过滤沉淀,并用DMF洗涤沉淀两次。将大量的DMF用旋转蒸发仪除去,并将剩余的溶液用大量的水进行沉淀,集沉淀并用水洗涤沉淀。将获得的沉淀重新用5mL乙酸乙酯进行溶解,并分别用1N盐酸、水以及饱和食盐水进行萃取处理,最后无水硫酸钠干燥浓缩获得N-(2-溴乙基氧基)邻苯二甲酰亚胺S2a(977.4mg,产率52%)。
1H NMR(400MHz,氘代二氯甲烷)δ7.89(m,2H ArH),7.80(m,2H ArH),4.51(t,2H),3.67(t,2H).HRMS计算值[M+Na]+291.9585,实测值[M+Na]+291.9569.
N-(2-叠氮乙氧基)邻苯二甲酰亚胺(S3)的合成
将获得的化合物S2a(500mg,1.85mmol,1.0eq)溶于10mL丙酮中,冰浴,向该溶液中逐滴滴加预先溶于2mL水中的叠氮化钠(NaN3,360mg,5.55mmol,3.0eq)。待滴加完毕于冰浴孵育30分钟后将反应液置于60℃并TLC监测反应的进程。当S2a完全耗尽后用30mL二氯甲烷进行稀释并分别用水以及饱和食盐水进行萃取处理。最后无水硫酸钠干燥并浓缩获得产物S3(184mg,产率79%)。1H NMR(400MHz,氘代二氯甲烷)δ7.89(dd,J=5.5,3.1Hz,2H),7.80(dd,J=5.5,3.1Hz,2H),4.42–4.35(m,2H),3.73–3.66(m,2H).HRMS计算值[M+Na]+255.0494,实测值[M+Na]+255.0476.
O-(2-叠氮基乙基)羟胺盐酸盐(M1)的合成
将产物S3(103mg,0.45mmol,1.0eq)置于茄形瓶中,加入水合肼(45mL,0.67mmol,
1.5eq)并磁子搅拌固体-胶状物30分钟,随后在20mL乙醚中继续搅拌2小时,反应液由淡黄色逐渐变为白色固体-液体混合液。离心去除沉淀并用无水乙醚进行洗涤,获取乙醚部分后加入1N HCl(2.0eq)并于室温搅拌两小时后离心获得白色沉淀M1。
1H NMR(400MHz,氘代二甲基亚砜)δ11.05(s,3H),4.15(q,J=5.2,4.4Hz,2H),3.59(t,J=4.4Hz,2H).13C NMR(126MHz,DMSO-d6)δ73.42,48.77.
O-(2-炔丙基)羟胺盐酸盐(M2)的合成
N-(2-炔丙氧基)邻苯二甲酰亚胺(S2b)的合成与O-(2-炔丙基)羟胺盐酸盐(M2)同上述S2a与M1的合成。
O-(2-炔丙基)羟胺盐酸盐(M2)。1H NMR(400MHz,氘代二甲基亚砜)δ11.07(s,3H),4.73(dd,J=5.5,2.2Hz,2H),3.86(t,J=2.4Hz,1H).
2-(Boc-氨氧基)-N-羟基琥珀酰亚胺乙酸酯(S5)的合成
N-Boc-氨氧基乙酸(1.0g,5.23mmol,1.0eq)溶于20mL二氯甲烷中,并置于冰浴中搅拌,同时往搅拌液中加入N-羟基琥珀酰亚胺(NHS,662.5mg,5.75mmol,1.1eq)以及二环己基碳二亚胺(DCC,1.3g,6.28mmol,1.2eq),置于并于搅拌10分钟后室温反应2小时。随后,反应液用饱和碳酸氢钠、饱和食盐水分别萃取,收集有机溶剂部分并浓缩得到白色固体S5(1.42g,产率94%),无需进一步纯化。1H NMR(400MHz,氘代二氯甲烷)δ8.37(s,1H),7.57(s,1H),4.34(s,2H),3.42(p,4H),1.86(p,2H),1.51(s,9H).HRMS计算值[M+Na]+296.1335,实测值[M+Na]+296.1347.
2-(Boc-氨氧基)-N-(3-叠氮丙基)乙酰胺(S6a)的合成
将S5(100mg,0.35mmol,1.0eq)溶于10mL二氯甲烷中,加入3-叠氮基-1-丙胺(40mg,0.38mmol,1.1eq)以及三乙胺(72mL,2.0eq)。将反应置于室温搅拌2小时,并用TLC进行监测。但反应完成后,反应液分别用水以及饱和食盐水进行萃取,无水硫酸钠干燥后浓缩得到淡黄色产物S6a(90mg,产率95%)。1H NMR(400MHz,氘代二氯甲烷)δ8.37(s,1H),7.57(s,1H),4.34(s,2H),3.42(p,4H),1.86(p,2H),1.51(s,9H).HRMS计算值[M+Na]+296.1335,实测值[M+Na]+296.1347.
2-氨氧基-N-(3-叠氮丙基)乙酰胺三氟乙酸盐(M3)的合成
将化合物S6a用8mL二氯甲烷进行溶解,并加入2mL三氟乙酸获得20%三氟乙酸反应液,室温搅拌1小时后反应完全,真空浓缩得到黄褐色油状物后,用2mL水进行溶解,并用2mL二氯甲烷萃取两次,收集水溶液部分并浓缩得到无色油状产物M3。HRMS计算值[M+Na]+196.0805,实测值[M+Na]+196.0814.1H NMR(400MHz,氘代二甲基亚
砜)δ8.24(t,J=5.6Hz,1H),4.32(s,2H),3.38(t,J=6.8Hz,2H),3.18(q,J=6.7Hz,2H),1.69(p,J=6.8Hz,2H).13C NMR(101MHz,DMSO-d6)δ167.85,72.37,48.76,36.12,28.71.
2-(Boc-氨氧基)-N-炔丙基乙酰胺(S6b)的合成
将S5(650mg,2.26mmol,1.0eq)溶于10mL二氯甲烷中并依次加入炔丙胺(186.17mg,3.38mmol,1.5eq),三乙胺(629mL,2.0eq)。室温搅拌2小时后TLC检测反应完全,用水以及饱和食盐水分别进行萃取,收集有机相并用无水硫酸钠进行干燥处理,浓缩得到S6b(462.8mg,产率90%)。HRMS计算值[M+Na]+251.1002,实测值[M+Na]+251.1004.
2-氨氧基-N-炔丙基乙酰胺三氟乙酸盐(M4)的合成
将S6b(462.8mg)溶于16mL二氯甲烷中,并加入4mL三氟乙酸得到20%三氟乙酸溶液。室温下搅拌1小时后TLC检测反应完全。真空干燥获得棕色固体化合物M4。1H NMR(400MHz,氘代二甲基亚砜)δ8.59(t,J=5.4Hz,1H),4.36(s,2H),3.93(dd,J=5.5,2.5Hz,2H),3.18(t,J=2.5Hz,1H).13C NMR(101MHz,氘代二甲基亚砜)δ167.90,81.12,73.78,72.56,28.11.HRMS计算值[M+H]+129.0658,实测值[M+H]+129.0659.
N-(2-氨乙基)马来酰亚胺(M5)的合成
N-Boc-乙二胺(S7)的合成
乙二胺(7mL,100mmol)溶于100mL氯仿中并置于冰浴中,慢慢往里滴加(滴加2小时)预先溶于氯仿的二碳酸二叔丁酯(2.185g,10mmol)。反应液继续在冰浴下搅拌2小时后,于室温下继续搅拌16个小时。但反应结束后,过滤掉沉淀,有机相继续用饱和食盐水和水分别进行萃取,随后有机相用无水硫酸钠干燥后浓缩得到产物S7(1.4g,产率87.5%)。1H NMR(400MHz,氘代二氯甲烷)δ4.91(s,1H),3.18(t,J=6.1Hz,2H),2.81(td,J=6.0,1.5Hz,2H),1.46(d,J=1.4Hz,9H),1.35(s,2H).HRMS计算值[M+H]+161.1285,实测值[M+H]+161.1279.
N-(2-Boc氨乙基)马来酰亚胺(S8)的合成
第一步:S7(320mg,2mmol,1.2eq),三乙胺(278mL,1.2eq)溶于30mL无水乙醇中,冰浴搅拌,并逐渐滴加预先溶于10mL无水乙醇中的马来酸酐(163mg,1.66mmol,1.0eq),滴加完成后继续在冰浴中搅拌4小时,TLC检测显示所有原料消耗完全。
第二步:待第一步完成后,将反应液中的有机溶剂在真空下悬蒸去除,并用8mL乙酸酐重新溶解,同时加入碳酸钠(193mg,1.1eq)。将反应液加热到65℃继续反应,TLC检测,但反应完成后,温度降至室温,加入一定体积的水后用乙酸乙酯进行萃取。收集有机相并分别用饱和碳酸氢钠溶液和饱和食盐水进行萃取,有机相用无水硫酸钠进行干燥后浓缩得到白色固体S8(312.5mg,78.3%)。1H NMR(400MHz,氘代二氯甲烷)δ6.73(s,2H),4.74(s,1H),3.68(t,J=8Hz,2H),3.35(q,J=4Hz,2H),1.43(s,9H).
N-(2-氨乙基)马来酰亚胺三氟乙酸盐(M5)的合成
将S8(300mg)溶于8mL二氯甲烷中,并滴加2mL三氟乙酸,室温搅拌1小时后真空悬蒸去除有机溶剂得到黄色的油状液体。重新用1mL甲醇溶解黄色油状液体,用过量的乙醚进行沉淀处理,收集沉淀并真空干燥得到化合物M5(272.7mg,产率92%)。1H NMR(400MHz,氘代二甲基亚砜)δ7.79(s,3H),7.09(s,2H),3.66(t,J=6.0Hz,2H),2.99(t,J=5.8Hz,2H).
去糖基化抗体6的制备
38mg野生型赫赛汀与1.5μg野生型糖苷内切酶EndoS溶于1mL磷酸缓冲液(1mL,pH 8.0)中,并于37℃孵育24小时。反应完成后将去糖基化赫赛汀吸附于Protein A凝胶柱上,依次用磷酸缓冲液A(50mM,pH 8.0)、磷酸缓冲液B(50mM,pH 5.0)、甘氨酸缓冲液C(100mM,pH 2.5)进行洗脱,收集缓冲液C洗脱组分并迅速调节至中性偏弱碱性(pH约7.5)。将收集的组分进行浓缩并浓度鉴定。
通用操作1:羟胺类化合物与醛基化唾液酸糖肽的反应
化合物2(10mg,3.65μmol,1.0eq)溶于100mL磷酸缓冲液(50mM,pH 7.5)中,往该溶液中加入羟胺类化合物M1-4(14.6μmol,4.0eq),室温反应2小时后进行制备高效液相色谱分离,分别得到产物:实施例1-化合物3a,实施例3-化合物3c,实施例4-化合物3d,实施例5-化合物3e。
通用操作2:化合物2的还原胺化反应
化合物2(10mg,3.65μmol)溶于100mL甲醇/磷酸缓冲液(50mM,pH 6.0,1:1v/v)中,随后加入伯胺类化合物(109.5μmol,30eq)以及氰基硼氢化钠(NaCNBH3,4.6mg,73μmol,20eq)并于冰浴中搅拌三个小时,制备分离得到产物:实施例2-化合物3b,实施例6-化合物3f。
通用操作3:“一锅法”酶催化糖基化转移制备官能团标记的抗体
制备得到的衍生化SGP(即,化合物3a-e)(10mg),野生型糖苷酶切酶EndoM(50
μg)共同溶于磷酸缓冲液(100μL,50mM,pH 6.25)中,30℃孵育3-4小时,高相液相色谱检测。待水解完全后,加水稀释至非天然寡糖的设定浓度50mM,同时加入2-氯-1,3-二甲基氯化咪唑啉(DMC,15eq)和氢氧化钠(40eq)。待反应2-3小时之后,在三羟甲基氨基甲烷盐酸缓冲液(Tris-Cl,50mM,pH7.4)中,依次加入糖基转移底物恶唑啉、去糖基化赫赛汀6、突变糖苷酶EndoS-D233Q,使各项的浓度依次为1.5mM、5.0mg/mL、0.15mg/mL,并于30℃孵育2小时左右,得到实施例9-13(即,抗体7a-7e)。
通用操作4:定点连接的抗体-药物偶联物制备
具有叠氮标记基团的抗体7a(实施例9)或者7b(实施例10)(10.0mg),与DBCO衍生细胞毒小分子8a-e(130μM,20eq.)共同在10mL含10%DMSO的磷酸缓冲液(50mM,pH7.5)中孵育过夜,聚丙烯酰氨凝胶电泳或LC-MS进行监测。待反应结束后,立即用ProteinA树脂进行纯化。分别得到实施例14-20(即,化合物9a-9f)。
实施例1:衍生化SGP-化合物3a,为化合物2与M1经通用操作1制备完成。
产率92.3%.1H NMR(500MHz,氧化氘)δ7.41(1.6H,dd,J=7.2,3.3Hz,H1of oxime),6.85(0.4H,dd,J=6.7,2.6Hz,H1of oxime),5.07(1H,s,H1c),4.96(1H,d,J=9.6Hz,H1a’),4.96(0.4H,H6f,H6f’),4.88(1H,s,H1c’),4.76(1H,s,H1b),4.59(1H,t,Asn Hα),4.53(3H,d,J=6.7Hz,H1a,H1d.H1d’),4.39(1H,d,J=3.5Hz,Thr Hα),4.37(1H,d,J=2.5Hz,H1e),4.35(1H,d,J=3Hz,H1e’),4.34-4.28(2H,m,Lys1Hα,Τhr Hβ),4.24(1H,q,J=7Hz,Ala Hα),4.17(5H,m,H2b,H2of oxime),4.17(1.6H,m,H6f,H6f’),4.12(1H,d,J=2Hz,H2c),4.08(1H,d,J=8Hz,Val Hα),4.04(1H,d,J=2Hz,H2c’),3.99(1H,t,J=6.5Hz,Lys2Hα),2.92(4H,q,J=7.5Hz,Lys Hε),2.81-2.61(2H,m,Asn Hβ),2.61-2.51(2H,dd,J=12.5Hz,2.5Hz,H3feq,H3f’eq),2.03-1.88(19H,m,6x 3Ac,Val Hβ),1.87-1.66(6H,m,Lys Hβ,H3fax,H3f’ax),1.66(4H,m,J=7.5Hz,Lys Hδ),1.43-1.32(4H,m,Lys Hγ),1.3(3H,d,J=7.5Hz,
Ala Hβ),1.13(3H,J=6.5Hz,Thr Hγ),0.89(6H,d,7.0Hz,Val Hγ).HSQC((1H,500MHz)/(13C,126MHz),氧化氘)7.41/149.38(H1g/C1g),6.85/149.11(H1g/C1g),5.06/99.43(H1c/C1c),4.96/78.27(H1a’/C1a’),4.95/67.43(H6f/C6f,H6f’/C6f’),4.87/96.94(H1c’/C1c’),4.69/100.39(H1b/C1b),4.59/49.87(Hα Asn/Cα Asn),4.52/99.60(H1a/C1a,H1d/C1d,H1d’/C1d’),4.39/57.76(Hα Thr/Cα Thr),4.36/103.72(H1e/C1e,H1e’/C1e’),4.33/53.57(Hα Lys/Cα Lys),4.32/67.08(Hβ Thr/Cβ Thr),4.23/49.46(Hα Ala/Cα Ala),4.17/72.10(H2b/C2b,H2g/C2g,H6f/C6f,H6f’/C6f’),4.12/76.41(H2c/C2c),4.07/59.53(Hα Val/Cα Val),4.04/76.23(H2c’/C2c’),3.99/52.63(Hα Lys/Cα Lys),3.46/49.76(H3g/C3g),2.92/39.12(Hε Lys/Cε Lys),2.64/36.66(Hβ Asn/Cβ Asn),2.58/39.18(H3f/C3f,H3f’/C3f’),1.97/30.60(Hβ Val/Cβ Val),1.81/39.05(H3f/C3f,H3f’/C3f’),1.77/30.39(Hβ Lys/Cβ Lys),1.62/26.13(Hδ Lys/Cδ Lys),1.37/21.38(Hγ Lys/Cγ Lys),1.34/16.54(Hβ Ala/Cβ Ala),1.16/18.82(Hγ Thr/Cγ Thr),0.85/17.73(Hγ Val/Cγ Val).HRMS,计算值C112H185N23O66[M+3H]3+970.4020,实测值970.3925.[M+2H]2+1455.0991,实测值1455.0845.
实施例2:衍生化SGP-化合物3b,为化合物2与3-叠氮基丙胺经通用操作2制备完成。
产率84.8%.1H NMR(500MHz,氧化氘)δ5.03(1H,s,H1c),4.93(1H,d,J=9.6Hz,H1a’),4.83(1H,s,H1c’),4.70(1H,s,H1b),4.56(1H,t,J=6.7Hz,Asn Hα),4.50(3H,dd,J=6.4,5.1Hz,H1a,H1d,H1d’),4.37(1H,d,J=3.5Hz,Thr Hα),4.35(1H,d,J=2.5Hz,H1e),4.34(1H,d,J=3Hz,H1e’),4.30(2H,m,Lys1Hα,Τhr Hβ),4.21(1H,q,J=7.2Hz,Ala Hα),4.14(1H,s,H2b),4.09(1H,d,J=2.7Hz,H2c),4.05(1H,d,J=7.6Hz,Val Hα),4.01(1H,d,J=3.5Hz,H2c’),4.00-3.90(3H,m,Lys Hα,H6f,H6f’),3.24-3.04(8H,m,-2x-CH2-NH-CH2-),2.92(4H,q,J=7.5Hz,Lys Hε),2.81-2.61(2H,m,Asn Hβ),2.61-2.51(2H,dd,J=12.5Hz,2.5Hz,H3feq,H3f’eq),2.03-1.90(19H,m,6x 3Ac,Val Hβ),1.90-1.84(4H,m,H3g),1.84-1.65(6H,m,Lys Hβ,H3fax,H3f’ax),1.59(4H,m,J=7.5Hz,Lya Hδ),1.40-1.29(4H,m,Lys Hγ),1.27(3H,d,J=7.5Hz,Ala Hβ),1.10(3H,J=6.5Hz,Thr Hγ),0.86(6H,d,7.0Hz,Val Hγ).
HSQC((1H,500MHz)/(13C,126MHz),氧化氘)5.05/99.53(H1c/C1c),4.96/78.22(H1a’/C1a’),4.85/96.87(H1c’/C1c’),4.69/100.38(H1b/C1b),4.59/49.84(Hα Asn/Cα Asn),4.53/99.58(H1a/C1a,H1d/C1d,H1d’/C1d’),4.38/58.04(Hα Thr/Cα Thr),4.37/103.51(H1e/C1e,H1e’/C1e’),4.32/53.51(Hα Lys/Cα Lys),4.31/67.02(Hβ Thr/Cβ Thr),4.23/49.46(Hα Ala/Cα Ala),4.17/70.16(H2b/C2b),4.11/76.42(H2c/C2c),4.07/59.39(Hα Val/Cα Val),4.03/76.11(H2c’/C2c’),3.99/52.73(Hα Lys/Cα Lys),3.43/48.19(H4g/C4g),3.21/48.59(H2g/C2g),3.16/46.14(H1g/C1g),2.91/39.01(Hε Lys/Cε Lys),2.76/36.86(Hβ Asn/Cβ Asn),2.59/39.43(H3f/C3f,H3f’/C3f’),1.98/30.12(Hβ Val/Cβ Val),1.80/30.30(Hβ Lys/Cβ Lys),1.75/39.63(H3f/C3f,H3f’/C3f’),1.59/26.28(Hδ Lys/Cδ Lys),1.36/21.42(Hγ Lys/Cγ Lys),1.29/16.67(Hβ Ala/Cβ Ala),1.12/19.03(Hγ Thr/Cγ Thr),0.88/18.21(Hγ Val/Cγ Val).HRMS,计算值C114H193N23O64[M+4H]4+728.0717,实测值728.0717;[M+3H]3+970.4263,实测值970.4254;[M+2H]2+1455.1356,实测值1455.1381.
实施例3:衍生化SGP-化合物3c,为化合物2与M3经通用操作1制备完成。
产率94.2%.1H NMR(500MHz,氧化氘)δ7.48(1.6H,dd,J=7.0,3.7Hz,H1g),6.89(0.4H,dd,J=7.0,2.5Hz,H1g),5.05(1.4H,s,H1c,H6f,H6f’),4.95(1H,d,J=9.7Hz,H1a’),4.87(1H,s,H1c’),4.67(1H,s,H1b),4.60(1H,t,Asn Hα),4.52(3H,d,J=7.1Hz,H1a,H1d,H1d’),4.49-4.41(4H,m,H2g),4.38(1H,d,Thr Hα),4.36(2H,dd,H1e,H1e’),4.33-4.29(2H,m,Lys Hα,Thr Hβ),4.24(1H,q,J=7.2Hz,Ala Hα),4.18(2.6H,m,H1b,H6f,H6f’),4.11(1H,d,J=2.5Hz,H2c),4.06(1H,d,J=7.6Hz,Val Hα),4.03(1H,d,J=2Hz,H2c’),3.98(1H,t,J=6.7Hz,Lys Hα),3.37-3.15(8H,m,H4g,H6g),2.91(4H,q,J=7.6Hz,Lys Hε),2.70(2H,ddd,J=62.2,16.4,6.7Hz,Asn Hβ),2.56(2H,dd,J=11.0Hz,H3feq,H3f’eq),1.80(4H,m,Lys Hβ)1.72(6H,m,H5g,H3fax,H3f’ax),1.43-1.31(4H,m,Lys Hγ),1.29(3H,d,Ala Hβ),1.11(3H,d,J=6.4Hz,Thr Hγ),0.88(6H,d,J=6.7Hz,Val Hγ).HSQC((1H,500MHz)/(13C,126MHz),氧化氘)7.49/150.77(H1g/C1g),6.90/150.47(H1g/C1g),5.06/99.43(H1c/C1c),5.05/67.26(H6f/C6f,
H6f’/C6f’),4.96/78.16(H1a’/C1a’),4.88/97.00(H1c’/C1c’),4.70/100.37(H1b/C1b),4.60/49.86(Hα Asn/Cα Asn),4.53/99.51(H1a/C1a,H1d/C1d,H1d’/C1d’),4.48/72.14(H2g/C2g),4.39/57.99(Hα Thr/Cα Thr),4.37/103.58(H1e/C1e,H1e’/C1e’),4.33/53.62(Hα Lys/Cα Lys),4.32/67.14(Hβ Thr/Cβ Thr),4.24/49.53(Hα Ala/Cα Ala),4.18/72.35(H2b/C2b,H6f/C6f,H6f’/C6f’),4.12/76.11(H2c/C2c),4.08/59.56(Hα Val/Cα Val),4.04/76.01(H2c’/C2c’),4.00/52.70(Hα Lys/Cα Lys),3.32/36.54(H4g/C4g),3.31/48.73(H6g/C6g),3.23/36.58(H4g/C4g),2.93/39.11(Hε Lys/Cε Lys),2.77/36.47(Hβ Asn/Cβ Asn),2.58/39.32(H3f/C3f,H3f’/C3f’),2.04/29.84(Hβ Val/Cβ Val),1.87/30.45(Hβ Lys/Cβ Lys),1.78/39.39(H3f/C3f,H3f’/C3f’),1.71/27.53(H5g/C5g),1.63/26.18(Hδ Lys/Cδ Lys),1.37/21.65(Hγ Lys/Cγ Lys),1.34/16.85(Hβ Ala/Cβ Ala),1.09/18.70(Hγ Thr/Cγ Thr),0.86/17.86(Hγ Val/Cγ Val).HRMS,计算值C118H195N25O68[M+3H]3+1017.7601,实测值1017.7606;[M+2H]2+1526.1363,实测值1526.1346.
实施例4:衍生化SGP-化合物3d,为化合物2与M4经通用操作1制备完成。
产率88%.1H NMR(500MHz,氧化氘)δ7.49(1.7H,dd,J=7.0,4.1Hz,H1g),6.90(0.3H,dd,J=7.0,3.9Hz,H1g),5.07(1.3H,s,H1c,H6f,H6f’),4.96(1H,d,J=9.6Hz,H1a’),4.88(1H,s,H1c’),4.67(1H,s,H1b),4.60(1H,t,J=6.5Hz,1H,Asn Hα),4.51(7H,m,H1a,H1d,H1d’,H2g),4.40(1H,d,Thr Hα),4.38(2H,dd,H1e,H1e’),4.33-4.30(2H,m,Lys Hα,Thr Hβ),4.24(1H,q,J=7.2Hz,Ala Hα),4.18(2.7H,m,H1b,H6f,H6f’),4.12(1H,d,J=2.5Hz,H2c),4.06(1H,d,J=7.6Hz,Val Hα),4.03(1H,d,J=2Hz,H2c’),3.98(1H,t,J=6.7Hz,Lys Hα),2.92(q,J=7.5Hz,4H,Lys Hε),2.81-2.61(ddd,J=62.3,16.4,6.7Hz,2H,Asn Hβ),2.60-2.51(3H,m,H3feq,H3f’eq,H6g),2.05-1.87(19H,m,Ac,Val Hβ),1.87-1.66(6H,m,Lys Hβ,H3fax,H3f’ax),1.62(4H,m,J=7.5Hz,Lya Hδ),1.38(4H,d,J=5.9Hz,Lys Hγ),1.30(3H,d,J=7.2Hz,Ala Hβ),1.12(3H,d,J=6.4Hz,Thr Hγ),0.89(6H,d,J=6.7Hz,Val Hγ).HSQC((1H,500MHz)/(13C,126MHz),氧化氘)7.49/150.71(H1g/C1g),6.90/150.28
(H1g/C1g),5.06/99.53(H1c/C1c),5.03/67.24(H6f/C6f,H6f’/C6f’),4.96/78.25(H1a’/C1a’),4.88/96.92(H1c’/C1c’),4.70/100.20(H1b/C1b),4.60/49.87(Hα Asn/Cα Asn),4.53/99.93(H1a/C1a,H1d/C1d,H1d’/C1d’),4.51/72.14(H2g/C2g),4.39/58.01(Hα Thr/Cα Thr),4.37/103.63(H1e/C1e,H1e’/C1e’),4.33/53.61(Hα Lys/Cα Lys),4.32/67.03(Hβ Thr/Cβ Thr),4.24/49.50(Hα Ala/Cα Ala),4.18/72.67(H2b/C2b,H6f/C6f,H6f’/C6f’),4.12/76.11(H2c/C2c),4.08/59.45(Hα Val/Cα Val),4.04/76.17(H2c’/C2c’),3.99/52.61(Hα Lys/Cα Lys),3.98/28.50(H4g/C4g),2.92/38.96(Hε Lys/Cε Lys),2.76/36.56(Hβ Asn/Cβ Asn),2.58/39.41(H3f/C3f,H3f’/C3f’),2.56/72.13(H6g/C6g),2.03/30.15(Hβ Val/Cβ Val),1.86/30.31(Hβ Lys/Cβ Lys),1.79/39.19(H3f/C3f,H3f’/C3f’),1.62/26.04(Hδ Lys/Cδ Lys),1.37/21.44(Hγ Lys/Cγ Lys),1.34/16.69(Hβ Ala/Cβ Ala),1.16/18.82(Hγ Thr/Cγ Thr),0.92/17.91(Hγ Val/Cγ Val)HRMS,计算值C114H183N17O66[M+3H]3+949.7240,实测值949.7164;[M+2H]2+1424.0821,实测值1424.0687.
实施例5:衍生化SGP-3e,为化合物2与M2经通用操作1制备完成。
产率86.6%.1H NMR(500MHz,氧化氘)δ7.39(1.6H,dd,J=7.2,3.3Hz,H1of oxime),6.85(0.4H,dd,J=6.7,2.6Hz,H1of oxime),5.07(1H,s,H1c),4.96(1.4H,d,J=9.6Hz,H1a’,H6f/f’),4.88(1H,s,H1c’),4.76(1H,s,H1b),4.61(5H,m,H2g,Asn Hα),4.54(3H,d,J=6.7Hz,H1a,H1d.H1d’),4.39(1H,d,J=3.5Hz,Thr Hα),4.37(1H,d,J=2.5Hz,H1e),4.35(1H,d,J=3Hz,H1e’),4.34-4.28(2H,m,Lys1Hα,Τhr Hβ),4.24I(1H,q,J=7Hz,Ala Hα),4.18(1H,s,H2b),4.16(1.6H,dd,J=10.0Hz,7.5Hz,H6f/f’),4.12(1H,d,J=2Hz,H2c),4.08(1H,d,J=8Hz,Val Hα),4.04(1H,d,J=2Hz,H2c’)3.99(1H,t,J=6.5Hz,Lys2Hα),2.92(4H,q,J=7.5Hz,Lys Hε),2.86(2H,q,H3g),2.81-2.61(2H,m,Asn Hβ),2.61-2.51(2H,dd,J=12.5Hz,2.5Hz,H3feq,H3f’eq),2.03-1.88(19H,m,6x 3Ac,Val Hβ),1.87-1.66(6H,m,Lys Hβ,H3fax,H3f’ax),1.66(4H,m,J=7.5Hz,Lya Hδ),1.43-1.32(4H,m,Lys Hγ),1.3(3H,d,J=7.5Hz,Ala Hβ),1.13(3H,J=6.5Hz,Thr Hγ),0.89(6H,d,7.0Hz,Val Hγ).HSQC((1H,500
MHz)/(13C,126MHz),Deuterium Oxide)7.41/150.05(H1g/C1g),6.88/149.59(H1g/C1g),5.09/99.34(H1c/C1c),4.98/78.34(H1a’/C1a’),4.97/67.43(H6f/C6f,H6f’/C6f’),4.91/96.89(H1c’/C1c’),4.72/100.33(H1b/C1b),4.63/61.46(H2g/C2g),4.62/49.85(Hα Asn/Cα Asn),4.55/99.74(H1a/C1a,H1d/C1d,H1d’/C1d’),4.42/58.25(Hα Thr/Cα Thr),4.39/103.91(H1e/C1e,H1e’/C1e’),4.35/53.53(Hα Lys/Cα Lys),4.34/67.11(Hβ Thr/Cβ Thr),4.26/49.43(Hα Ala/Cα Ala),4.20/70.52(H2b/C2b,H6f/C6f,H6f’/C6f’),4.14/76.54(H2c/C2c),4.10/59.64(Hα Val/Cα Val),4.06/76.25(H2c’/C2c’),4.02/52.83(Hα Lys/Cα Lys),2.94/39.16(Hε Lys/Cε Lys),2.89/76.16(H4g/C4g),2.78/37.03(Hβ Asn/Cβ Asn),2.58/39.45(H3f/C3f,H3f’/C3f’),2.01/30.19(Hβ Val/Cβ Val),1.84/30.59(Hβ Lys/Cβ Lys),1.79/39.37(H3f/C3f,H3f’/C3f’),1.65/26.40(HδLys/CδLys),1.43/21.61(Hγ Lys/Cγ Lys),1.32/16.78(Hβ Ala/Cβ Ala),1.14/19.15(Hγ Thr/Cγ Thr),0.91/18.15(Hγ Val/Cγ Val).HRMS,计算值C118H189N19O68[M+3H]3+987.7383,实测值987.7380;[M+2H]2+1481.1036,实测值1481.1070.
实施例6:衍生化SGP-化合物3f,为化合物2与炔丙胺经通用操作2制备完成。
(8.5mg,3.0μmol,产率82.2%).1H NMR(500MHz,氧化氘)δ5.15(1H,s,H1c),5.05(1H,d,J=9.6Hz,H1a’),4.95(1H,s,H1c’),4.84(1H,s,H1b),4.67(1H,t,J=6.7Hz,Asn Hα),4.62(3H,dd,J=6.4,5.1Hz,H1a,H1d,H1d’),4.46(2H,dd,J=7Hz,H1e,H1e’),4.41(1H,t,Lys1Hα),4.31(1H,q,J=7.2Hz,Ala Hα),4.26(1H,s,H2b),4.22(1H,m,Thr Hβ)4.21(1H,m,Thr Hα),4.17-4.10(3H,m,H2c,Val Hα,H2c’),4.01(4H,H2g),3.43-3.16(4H,m,H1g),3.01(6H,m,Lys Hε,Η4g),2.93-2.69(2H,m,Asn Hβ),2.69-2.59(2H,dd,J=12.5Hz,2.5Hz,H3feq,H3f’eq),2.15ii-1.90(16iH,m,5x 3Ac,Val Hβ),1.97-1.81(7H,m,Lys Hβ,Αc)1.80-1.64(6H,m,Lys Hβ,H3fax,H3f’ax),1.66(4H,m,J=7.5Hz,Lya Hδ),1.43–1.32(4H,m,Lys Hγ),1.3(3H,d,J=7.5Hz,Ala Hβ),1.13(3H,J=6.5Hz,Thr Hγ),0.89(6H,d,7.0Hz,Val Hγ).HSQC((1H,500MHz)/(13C,126MHz),氧化氘)5.06/100.47(H1c/C1c),4.96/79.04
(H1a’/C1a’),4.86/97.82(H1c’/C1c’),4.70/101.42(H1b/C1b),4.59/50.87(Hα Asn/Cα Asn),4.53/100.62(H1a/C1a,H1d/C1d,H1d’/C1d’),4.37/103.51(H1e/C1e,H1e’/C1e’),4.32/54.42(Hα Lys/Cα Lys),4.31/67.02(Hβ Thr/Cβ Thr),4.22/50.36(Hα Ala/Cα Ala),4.17/71.01(H2b/C2b),4.12/77.38(H2c/C2c),4.06/60.90(Hα Val/Cα Val),4.04/77.09(H2c’/C2c’),3.92/53.74(Hα Lys/Cα Lys),3.91/37.48(H2g/C2g),3.28/48.52(H1g/C1g),2.90/39.89(Hε Lys/Cε Lys),2.79/39.26(Hβ Asn/Cβ Asn),2.60/40.92(H3f/C3f,H3f’/C3f’),1.97/30.09(Hβ Val/Cβ Val),1.79/31.46(Hβ Lys/Cβ Lys),1.69/41.18(H3f/C3f,H3f’/C3f’),1.65/27.10(Hδ Lys/Cδ Lys),1.35/22.40(Hγ Lys/Cγ Lys),1.30/17.64(Hβ Ala/Cβ Ala),1.09/20.15(Hγ Thr/Cγ Thr),0.89/19.16(Hγ Val/Cγ Val).HRMS,计算值C114H187N17O64[M+4H]4+705.5554,实测值705.5570;[M+3H]3+940.4045,实测值940.4066;[M+2H]2+1410.1029,实测值1410.1140.
实施例7
将2-叠氮乙基肟唾液酸糖肽(化合物SGP-化合物3a,8.7mg)溶于磷酸缓冲液(50mM,pH6.5,87mL)中,加入43.5mg糖苷酶切酶后在30℃孵育过夜。检测反应完成后,制备高效液相色谱分离得到白色粉末化合物4(5.5mg,产率88.87%)。
1H NMR(500MHz,氧化氘)δ7.39(dd,J=7.2,4.8Hz,1.3H,–N=CH-),6.83(dd,J=6.6,4.0Hz,0.7H,–N=CH-),5.11(d,J=3.1Hz,0.8H,H1a(α)),5.05(s,1H,H1c),4.94(dd,J=9.0,6.6Hz,0.7H,H6f/f’),4.86(s,1H,H1c’),4.50(d,J=7.3Hz,2H,H1d/d’),4.33(d,J=7.9Hz,2H,H1e/e’),4.22-4.12(m,6.3H,H2b,-CH2-O-N=C,H6f/f’),4.09(s,1H,H2c),4.02(s,1H,H2c’),3.92-3.76(m,14H,H2a,H6’b,H3c,H3c’,H6’c,H6’c’,H6d,H6d’,H6’d,H6’d’,H4e,H4e’,H6’e,H6’e’),3.68(m,20H),3.58-3.38(m,21H),1.99-1.87(m,15H),2.55(d,J=11.8Hz,2H,H3feq/f’eq),2.00-1.85(m,15H,Ac),1.77(t,J=11.4Hz,2H,H3fax/f’ax).HSQC((1H,500MHz)/(13C,126MHz),氧化氘)7.39/149.30(H1g/C1g),6.83/149.30(H1g/C1g),5.11/90.40(H1aa/C1aa),5.05/99.56(H1c/C1c),4.95/67.23(H6f/C6f,H6f’/C6f’),4.86/96.91(H1c’/C1c’),
4.68/100.36(H1b/C1b),4.62/94.73(H1ab/C1ab),4.50/99.20(H1d/C1d,H1d’/C1d’),4.34/103.64(H1e/C1e,H1e’/C1e’),4.16/72.65(H2b/C2b,H2g/C2g,H6f/C6f,H6f’/C6f’),4.10/76.23(H2c/C2c),4.03/76.06(H2c’/C2c’),3.44/49.89(H3g/C3g),2.54/39.18(H3f/C3f,H3f’/C3f’),1.77/38.84(H3f/C3f,H3f’/C3f’).HRMS,计算值C76H121N13O53[M+2H]2+1032.8664,实测值[M+2H]2+1032.8665,[M+3H]3+688.9136,实测值688.9182.
实施例8
将化合物4(5mg,2.42μmol)溶于34mL纯水中,然后加入三乙胺(15μL)、2-氯-1,3-二甲基氯化咪唑啉(DMC,36.3μmol),并将反应液置于冰浴中反应2小时。反应完成后G25纯化,用0.2%三乙胺水溶液进行洗脱。所得产物组分冷冻干燥得到白色粉末化合物5(4.5mg,91%)。
1H NMR(500MHz,氧化氘)δ7.37(d,J=7.2Hz,1.6H,H1g),6.80(d,J=6.6Hz,0.4H,H1g),6.01(d,J=7.2Hz,1H,H1of oxazoline),5.11-4.96(m,1.4H,H1c,H6f,H6f’),4.89(s,1H,H1c’),4.66(s,1H,H1b),4.53(dd,J=5.7Hz,2H,H1d,H1d’),4.35(d,J=6.6Hz,2H,H1e,H1e’),4.30(s,1H,H3a),4.26-4.18(dd,J=,1.6H,H6f,H6f’),4.15(t,J=4.6Hz,4H,H2g),4.11(m,1H,H2a),4.09(m,1H,H2b),2.55(d,J=13.2Hz,2H,H3feq,H3f’eq),2.13–1.83(m,15H,Ac),1.67(t,2H,H3fax,H3f’ax).HSQC((1H,500MHz)/(13C,126MHz),氧化氘)7.39/150.81(H1g/C1g),6.82/150.84(H1g/C1g),6.02/100.66(H1a/C1a oxa),5.07/100.38(H1c/C1c),4.90/97.41(H1c’/C1c’),4.67/102.13(H1b/C1b),4.54/100.31(H1d/C1d,H1d’/C1d’),4.36/104.59(H1e/C1e,H1e’/C1e’),4.32/70.15(H3a/C3a,oxa),4.23/73.66(H6f/C6f,H6f’/C6f’),4.17/73.32(H2g/C2g),4.13/66.06(H2a/C2a,oxa),4.12/77.28(H2c/C2c),4.09/77.06(H2c’/C2c’),4.08/71.19(H2b/C2b),3.47/50.52(H3g/C3g),2.57/41.16(H3f/C3f,H3f’/C3f’),1.68/41.06(H3f/C3f,H3f’/C3f’).
实施例9
非天然糖基化抗体7a由去糖基化抗体6与化合物SGP-3a经通用操作3制备完成;或由化合物5与去糖基化抗体6在EndoS突变酶的作用下制备完成,参考通用操作3完成。
实验结果如图2A-C所示。
实施例10
非天然糖基化抗体7b由去糖基化抗体6与化合物SGP-化合物3b经通用操作3制备完成。
实验结果如图4A-C所示。
实施例11
非天然糖基化抗体7c由去糖基化抗体6与化合物SGP-化合物3c经通用操作3制备完成。
实验结果如图6A-C所示。
实施例12
非天然糖基化抗体7d由去糖基化抗体6与化合物SGP-化合物3d经通用操作3制备完成。
实验结果如图8A-C所示。
实施例13
非天然糖基化抗体7e由去糖基化抗体6与化合物SGP-化合物3e经通用操作3制备完成。
实验结果如图10A-C所示。
实施例14
ADC-9a由叠氮基抗体7a与小分子细胞毒化合物8a经通用操作4制备完成。
实验结果如图11所示。
实施例15
ADC-9b由叠氮基抗体7a与小分子细胞毒化合物8b经通用操作4制备完成。
实验结果如图12所示。
实施例16
ADC-9c由叠氮基抗体7b与小分子细胞毒化合物8b经通用操作4制备完成。
实验结果如图13所示。
实施例17
ADC-9d由叠氮基抗体7a与小分子细胞毒化合物8c经通用操作4制备完成。
实验结果如图14所示。
实施例18
ADC-9e由叠氮基抗体7b与小分子细胞毒化合物8c经通用操作4制备完成。
实验结果如图15所示。
实施例19
ADC-9f由叠氮基抗体7b与小分子细胞毒化合物8e经通用操作4制备完成。
实验结果如图16所示。
实施例20
ADC-9g由叠氮基抗体7b与小分子细胞毒化合物8d经通用操作4制备完成。
实验结果如图17所示。
实施例21
“双药”ADC化合物11的制备
非天然寡糖赫赛汀7a(1.0mg/mL,5mg,1.0eq),DM1-SMCC(100μM,15.0eq)共同溶于10%DMSO的磷酸缓冲液中(50mM,pH 7.5),并于37℃孵育1小时。LC-MS显示,经过1小时后随即连接DM1的数量主要在2-3个之间,DAR值约为2.6。混合液立即进行ProteinA树脂纯化,得到具有叠氮基团的ADC化合物N3-T-DM1(化合物10)。化合物10(实施例21)的结构以及质谱数据如图18所示。
实施例22
将得到的化合物10(0.5mg/mL,2mg,1.0eq.)与DBCO-PEG4-VC-PAB-MMAE(66.7μM,20.0eq.)在30℃共同孵育。SDS-PAGE以及LC-MS显示过夜反应后偶联完全,并将混合液进行ProteinA树脂纯化得到双药ADC化合物11(实施例22)。化合物11的结构和质谱数据如图19所示。
实施例23
抗肿瘤细胞生长抑制实验
乳腺癌肿瘤细胞SK-BR-3细胞(中科院上海细胞研究所)的生长抑制检测采用SRB方法。具体步骤如下:处于对数生长期的细胞按合适密度接种至96孔培养板,每孔100μL,培养过夜后,加入不同浓度的药物作用72h,每个浓度设三复孔,并设相应浓度的生理盐水溶媒对照及无细胞调零孔。作用结束后,贴壁细胞倾去培养液,加入10%(w/v)三氯乙酸(100μL/孔)于4℃固定1h,随后用蒸馏水冲洗五次,室温干燥后,每孔加入
SRB溶液(Sigma,St.Louis,MO,U.S.A)(4mg/mL,溶于1%冰乙酸)100μL,室温下孵育染色15min后,用1%冰乙酸冲洗五次洗去未结合的SRB,室温干燥后,每孔加入10mM Tris溶液100μL,VERSMax酶标仪测定560nm波长下的光密度(OD值)。抗体-药物偶联物实施例17、实施例18、实施例19、实施例22的抗肿瘤活性如图20所示。如图20所示,显示了实施例17、实施例18、实施例19、实施例22良好ADCs化合物的活性。
Claims (10)
- 如权利要求1所述的式(I)所示的寡糖连接子的制备方法,如以下流程图所示:其中,n、X和Y的定义与上述式(I)中的定义相同,所述方法包括以下步骤:经蛋黄提取得到的唾液酸糖肽通过氧化得到具有醛基活性基团的唾液酸糖肽,该醛基化的唾液酸糖肽经过还原胺化反应,引入其他具有生物正交反应 价值的官能团,并在糖苷内切酶的作用下释放还有特殊标记基团的寡糖链,并经过环合过程获得寡糖连接子;或者,所述方法包括以下步骤:经蛋黄提取得到的唾液酸糖肽通过糖苷酶水解产物末端唾液酸寡糖链,经过氧化、还原胺化反应、环合过程制备获得寡糖连接子;或者,所述方法包括以下步骤:经蛋黄提取得到的唾液酸糖肽通过氧化得到具有醛基活性基团的唾液酸糖肽,该醛基化的唾液酸糖肽经过糖苷酶水解、经过还原胺化反应,引入其他具有生物正交反应价值的官能团,并在糖苷内切酶的作用下释放还有特殊标记基团的寡糖链,并经过环合过程获得寡糖连接子。
- 一种如下式(II)所示的基于抗体Fc区域N-糖基化位点的定点连接的抗体-药物偶联物:其中:n为选自0-6之间的整数;m、p和q分别独立地选自0或1;Ab为抗体;X选自-NHCH2-、-ONH=CH-、-CONHCH2-、-NHCO-CH=CH-;Y基团为D-L-Z-,其中,Z选自以下基团:其中,R1和R2分别独立选自H或CH3,其中,L选自以下基团:其中,a、c和d分别独立地为选自2-6之间的整数;b为选自3-20之间的整数,其中,R3和R4分别独立选自CH3-、CH3CH(CH3)-、PhCH2-、NH2(CH2)4-、NH2CONH(CH2)3-,D为小分子药物或小分子活性化合物,
- 如权利要求5所述的式(II)所示的抗体-药物偶联物的制备方法,包括以下步骤:a)将抗体通过糖苷酶切除其N-糖链,得到其Fc区域N-糖基化位点为乙酰葡萄糖胺单糖或岩藻糖α1,6乙酰葡萄糖胺二糖修饰的抗体;b)将式(I)所示的寡糖连接子通过糖苷酶或其突变体催化,连接到步骤a)所得到的乙酰葡萄糖胺单糖或岩藻糖α1,6乙酰葡萄糖胺二糖修饰的抗体上,制备包含特殊反应性基团的式(I)所示的寡糖连接子修饰的抗体;c)将步骤b)所得到的包含特殊反应性基团的式(I)所示的寡糖连接子修饰的抗体,与带有能与抗体糖链上该特殊反应性基团进行特异性偶联反应的对应基团修饰的小分子药物进行偶联,制备得到式(II)所示的抗体-药物偶联物。
- 如权利要求7所述的式(II)所示的抗体-药物偶联物的制备方法,其中,步骤a)和b)中,所述糖苷酶或其突变体为岩藻糖水解酶、N-乙酰葡萄糖胺内切水解酶或它们的突变体,更优选地,所述N-乙酰葡萄糖胺内切水解酶包括选自Endo-S(酿脓链球菌内切糖苷酶-S)、Endo-F3(Elizabethkingia miricola内切糖苷酶-F3)、Endo-S2(Endoglycosidase-S2, 酿脓链球菌内切糖苷酶-S2)、Endo-Sd(Endoglycosidase-Sd,酿脓链球菌内切糖苷酶-Sd)和Endo-CC(Endoglycosidase-CC,酿脓链球菌内切糖苷酶-CC)中的至少一种;和/或,优选地,步骤b)和c)所述的特殊反应性基团和能与该基团进行特异性偶联反应的对应基团分别为叠氮与炔基、巯基与马来酰亚胺、巯基与巯基或巯基活化形式、醛基与氨基、醛基与羟胺或肼基等两两对应组合。
- 一种双药偶联的抗体-药物偶联物,其为在如权利要求5所述的式(II)定点连 接的抗体-药物偶联物基础上,在其抗体上所含赖氨酸的氨基与另一不同小分子药物连接,形成两个药物偶联的抗体-药物偶联物。
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---|---|---|---|---|
WO2020263943A1 (en) * | 2019-06-24 | 2020-12-30 | University Of Georgia Research Foundation, Inc. | Dual drug antibody-drug conjugates |
CN113358865A (zh) * | 2020-03-03 | 2021-09-07 | 深圳先进技术研究院 | 一种循环肿瘤细胞检测方法 |
WO2024002330A1 (zh) * | 2022-07-01 | 2024-01-04 | 启德医药科技(苏州)有限公司 | 固定化的内切糖苷酶融合蛋白及其应用 |
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CN113943330A (zh) * | 2020-07-17 | 2022-01-18 | 中国科学院上海药物研究所 | 一类含糖结构化合物、其制备方法、药物组合物和用途 |
CA3211180A1 (en) | 2021-02-22 | 2022-08-25 | Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences | Disaccharide linker, disaccharide-small molecule drug conjugate and sugar chain fixed-point antibody-drug conjugate, and preparation method therefor and use thereof |
WO2022211075A1 (ja) * | 2021-03-31 | 2022-10-06 | 日本マイクロバイオファーマ株式会社 | 抗体薬物複合体の製造方法及びそれに用いる酵素 |
AU2021225125B9 (en) * | 2021-07-19 | 2023-02-02 | Mabplex International Co., Ltd. | Antibody drug conjugate loaded with binary toxins and its application |
CN115703845A (zh) * | 2021-08-05 | 2023-02-17 | 中国科学院上海药物研究所 | 寡糖连接子与侧链亲水性片段组合的糖链定点抗体-药物偶联物,其制法及用途 |
WO2024056101A1 (zh) * | 2022-09-16 | 2024-03-21 | 上海君实生物医药科技股份有限公司 | 用于抗体药物偶联物的连接子及其应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102037004A (zh) * | 2008-01-08 | 2011-04-27 | 生物种属学股份公司 | 使用寡糖基转移酶的多肽的糖缀合 |
CN104220603A (zh) * | 2012-02-10 | 2014-12-17 | 马里兰大学,巴尔的摩 | 抗体及其Fc片段的化学酶法糖基化工程 |
WO2015184004A1 (en) * | 2014-05-27 | 2015-12-03 | Academia Sinica | Anti-cd20 glycoantibodies and uses thereof |
WO2016123593A1 (en) * | 2015-01-30 | 2016-08-04 | Academia Sinica | Compositions and methods relating to universal glycoforms for enhanced antibody efficacy |
-
2016
- 2016-08-22 CN CN201610703219.7A patent/CN107778372B/zh active Active
-
2017
- 2017-08-15 WO PCT/CN2017/097439 patent/WO2018036403A1/zh active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102037004A (zh) * | 2008-01-08 | 2011-04-27 | 生物种属学股份公司 | 使用寡糖基转移酶的多肽的糖缀合 |
CN104220603A (zh) * | 2012-02-10 | 2014-12-17 | 马里兰大学,巴尔的摩 | 抗体及其Fc片段的化学酶法糖基化工程 |
WO2015184004A1 (en) * | 2014-05-27 | 2015-12-03 | Academia Sinica | Anti-cd20 glycoantibodies and uses thereof |
WO2016123593A1 (en) * | 2015-01-30 | 2016-08-04 | Academia Sinica | Compositions and methods relating to universal glycoforms for enhanced antibody efficacy |
Non-Patent Citations (1)
Title |
---|
KUROGOCHI, M. ET AL.: "Sialic Acid-focused Quantitative Mouse Serum Glycopro-teomics by Multiple Reaction Monitoring Assay", MOLECULAR & CELLULAR PROTEOMICS, vol. 9, no. 11, 22 June 2010 (2010-06-22), pages 2354 - 2368, XP055468851, ISSN: 1535-9476 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020263943A1 (en) * | 2019-06-24 | 2020-12-30 | University Of Georgia Research Foundation, Inc. | Dual drug antibody-drug conjugates |
CN113358865A (zh) * | 2020-03-03 | 2021-09-07 | 深圳先进技术研究院 | 一种循环肿瘤细胞检测方法 |
WO2024002330A1 (zh) * | 2022-07-01 | 2024-01-04 | 启德医药科技(苏州)有限公司 | 固定化的内切糖苷酶融合蛋白及其应用 |
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