CN110590877B - Linker, drug-loaded linker, cell-penetrating peptide conjugate drug, antibody conjugate drug, and method for producing same - Google Patents

Linker, drug-loaded linker, cell-penetrating peptide conjugate drug, antibody conjugate drug, and method for producing same Download PDF

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CN110590877B
CN110590877B CN201910744469.9A CN201910744469A CN110590877B CN 110590877 B CN110590877 B CN 110590877B CN 201910744469 A CN201910744469 A CN 201910744469A CN 110590877 B CN110590877 B CN 110590877B
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linker
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周传政
臧传龙
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Nankai University
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention relates to the field of drug delivery, and discloses a linker, a drug-loaded linker, a cell-penetrating peptide conjugated drug, an antibody conjugated drug and a preparation method thereof, wherein the linker has a structure shown in formula (I), and the photoresponse autocatalytically cleaved linker and the drug-loaded linker provided by the invention can couple the drug to a polypeptide or protein delivery carrier, so that the controllable delivery of the drug to cells can be realized.
Figure DDA0002165101590000011

Description

Linker, drug-loaded linker, cell-penetrating peptide conjugate drug, antibody conjugate drug, and method for producing same
Technical Field
The invention relates to the field of drug delivery, in particular to a photoresponse autocatalytic rupture type connector, a photoresponse autocatalytic rupture type drug-loaded connector and a preparation method thereof, a method for preparing a cell penetrating peptide coupled drug, a cell penetrating peptide coupled drug prepared by the method, a method for preparing an antibody coupled drug and the antibody coupled drug prepared by the method.
Background
Prodrug strategies are widely used in drug design and development with the aim of improving the pharmacokinetic properties, especially the targeted delivery capacity, of the parent drug (nat. rev. drug Discov.2108,17, 559-.
In general, prodrugs are constructed by coupling a drug molecule to a carrier via a linker containing a "trigger switch". After the prodrug is delivered to target cells or tissues, the linker is cleaved under the action of endogenous stimuli (pH value change, enzymes, redox reactions) or exogenous stimuli (small-molecule photoinitiators), so that the active drug is released and the drug effect is exerted. Therefore, rational design of the linker is key to achieving efficient, controlled delivery of the prodrug.
In some cases, the spatial distance between the drug and the carrier may disrupt cleavage of the linker, resulting in incomplete release of the drug.
To overcome this problem, researchers developed "self-immolative" linkers (Angew. chem. int. Ed.2003,42, 4494-. The self-sacrifice linker can efficiently release active drugs through a series decomposition reaction triggered by a stimulus factor.
To date, only two classes of "self-immolative" linkers have been widely used; one is via a tandem elimination mechanism and the other is via a cyclization mechanism (Angew. chem. int. Ed.2015,54, 7492-7509; chem. Eng. J.2018,340, 24-31). However, both types of linkers may produce products with toxic side effects, such as quinone methides, during cleavage, resulting in additional side effects.
Therefore, the development of new, biocompatible, highly efficient "self-immolative" linkers is essential for the design and development of prodrugs.
Disclosure of Invention
One of the objectives of the present invention is to overcome the drawbacks of the prior art and to provide a novel photoresponsive autocatalytic cleavage type linker and a drug-loaded linker.
The invention also aims to provide a cell penetrating peptide coupling drug and an antibody coupling drug, so as to realize the targeted and controllable delivery of the anticancer drug to cancer cells.
In order to achieve the above object, a first aspect of the present invention provides a linker of a photoresponsive autocatalytic cleavage type, the linker having a structure represented by formula (I),
Figure BDA0002165101570000021
wherein, in the formula (I),
R1is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl;
R2is selected from N, O, S with or withoutAt least one hetero atom chain alkyl group in (1) to form R2The number of atoms in the main chain of the chain alkyl group is 10 to 60, the main chain of the chain alkyl group contains or does not contain a saturated ring structure, contains or does not contain an unsaturated ring structure, contains or does not contain an alkenyl group, contains or does not contain an alkynyl group, and the main chain of the chain alkyl group is an unsubstituted group or is selected from C1-3Or a double bond structure is formed between at least one C atom and an O atom on the main chain of the chain alkyl group.
The second aspect of the present invention provides a drug-carrying linker of a photoresponsive autocatalytic cleavage type, which has a structure represented by formula (II), wherein, in formula (II), R1And R2Is as defined in claim 1 or 2, M is a group containing a group selected from-NH2At least one group capable of reacting among-SH, -OH, Y is-NH-, -S-or-O-provided by M;
Figure BDA0002165101570000031
a third aspect of the invention provides a method of preparing a drug-carrying linker of formula (II1), the method comprising:
Figure BDA0002165101570000032
(1) reacting a compound shown in a formula (1) with triphosgene in the presence of diisopropylethylamine and a solvent to obtain a compound shown in a formula (2);
(2) reacting the compound shown in the formula (2) with adriamycin in the presence of diisopropylethylamine and a solvent to obtain a compound shown in a formula (3);
(3) reacting the compound shown in the formula (3) with azido polyethylene glycol maleimide shown in the formula (4) and ascorbate in the presence of copper sulfate, dimethylformamide and water to obtain the drug-loaded linker;
Figure BDA0002165101570000041
wherein, in the formula (II1), the formula (1), the formula (2) and the formula (3), R1Is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl.
In a fourth aspect, the present invention provides a method for preparing a cell-penetrating peptide-conjugated drug, the method comprising:
(1) dissolving a drug-loaded linker described herein in a solvent to obtain a drug-loaded linker stock;
(2) and carrying out addition reaction on the drug-loaded linker storage solution and cell penetrating peptide H3-V35C to obtain the cell penetrating peptide coupling drug.
A fifth aspect of the invention provides a cell penetrating peptide conjugate drug prepared by the method as hereinbefore described.
A sixth aspect of the present invention provides a method for preparing an antibody-conjugated drug, the method comprising:
(1) dissolving the drug-loaded linker in a solvent to obtain a drug-loaded linker storage solution; and carrying out reduction reaction on the monoclonal antibody and a reducing agent solution to obtain a solution after the antibody is reduced;
(2) and carrying out addition reaction on the drug-carrying linker storage solution and the solution obtained after the reduction of the antibody to obtain the antibody conjugated drug.
The seventh aspect of the present invention provides an antibody-conjugated drug prepared by the method described above.
The photoresponse autocatalytic cleavage linker and the drug-loaded linker provided by the invention can couple a drug to a polypeptide or protein delivery carrier, and can realize controllable delivery of the drug to cells.
Drawings
FIG. 1 is a confocal laser microscopy analysis of the localization of the drug Doxorubicin (DOX) in HeLa cells;
FIG. 2 is a toxicity study of cell penetrating peptide coupling drug H3-PC4AP-DOX in HeLa cells;
FIG. 3 shows the efficiency of the antibody-conjugated drug Trastuzumab-PC4P-DOX to release drug DOX in serum after light irradiation;
FIG. 4 is a confocal laser microscopy analysis of the binding affinity of Trastuzumab and the antibody conjugate Trastuzumab-PC4P-DOX to breast cancer cells with different expression levels of HER2, wherein FIG. 4a shows that both Trastuzumab and the antibody conjugate Trastuzumab-PC4P-DOX are able to recognize and bind to SK-RB-3 cells with high expression of HER2, and FIG. 4b shows that Trastuzumab and the antibody conjugate Trastuzumab-PC4P-DOX are unable to recognize and bind to MCF7 cells with low expression of HER 2;
FIG. 5 is a graph of the toxicity of the antibody conjugate drug Trastuzumab-PC4AP-DOX in breast cancer cells with different levels of HER2 expression, wherein FIG. 5a shows that the toxicity of the antibody conjugate drug Trastuzumab-PC4AP-DOX is dose-dependent in SK-BR-3 cells with high expression of HER2, and FIG. 5b compares the toxicity difference of the antibody conjugate drug Trastuzumab-PC4AP-DOX in SK-BR-3 cells with high expression of HER2 with MCF7 cells with low expression of HER 2.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
As described above, the first aspect of the present invention provides a linker of a photoresponsive autocatalytic cleavage type having a structure represented by formula (I),
Figure BDA0002165101570000061
wherein, in the formula (I),
R1is selected from 4, 5-dimethoxy-At least one photosensitive protecting group selected from 2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl;
R2is a chain alkyl group containing or not containing at least one heteroatom selected from N, O, S to form R2The number of atoms in the main chain of the chain alkyl group is 10 to 60, the main chain of the chain alkyl group contains or does not contain a saturated ring structure, contains or does not contain an unsaturated ring structure, contains or does not contain an alkenyl group, contains or does not contain an alkynyl group, and the main chain of the chain alkyl group is an unsubstituted group or is selected from C1-3Or a double bond structure is formed between at least one C atom and an O atom on the main chain of the chain alkyl group.
According to a preferred embodiment, in formula (I), the linker has a structure represented by formula (I1),
Figure BDA0002165101570000062
wherein, in the formula (I1),
R1is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl;
n is a positive integer of 2 or more.
According to another preferred embodiment, in formula (I1),
R1is 4, 5-dimethoxy-2-nitrobenzyl, and n is 3.
According to a particularly preferred embodiment, the linker has the structure shown in formula (I2), and in formula (I2), R1Is 4, 5-dimethoxy-2-nitrobenzyl;
Figure BDA0002165101570000071
as mentioned above, the second aspect of the present invention provides a photoresponsive autocatalytic cleavage type drug-loaded linkerA linker having a structure represented by formula (II), wherein R in formula (II)1And R2Is as defined in claim 1 or 2, M is a group containing a group selected from-NH2At least one group capable of reacting among-SH, -OH, Y is-NH-, -S-or-O-provided by M;
Figure BDA0002165101570000072
according to a preferred embodiment, the drug-carrying linker has the structure shown in formula (II1), in formula (II1) DOX is a group provided by doxorubicin,
Figure BDA0002165101570000081
as mentioned above, the third aspect of the present invention provides a method for preparing a drug-carrying linker of formula (II1), the method comprising:
Figure BDA0002165101570000082
(1) reacting a compound shown in a formula (1) with triphosgene in the presence of diisopropylethylamine and a solvent to obtain a compound shown in a formula (2);
(2) reacting the compound shown in the formula (2) with adriamycin in the presence of diisopropylethylamine and a solvent to obtain a compound shown in a formula (3);
(3) reacting the compound shown in the formula (3) with azido polyethylene glycol maleimide shown in the formula (4) and ascorbate in the presence of copper sulfate, dimethylformamide and water to obtain the drug-loaded linker;
Figure BDA0002165101570000083
Figure BDA0002165101570000091
wherein, in the formula (II1), the formula (1), the formula (2) and the formula (3), R1Is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl.
Preferably, in the step (1), the compound represented by the formula (1) is used in a molar ratio of 1: (2-3): (1-1.5).
Preferably, in step (1), the solvent is tetrahydrofuran.
Preferably, in step (1), the reaction conditions include: the temperature is 10-40 ℃, the time is 4-24h, and the stirring speed is 50-1000 rpm.
Preferably, in the step (2), the molar ratio of the compound shown in the formula (2) to the diisopropylethylamine and the drug is 1: (2-3): (1-1.5).
Preferably, in step (2), the solvent is dichloromethane.
Preferably, in step (2), the reaction conditions include: the temperature is 10-40 ℃, the time is 1-15h, and the stirring speed is 50-1000 rpm.
Preferably, in the step (3), the compound shown in the formula (3) and the azido polyethylene glycol maleimide, ascorbate and copper sulfate shown in the formula (4) are used in a molar ratio of 1: (1-1.5): (0.8-1.2): (0.4-0.6).
Preferably, in step (3), the reaction conditions include: the temperature is 10-40 ℃, the time is 2-30h, and the stirring speed is 50-1000 rpm.
The aforementioned step (1), step (2) and step (3) of the present invention may be further combined with various post-treatment means conventionally used in the art to perform post-treatment on the obtained intermediates and the like, and the present invention is not described in detail herein, and those skilled in the art should not be construed as limiting the present invention.
The method for preparing the compound represented by formula (1) is not particularly limited in the present invention, and those skilled in the art can obtain the compound according to the structural formula of the compound represented by formula (1) by a conventional preparation method in the field of organic synthesis. The following exemplary embodiment of the present invention provides a preferred embodiment for preparing the compound represented by formula (1), and those skilled in the art should not be construed as limiting the present invention.
Figure BDA0002165101570000101
(1) Carrying out first mixing on the compound shown in the formula (a1), imidazole, tert-butyldiphenylchlorosilane and dimethylformamide to form a first mixed solution, washing, drying, filtering and carrying out column chromatography on an organic phase obtained after reaction to obtain the compound shown in the formula (a2), wherein R is R2Radicals provided for tert-butyldiphenylchlorosilane, R3Is benzoyl;
(2) secondly mixing the compound shown as the formula (a2) with sodium methoxide methanol solution and dichloromethane to form a second mixed solution, then performing rotary evaporation on the second mixed solution to remove the solvent, and performing column chromatography to obtain the compound shown as the formula (a 3);
(3) carrying out third mixing on the compound shown in the formula (a3), sodium hydride and tetrahydrofuran, and then cooling to form a third mixed solution; reacting the third mixed solution with propargyl bromide; then washing, drying, filtering and carrying out column chromatography on an organic phase obtained after the reaction to obtain a compound shown as a formula (a 4);
(4) reacting the compound shown as the formula (a4) with boron trifluoride diethyl etherate, propanedithiol and dichloromethane, and then washing, drying, filtering and carrying out column chromatography on an organic phase obtained after the reaction to obtain a compound shown as a formula (a 5);
(5) reacting the compound shown as the formula (a5) with oxalyl chloride, triethylamine and dimethyl sulfoxide, and then washing, drying, filtering and carrying out column chromatography on an organic phase obtained after the reaction to obtain a compound shown as a formula (a 6);
(6) reacting the compound shown as the formula (a6) with N-bromosuccinimide, R1Alcohol compound of the shown protective group, acetonitrile intoMixing to form a mixed solution, then performing rotary evaporation on the mixed solution to remove the solvent, and performing column chromatography to obtain a compound shown as a formula (a 7);
(7) and (2) reacting the compound shown as the formula (a7) with tetra-n-butylammonium fluoride and tetrahydrofuran, then performing rotary evaporation on the solution obtained after the reaction to remove the solvent, and performing column chromatography to obtain the compound shown as the formula (1).
Preferably, in the aforementioned preferred embodiment of the preparation of the compound represented by the formula (1), in the step (1), the compound represented by the formula (a1), imidazole, t-butyldiphenylchlorosilane and dimethylformamide are used in a ratio of 1mmol (3-4) mmol (1.2-1.5) mmol (50-70) mL; more preferably, the conditions of the first mixing include: the temperature is 18-25 ℃, the time is 10-12 hours, and the stirring speed is 400-600 revolutions per minute.
Preferably, in the aforementioned preferred embodiment of the preparation of the compound represented by the formula (1), in the step (2), the amount ratio of the compound represented by the formula (a2) to the sodium methoxide methanol solution and dichloromethane is 1mmol (0.3-0.4) mL (4-7) mL; more preferably, the conditions of the second mixing include: the temperature is 18-25 ℃, the time is 0.5-1 h, and the stirring speed is 400-600 r/min.
Preferably, in the aforementioned preferred embodiment for preparing the compound represented by the formula (1), in the step (3), the compound represented by the formula (a3) is used in an amount ratio to the propargyl bromide, the sodium hydride and the tetrahydrofuran of 1mmol (2-3) mmol: (3-5) mL. More preferably, the third mixing conditions include: the temperature is between 10 ℃ below zero and 10 ℃, the time is between 15 and 30 minutes, and the stirring speed is 400-600 revolutions per minute. Preferably, the conditions for reacting the third mixed solution with propargyl bromide include: the temperature is 18-25 ℃, the time is 4-6 hours, and the stirring speed is 400-600 revolutions per minute.
Preferably, in the aforementioned preferred embodiment for preparing the compound represented by the formula (1), in the step (4), the ratio of the compound represented by the formula (a4) to the amounts of propanedithiol, boron trifluoride etherate and dichloromethane is 1mmol (1.0-1.5) mmol (2.0-2.5) mmol (4-6) mL; preferably, the conditions of the reaction in step (4) include: the temperature is between-5 and 0 ℃, the time is between 10 and 30 minutes, the stirring speed is 400-600 revolutions per minute, the boron trifluoride diethyl etherate is added into the system in a dropwise manner, and the dropwise adding speed is between 10 and 15 drops per minute.
Preferably, in the aforementioned preferred embodiment of the preparation of the compound represented by the formula (1), in the step (5), the ratio of the amount of the compound represented by the formula (a5), oxalyl chloride, dimethyl sulfoxide, triethylamine and dichloromethane is 1mmol (1.0-1.5) mmol (2.0-2.5) mmol (4.0-6.0) mmol (4-6) mL; preferably, in step (5), the reaction process comprises: the reaction conditions of the first stage comprise that the temperature is-78 ℃, the time is 40-60 minutes, the stirring speed is 400-600 revolutions per minute, the dimethyl sulfoxide is added into the system in a dropwise adding mode, and the dropwise adding speed is 10-15 drops per minute; the reaction conditions of the second stage comprise that the temperature is-78 ℃, the time is 40-60 minutes, the stirring speed is 400-600 r/min, the compound shown in the formula (a5) is added into the system in a dropping way, and the dropping speed is 10-15 r/min; the reaction conditions of the third stage comprise that the temperature is-78 ℃, the time is 20-40 minutes, the stirring speed is 400-600 revolutions per minute, and the triethylamine is added into the system in a dropwise manner.
Preferably, in the aforementioned preferred embodiment of the preparation of the compound represented by formula (1), in step (6), the compound represented by formula (a6) is reacted with N-bromosuccinimide, R1The usage ratio of the alcohol compound of the protecting group is 1mmol (5-6) mmol (4-6) mmol (20-30) mL; preferably, in step (6), the mixing conditions include: the temperature is-10 ℃, the time is 40-60 minutes, and the stirring speed is 400-600 revolutions per minute.
Preferably, in the aforementioned preferred embodiment for preparing the compound represented by the formula (1), in the step (7), the ratio of the compound represented by the formula (a7) to the amount of tetra-n-butylammonium fluoride and tetrahydrofuran is 1mmol (2-3) mL (8-12) mL; preferably, in step (7), the reaction conditions include: the temperature is 18-25 ℃, the time is 1-3 hours, and the stirring speed is 400-600 revolutions per minute.
As previously described, a fourth aspect of the present invention provides a method for preparing a cell-penetrating peptide-conjugated drug, the method comprising:
(1) dissolving the drug-loaded linker in a solvent to obtain a drug-loaded linker storage solution;
(2) and carrying out addition reaction on the drug-loaded linker storage solution and cell penetrating peptide H3-V35C to obtain the cell penetrating peptide coupling drug.
Preferably, in the step (2), the molar ratio of the drug-loaded linker stock to the cell penetrating peptide H3-V35C is (1-1.5): 1.
preferably, in step (2), the conditions of the addition reaction include: the reaction temperature is 20-60 ℃, and the reaction time is 0.5-8 h.
Preferably, in step (2), the addition reaction is carried out in the presence of a HEPES buffer, preferably having a pH of 7.5.
The method of the invention preferably further comprises the step of further purifying the obtained cell penetrating peptide coupled drug, preferably by PD-MiniTrap-G25 gravity centrifugal desalination, collecting the required component peak NanoDrop concentration and storing at-80 ℃ for later use.
The cell penetrating peptide conjugate drug can optically control release of a loaded antitumor drug in a tumor cell, the tumor cell is preferably a HeLa cell, and the optically controlled condition is preferably 7mW/cm2The illumination time is preferably 5 to 7 minutes.
As mentioned above, a fifth aspect of the present invention provides a cell penetrating peptide conjugate drug prepared by the method as described above.
Preferably, the cell-penetrating peptide-conjugated drug has a structure represented by formula (5):
Figure BDA0002165101570000141
wherein, in the formula (5), Nv is 4, 5-dimethoxy-2-nitrobenzyl.
As previously described, a sixth aspect of the present invention provides a method for preparing an antibody-conjugated drug, the method comprising:
(1) dissolving the drug-loaded linker in a solvent to obtain a drug-loaded linker storage solution; and carrying out reduction reaction on the monoclonal antibody and a reducing agent solution to obtain a solution after the antibody is reduced;
(2) and carrying out addition reaction on the drug-carrying linker storage solution and the solution obtained after the reduction of the antibody to obtain the antibody conjugated drug.
Preferably, in step (1), the monoclonal antibody is a trastuzumab monoclonal antibody.
Preferably, the time of the reduction reaction is 2-3 hours, and the reaction temperature is 35-40 ℃, and more preferably 37 ℃.
Preferably, the temperature of the addition reaction is 35-40 ℃, and more preferably 37 ℃.
Preferably, the molar amount of the reducing agent is 4 times the molar amount of the antibody based on the molar amount of the antibody.
Preferably, the molar amount of the drug-carrying linker is 8 times the molar amount of the antibody based on the molar amount of the antibody.
The method of the invention preferably further comprises the step of further purifying the obtained antibody-conjugated drug, preferably by means of PD-MiniTrap-G25 gravity centrifugation for desalting, collecting the required component peak NanoDrop for concentration determination, and storing at-80 ℃ for later use.
As mentioned above, a seventh aspect of the present invention provides an antibody-conjugated drug prepared by the method described above.
The antibody-conjugated drug of the present invention preferably has a structure represented by formula (6):
Figure BDA0002165101570000151
wherein, in the formula (6), the mAb is Trastuzumab (Trituzumab monoclonal antibody), n is an integer more than 1 and less than or equal to 8, and Nv is 4, 5-dimethoxy-2-nitrobenzyl.
In the invention, the Trastuzumab monoclonal antibody is a recombinant humanized antibody which specifically binds to human HER2, and the molecular weight of the antibody is about 150 kDa. M is preferably anticancer drug adriamycin DOX, and the antibody conjugate drug is prepared by the Michael addition reaction of a maleimide group on a linker and an interchain disulfide bond of a Trastuzumab antibody.
The antibody conjugated drug can realize the targeted delivery of the drug in tumor cells. Wherein the tumor cells comprise HER2 positive SK-BR-3 cells and HER2 negative MCF7 cells.
The invention also has the following specific advantages:
(1) the drug-loaded linker has good stability under physiological conditions, and has low side effect caused by off-target release;
(2) after illumination, the drug can be efficiently and quickly released from the carrier, and the linker is cracked in an autocatalytic manner, so that toxic byproducts are not generated;
(3) the linker is photo-responsive, and has the advantages of high spatial and temporal resolution and non-invasiveness.
Based on the above, the novel photoresponsive autocatalytic cleavage type linker has a wide application prospect in the aspect of controllable drug delivery.
The present invention will be described in detail below by way of examples. In the following examples, various raw materials used are commercially available without specific description.
4, 5-dimethoxy-2-nitrobenzyl alcohol (as manufactured by Korea), tert-butyldiphenylchlorosilane (carbofuran, cat # 362315), imidazole (Adamas, cat # 45081D), propargyl bromide (Innochem, cat # A64659), sodium hydride (Adamas, cat # 81778A), boron trifluoride etherate (TCI, cat # A15275), propanedithiol (Adamas, cat # 13966C), N-bromosuccinimide (Allatin, cat # B105057), methylene chloride (Tianjin chemical reagent supplier, cat # 3975), tetrahydrofuran (Tianjin chemical reagent supplier, cat # 2287), diisopropylethylamine (Alfa, cat # A11801), doxorubicin hydrochloride (Soulebao, cat # D8740-25), triphosgene (Innochem, cat # A57532), sodium ascorbate (Micheln, cat # S8135), pentahydrate Michelin (Michelle # 807656, cat # C5356).
Example 1
1.0eq of a compound represented by the formula (a1) (wherein, in the formula (a1), "R" is weighed3Is benzeneFormyl) is dissolved by 60mL of anhydrous dimethylformamide under the protection of argon; then, 3.0eq of imidazole and 1.5eq of t-butyldiphenylchlorosilane were added to the reaction system. After stirring overnight at room temperature, the mixture was diluted with ethyl acetate, washed with water, and the separated organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and dried by suction to give a crude compound represented by the formula (a 2).
Taking the product of the last step (wherein, in the formula (a2), "R2"t-butyldiphenylsilyl group") was dissolved in 60mL of methylene chloride, and 7.0mL of a 25 mass% sodium methoxide methanol solution was added thereto and the mixture was stirred at room temperature for 1 hour. Concentrating under reduced pressure, and separating by column chromatography to obtain two isomers with two-step yield of 85%.
Isomer a:1H NMR(400MHz,CDCl3)δ7.65-7.62(m,4H),7.44-7.38(m,6H),5.12(dd,J＝5.6,2.0Hz,1H),4.51-4.47(m,1H),4.10-4.07(m,1H),3.42(dd,J＝12.0,2.8Hz,1H),3.33(s,3H),3.11(dd,J＝12.0,4.4Hz,1H),2.19(ddd,J＝14.0,5.6,4.8Hz,1H),2.04(ddd,J＝14.0,6.8,2.4Hz,1H),1.06(s,9H).13C NMR(101MHz,CDCl3)δ135.7,133.5,133.5,130.0,129.9,127.8,127.8,105.9,88.4,73.4,63.4,55.4,43.1,26.9,19.0.HRMS(ESI):C22H30NaO4Si,[M+Na]+calculated 409.1811, found 409.1803.
Isomer b:1H NMR(400MHz,CDCl3)δ7.67-7.64(m,4H),7.44-7.38(m,6H),4.91(dd,J＝5.6,2.4Hz,1H),4.21-4.17(m,1H),4.01-3.98(m,1H),3.57(dd,J＝12.0,2.8Hz,1H),3.38(s,3H),3.23(dd,J＝12.0,4.4Hz,1H),2.17-2.09(m,1H),1.91(ddd,J＝13.6,4.8,2.4Hz,1H),1.07(s,9H).13C NMR(101MHz,CDCl3)δ135.8,135.8,133.7,133.6,129.9,129.9,127.8,127.7,104.6,84.0,72.0,61.7,55.1,42.0,26.9,19.1.HRMS(ESI):C22H30NaO4Si,[M+Na]+calculated 409.1811, found 409.1803.
Under the protection of argon, 1.0eq of the compound of formula (a3) was dissolved in 36mL of dry tetrahydrofuran, 2.5eq of sodium hydride was added at 0 ℃ and after 15 minutes stirring was continued at room temperature for 30 minutes, 2.5eq of propargyl bromide was added and stirring was continued at room temperature for 5 hours. Quenching with water, concentrating, diluting with ethyl acetate, washing with saturated sodium chloride, drying the organic phase over anhydrous magnesium sulfate, filtering, concentrating, and separating by column chromatography to obtain the compound represented by the formula (a4) (yield 95%).
1H NMR(400MHz,CDCl3)δ7.69-7.64(m,8H),7.46-7.37(m,12H),5.06(dd,J＝5.2,2.0Hz,1H),4.93(dd,J＝6.0,2.4Hz,1H),4.38-4.33(m,1H),4.21-4.17(m,1H),4.15-4.09(m,2H),4.06-4.05(m,2H)4.02(dd,J＝4.8,2.4Hz,1H),3.53(dd,J＝11.8,2.8Hz,1H),3.39(s,2H),3.34-3.24(m,2H),3.29(s,6H),3.38-3.35(m,2H),2.16-2.05(m,2H),1.97(ddd,J＝13.2,6.8,2.0Hz,1H),1.86(ddd,J＝14.0,4.8,2.4Hz,1H),1.08(s,9H),1.07(s,9H).13C NMR(101MHz,CDCl3)δ135.88,135.86,135.79,129.83,129.79,127.74,127.70,127.68,105.43,104.64,85.18,82.62,79.63,79.43,74.60,74.40,73.85,72.53,71.40,68.91,58.45,58.29,55.09,54.98,41.93,41.66,26.91,26.89,19.13,19.06.HRMS(ESI):C25H32NaO4Si,[M+Na]+Calculated 447.1968, found 447.1975.
1.0eq. of the compound represented by the formula (a4) and 1.2eq. of propanedithiol were dissolved in 25.0mL of methylene chloride, and 2.2eq. of boron trifluoride diethyl ether was added dropwise under ice-cooling, and stirring was continued for 15 minutes. The reaction was quenched with saturated sodium bicarbonate, separated, washed with an organic phase saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and isolated by column chromatography to give the product (a5) (85% yield).
1H NMR(400MHz,CDCl3)δ7.75-7.70(m,4H),7.45-7.39(m,6H),4.11-4.03(m,3H),3.93(dd,J＝10.0,4.4Hz,1H),3.85-3.80(m,1H),3.51(dd,J＝10.0,4.0Hz,1H),3.42(dd,J＝9.6,7.6Hz,1H),2.71-2.63(m,3H),2.46-2.41(m,1H),2.39(t,J＝2.0Hz,1H),2.28(d,J＝4.0Hz,1H),2.09-2.02(m,1H),2.00-1.96(m,1H),1.87-1.72(m,2H),1.09(s,9H).13C NMR(101MHz,CDCl3)δ136.1,136.1,133.7,133.2,129.9,129.8,127.8,127.7,79.3,74.8,73.3,71.5,70.5,58.4,43.7,38.5,30.2,29.6,27.1,25.8,19.6.HRMS(ESI):C27H36NaO3S2Si,[M+Na]+Calculated 523.1773, found 523.1761.
Under the protection of argon, dropwise adding 2.4 eq.dimethyl sulfoxide into 1.3 eq.oxalyl chloride in dichloromethane at-78 ℃, continuing stirring for 45 minutes, dropwise adding 1.0 eq.5.0 eq.45 minutes later of the compound shown in the formula (a5), and heating to room temperature after 30 minutes. The reaction was quenched with saturated ammonium chloride, stirred for 15 minutes, then separated, the aqueous phase was extracted with dichloromethane, the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous magnesium sulfate, filtered, concentrated, and isolated by column chromatography to give the product (a6) (yield 85%).
1H NMR(400MHz,CDCl3)δ7.65-7.64(m,4H),7.47-7.37(m,6H),4.53(d,J＝18Hz,1H),4.51(t,J＝5.2Hz,1H),4.33(d,J＝18Hz,1H),4.12(d,J＝2.2Hz,2H),3.95(t,J＝7.6Hz,1H),2.80-2.56(m,4H),2.41(t,J＝2.2Hz,1H),2.36-2.27(m,1H),2.05-1.95(m,2H),1.88-1.80(m,1H),1.13(s,9H).13C NMR(101MHz,CDCl3)δ207.9,135.9,132.8,132.3,130.2,128.0,128.0,78.8,75.4,75.2,72.2,58.1,41.1,39.9,28.6,28.5,27.1,25.5,19.4.HRMS(ESI):C27H35O3S2Si,[M+H]+Calculated 499.1797, found 499.1800.
1.0 eq.5 eq.N-bromosuccinimide of the compound of the formula (a6) was dissolved in 42.0mL of dichloromethane/acetonitrile and 4.5 eq.4, 5-dimethoxy-2-nitrobenzyl alcohol was added at-10 ℃ and stirring was continued for 1 hour. And (3) raising the temperature to room temperature, quenching the reaction by saturated sodium thiosulfate, separating liquid, extracting the water phase for 2 times by using diethyl ether, combining organic phases, washing the saturated sodium bicarbonate and sodium chloride sequentially, drying the mixture by using anhydrous magnesium sulfate, filtering the dried mixture, concentrating the filtered mixture, and carrying out column chromatography to obtain the compound shown in the formula (a7) (the yield is 65 percent, and 4 isomers) by using the compound.
1.0 eq.1 part by weight of a compound represented by the formula (a7) (wherein, in the formula (2), "R" is1"4, 5-dimethoxy-2-nitrobenzyl group") was dissolved in 10.0mL of tetrahydrofuran, and 1.82mL of a 1M solution of tetra-n-butylammonium fluoride in tetrahydrofuran was added and stirred at room temperature for 2 hours. Concentrating under reduced pressure, and separating by column chromatography to obtain the product, namely the compound shown in the formula (1) (yield 89%). Preparing under high pressure (C18,10 μm,21.2mm × 250mm, flow rate 15mL/min, mobile phase A: water, B: acetonitrile, 0-10-20-27min, B: 5% -40% -70% -70%), and purifying to obtain nuclear magnetic identification sample:
1-1(RT＝22.0min)：1H NMR(400MHz,CDCl3)δ7.59(s,1H),7.53(s,1H),7.28(s,1H),6.84(s,1H),5.58(dd,J＝5.6,4.4Hz,1H),4.99(s,2H),4.93(s,2H),4.56-4.54(m,1H),4.24-4.21(m,2H),3.97-3.86(m,2H),3.92(s,3H),3.90(s,3H),3.89(s,3H),3.79(s,3H),2.50(t,J＝2.4Hz,1H),2.46-2.41(m,1H),2.36-2.31(m,1H).13C NMR(101MHz,CDCl3)δ153.39,153.15,147.74,147.23,139.67,138.73,130.90,128.61,110.18,109.84,109.81,107.97,107.39,105.68,78.78,76.45,75.83,67.79,66.75,60.76,58.72,56.30,56.24,56.21,56.14,40.07.HRMS(ESI):C26H30N2NaO13,[M+H]+calculated 601.1646, found 601.1643.
1-2(RT＝23.5min)：1H NMR(400MHz,CDCl3)δ7.67(s,1H),7.65(s,1H),7.25(s,1H),7.15(s,1H),5.33(d,J＝4.8Hz,1H),5.18-4.85(m,4H),4.61(dd,J＝9.6,7.2Hz,1H),4.15(t,J＝2.4Hz,2H),3.96-3.91(m,2H),3.97(s,3H),3.96(s,3H),3.92(s,6H),2.45-2.41(m,2H),2.24-2.17(m,1H).13C NMR(101MHz,CDCl3)δ153.55,153.50,147.70,139.49,139.35,130.13,129.74,110.18,109.84,107.94,107.90,104.10,102.17,78.89,75.30,73.49,70.08,66.69,61.45,58.69,56.37,39.40.HRMS(ESI):C26H30N2NaO13,[M+H]+Calculated 601.1646, found 601.1643.
1-3(RT＝24.5min)：1H NMR(400MHz,CDCl3)δ7.71(s,1H),7.68(s,1H),7.24(s,1H),7.23(s,1H),5.41(d,J＝5.6Hz,1H),5.19-5.06(m,2H),4.97-4.92(m,2H),4.33(m,1H),4.21(d,J＝2.4Hz,2H),4.01-3.87(m,2H),3.99(s,3H),3.98(s,3H),3.95(s,3H),3.94(s,3H),2.62-2.55(m,1H),2.45(t,J＝2.4Hz,1H),2.18–2.15(m,1H).13C NMR(101MHz,CDCl3)δ153.61,153.48,147.83,147.61,139.53,139.45,130.27,129.61,110.44,110.26,110.24,108.01,107.87,105.22,79.00,75.39,75.09,67.24,66.23,60.96,58.67,56.46,56.40,56.37,56.34,39.22.HRMS(ESI):C26H30N2NaO13,[M+H]+Calculated 601.1646, found 601.1643.
Under the protection of argon, 1.0 eq.1-1 is dissolved in 2.0mL of anhydrous tetrahydrofuran, 2.2 eq.diisopropylethylamine and 1.0 eq.triphosgene are added, and the mixture is stirred at room temperature overnight. Washing with water, extracting with dichloromethane, drying the organic phase with anhydrous magnesium sulfate, filtering, and concentrating to obtain a crude product, namely the compound shown in the formula (2). 1.0eq. of the compound represented by the above formula (2) was dissolved in 3.0mL of methylene chloride, and 2.4eq. diisopropylethylamine and 1.2eq. adriamycin were added thereto and stirred at room temperature for 4 hours. Concentrating, and separating by column chromatography to obtain the compound shown in the formula (3) (two-step yield 65%).
1H NMR(400MHz,CDCl3)δ14.0(s,1H),13.2(s,1H),8.00(d,J＝7.6Hz,1H),7.77(t,J＝8.0Hz,1H),7.58(s,1H),7.49(s,1H),7.37(d,J＝8.4Hz,1H),7.27(s,1H),6.82(s,1H),5.52-5.48(m,2H),5.35-5.34(m,1H),5.24(d,J＝8.8Hz,1H),4.96-4.91(m,4H),4.75(s,2H),4.54(s,1H),4.17-4.12(m,2H),4.10(d,J＝2Hz,2H),4.06(s,3H),3.91-3.81(m,2H),3.90(s,3H),3.86(s,3H),3.83(s,3H),3.75(s,3H),3.70-3.66(m,2H),3.24(d,J＝18.8Hz,1H),2.97(d,J＝18.8Hz,1H),2.53-2.49(m,1H),2.42-2.38(m,1H),2.38-2.30(m,2H),2.20-2.15(m,1H),1.92-1.79(m,2H),1.29(d,J＝6.4Hz,3H).13C NMR(101MHz,CDCl3)δ213.8,187.1,186.6,161.0,156.1,155.6,154.3,153.4,153.2,147.7,147.1,139.5,138.5,135.8,135.4,133.5,133.4,131.0,128.6,120.7,119.8,118.4,111.6,111.4,110.0,109.7,108.6,107.9,107.3,105.0,100.6,79.2,75.3,69.6,69.6,67.9,67.2,65.9,65.5,60.8,58.5,56.7,56.3,56.2,56.1,53.4,47.0,38.8,35.6,34.0,30.2,16.8.HRMS(ESI):C54H57N3NaO25,[M+Na]+Calculated 1170.3179, found 1170.3206.
1.0eq of the compound of formula (3) and 1.2eq of Mal-PEG3-N3Dissolving in 3mL (2:1) of dimethylformamide-water mixed solvent, adding 0.5eq copper sulfate pentahydrate and 1.0eq sodium ascorbate, stirring overnight at room temperature, monitoring the reaction by LC-MS, and directly high-pressure purifying to obtain red solid drug-loaded linker (yield 45%) after the raw materials are reacted. Separation conditions (C18,10 μm,21.2 mm. times.250 mm, flow rate 15mL/min, mobile phase A: water, B: acetonitrile, 0-18min, B: 45% -80%. RT ═ 10.5 min).
1H NMR(400MHz,CDCl3)δ13.99(s,1H),13.26(s,1H),8.03(d,J＝7.6Hz,1H),7.79(t,J＝8.0Hz,1H),7.72(s,1H),7.58(s,1H),7.49(s,1H),7.39(d,J＝8.4Hz,1H),7.36(s,1H),6.84(s,1H),6.69(s,2H),5.54-5.44(m,2H),5.35-5.32(m,2H),4.96-4.86(m,4H),4.78(s,2H),4.68-4.53(m,5H),4.17-4.15(m,1H),4.07(s,3H),3.92(s,3H),3.88(s,3H),3.84-3.82(m,3H),3.80(s,3H),3.78(s,3H),3.73-3.70(m,2H),3.65-3.50(m,10H),3.42-3.40(m,2H),3.30-3.26(m,1H),3.16-3.02(m,2H),2.52-2.49(m,3H),2.38-2.34(m,3H),2.22-1.85(m,5H),1.34-1.32(m,3H).HRMS(ESI):C69H81N8O31,[M+H]+Calculated 1517.5008, found 1517.5007.
The structure of the obtained drug-loaded linker (Mal-PC4AP-DOX) is as follows:
Figure BDA0002165101570000211
nv is 4, 5-dimethoxy-2-nitrobenzyl.
Example 2
This example is presented to illustrate the procedure for the preparation of cell penetrating peptide conjugated drugs by conjugation of the drug loaded linker prepared in example 1 with cell penetrating peptides.
The method comprises the following steps:
1) the drug-loaded linker Mal-PC4AP-DOX prepared in example 1 was dissolved in dimethylformamide to give a drug-loaded linker stock solution with a concentration of 1.5 mM. The H3-V45C protein was dissolved in sterile water to give a stock solution at a concentration of 300. mu.M.
2) 33.5 microliters of the drug-loaded linker stock was mixed with 160 microliters of H3-V35C protein in HEPES buffer at pH 7.5(20mM) and incubated at 37 ℃ for 1 hour.
3) After the reaction is finished, the obtained cell penetrating peptide coupling drug (H3-PC4AP-DOX, the structure is shown in formula 5) needs to be further purified, and is desalted by adopting a PD MiniTrap G-25 desalting column. The specific desalting method comprises the following steps: the sample is loaded on a desalting column by 200 microlitres, and the gravity centrifugation mode is adopted, wherein the centrifugation temperature is room temperature, the rotation speed is 1000 Xg, and the centrifugation time is 2 minutes. The concentration of the NanoDrop after desalting was measured and stored at-80 ℃.
Example 3
This example is intended to illustrate that the cell penetrating peptide-conjugated drug H3-PC4AP-DOX prepared in example 2 can be used for the light controlled release of doxorubicin DOX in HeLa cells.
3.1 confocal microscopy detection of cellular localization of DOX and H3-PC4AP-DOX
Taking HeLa cells in logarithmic phase, digesting with pancreatin, washing, centrifuging and preparing into single cell suspension. The counted cells were seeded in 24-well plates (density 1.0X 10)5One/well), 5% CO at 37 deg.C2The cells were cultured overnight in a constant temperature incubator. After overnight culture, cells were washed 2 times with PBS and complete medium containing 10. mu.M doxorubicin, H3-PC4AP-DOX, respectively, was added. After further incubation for various periods, the stock culture was aspirated, the cells were washed 2 times with PBS, and 300. mu.L of 4% paraformaldehyde was fixed for 20 minutes at room temperature. After 2 PBS rinses, 300. mu.L (10. mu.g/mL) of DAPI was added for staining for 7 minutes, followed by PBS rinse and mounting. Confocal laser microscopy (Nikon A1+, Nikon, Tokyo, Japan) for photographic observations (DAPI channel: excitation wavelength 408nm, emission wavelength: 425-: excitation wavelength 561nm, emission wavelength: 570-620 nm).
After 3 hours incubation in the light group H3-PC4AP-DOX, fresh medium, 365nm (7.0mW cm)-2) After 5 minutes of illumination, incubation was continued at 37 ℃ for a certain period of time. The rest of the operation is the same as the above process.
As shown in fig. 1: fluorescence (red) of DOX was clearly observed in HeLa cell nuclei after H3-PC4AP-DOX was irradiated with light. Thus, it can be seen that DOX can be released from the cell penetrating peptide-coupled drug H3-PC4AP-DOX designed and synthesized in example 2 after light irradiation.
Cell killing Activity of 2H 3-PC4AP-DOX against HeLa cells
Taking HeLa cells in logarithmic phase, digesting with pancreatin, washing, centrifuging and preparing into single cell suspension. mu.L of the suspension was inoculated into a 96-well plate (3000/well), and the cell culture plate was incubated at 37 ℃ with 5% CO2Overnight incubation in a cell culture incubator. Complete medium of different concentration gradients H3-PC4AP-DOX was added, after further incubation for 40 minutes, the cells were washed 2 times with PBS and fresh medium was added. Illumination groups 96 well plates were placed on UV-LED (7 mW/cm)2365nm) for 7 minutes, and the control group was incubated for the same time at room temperature in the dark. After the culture is continued in the incubator for 48 hours, 10 mu L of CCK-8 is added into each hole, after the culture is continued for 1 hour, the absorbance at 450nm is measured by using an enzyme-linked immunosorbent assay, and the growth inhibition rate of the drug to the cells under different conditions is calculated. A blank group (without cells) and a negative control group (without drug treatment) are respectively arranged in the experimental process, and the inhibition rate of the cells is calculated according to the following formula: the cell inhibition rate was (control a450 value-medicated a450 value)/(control a450 value-blank a450 value) × 100%. IC (integrated circuit)50Values were analyzed by GraphPad Prism software processing. The above experiment was independently repeated 3 times with 4 replicates per concentration. As shown in fig. 2: CCK-8 experiments show that compared with a control group, the cell survival rate of Hela cells incubated by H3-PC4AP-DOX is obviously reduced after the Hela cells are illuminated, so that the photosensitive Mal-PC4AP-DOX linker obtained by the invention can be used for a drug delivery system taking cell penetrating peptide as a carrier.
Example 4
This example is intended to illustrate that the drug-loaded linker Mal-PC4AP-DOX prepared in example 1 can be used to conjugate drugs with the preparation antibody to achieve targeted delivery of the drug.
4.1 preparation of antibody drug conjugate Trastuzumab-PC4AP-DOX (structure shown in formula 6)
The method comprises the following steps:
1) TCEP reducing agent is dissolved in sterile water to prepare a reducing agent storage solution with the concentration of 1.0 mM. The antibody Trastuzumab was dissolved in PBS buffer to prepare a stock solution having a concentration of 5 mg/mL.
2) mu.L of TCEP was added to 20. mu.L of Trastuzumab solution, and the mixture was incubated at 37 ℃ for 2 hours to obtain a solution after antibody reduction. The molar amount of TCEP reducing agent added is 4 times the molar amount of Trastuzumab antibody.
3) 3.8 mu L of the drug-loaded linker Mal-PC4AP-DOX prepared in example 1 was added to the reduced antibody solution and incubation was continued for 1 hour to obtain the antibody conjugate drug. The linker-cytotoxin drug solution added was 8 times the molar amount of antibody. After the reaction was completed, the antibody conjugate was purified by desalting by PD SpinTrap G-25(GE Healthcare,28-9180-04) by gravity centrifugation. The specific desalination method is as follows: the desalting column was loaded to 100. mu.L and centrifuged by gravity at room temperature at 800 Xg for 2 min. After desalting was complete, the concentration was measured by NanoDrop and stored at-80 ℃.
4.212% SDS-PAGE detection of light release efficiency of antibody drug conjugate Trastuzumab-PC4AP-DOX in serum
mu.L of 3. mu.M Trastuzumab-PC4AP-DOX was mixed with 18. mu.L of human serum at 365nm (7 mW/cm)2) After 5 minutes of light, incubation was carried out at 37 ℃. mu.L of samples were taken at different time points and quenched with 20mM sodium cyanoborohydride, mixed with SDS loading buffer (containing 50mM Tris, pH6.8, glycerol 10% (v/v), SDS 2% (w/v), bromophenol blue 0.1% (w/v)) and analyzed by 12% SDS-PAGE. The resulting Gel was first imaged by GE Typhoon Gel Imaging Scanner fluorescence (excitation wavelength 532nm, emission wavelength 570nm) and then stained with Coomassie Brilliant blue.
As shown in FIG. 3, after light irradiation and incubation for only 10 minutes, DOX was released from the heavy chain and light chain of the antibody, respectively, with a total release efficiency of about 70%. The total release efficiency can reach about 85% after the time is prolonged to 1 hour, so the drug-carrying linker Mal-PC4AP-DOX can be used for drug delivery with an antibody as a carrier.
4.3 detection of the binding Activity of the antibody drug conjugate Trastuzumab-PC4AP-DOX with Breast cancer cells by confocal laser microscopy
Respectively inoculating counted breast cancer cells SK-BR-3(HER2 high expression) and MCF-7(HER2 low expression) cells into a 24-well plate (density is 1.0 × 10)5One/well), 5% CO at 37 deg.C2The cells were cultured overnight in a constant temperature incubator. After overnight incubation, cells were washed 2 times with PBS and fixed in 4% paraformaldehyde for 20 min at room temperature. After 2 washes with PBS, 10% goat serum was blocked for 30 min at room temperature. Serum was removed, cells were incubated with 500nM Trastuzumab or Trastuzumab-PC4AP-DOX for 30 min at room temperature, and after 2 washes with PBS, goat anti-human AF488 antibody (1:200) was incubated for a further 30 min. DAPI (10. mu.g/mL) staining was performed for 7 minutes by PBS washing, mounting, and photographing observation was performed by a laser confocal microscope (Nikon A1+, Nikon, Tokyo, Japan) (DAPI channel: excitation wavelength 408nm, emission wavelength: 425-475nm), AF488 channel: excitation wavelength 486nm, emission wavelength: 500-550 nm). The results are shown in FIG. 4a, and both Trastuzumab and the antibody-conjugated drug Trastuzumab-PC4AP-DOX can specifically bind to the HER2 highly expressed breast cancer cell SK-BR. However, none of them could bind to MCF7, a breast cancer cell with low expression of HER2 (fig. 4 b).
4.4 cell killing Activity of Trastuzumab-PC4AP-DOX against tumor cells at different HER2 expression levels
SK-BR-3 and MCF-7 cells in logarithmic phase are taken and are prepared into single cell suspension after being digested, washed and centrifuged by pancreatin. 100 μ L of the cell suspension was inoculated into 96-well plates (7000 cells/well for SK-BR-3 and 3000 cells/well for MCF-7), and the cell culture plates were placed at 37 ℃ with 5% CO2Overnight incubation in a cell culture incubator. Adding drug-loaded linker, antibody drug conjugate and DOX complete culture medium with different concentration gradients, continuously culturing for 6 hours, washing cells for 2 times with PBS, and adding fresh culture medium. Illumination groups 96 well plates were placed on UV-LED (7 mW/cm)2365nm) for 5 minutes, and the control group was incubated at room temperature in the dark for the same time. After the culture is continued in the incubator for 48 hours, 10 mu L of CCK-8 is added into each hole, after the culture is continued for 1 hour, the absorbance at 450nm is measured by using an enzyme-labeling instrument, and the growth survival rate of the cells under the conditions of different drug concentrations is calculated. The cell viability was calculated according to the following formula: cell viability ═ 100% (medicated a450 value-blank a450 value)/(control a450 value-blank a450 value). The above experiment was independently repeated 3 times, with 4-5 replicates per concentration. As shown in FIG. 5a, for SK-BR-3 cells with over-expression of HER2, the survival rate of SK-BR-3 cells incubated with antibody conjugated drugs is obviously reduced under the condition of illumination, the efficacy of the SK-BR-3 cells is equivalent to that of free adriamycin and is dose-dependent; in contrast, in the non-illuminated group, the growth of the cells was not significantly inhibited. SK-BR-3 cells incubated with the drug-loaded linker did not cause toxicity to the cells regardless of the presence of light. This indicates that the single light irradiation does not cause the release of DOX from the antibody, which further verifies that the drug release mechanism is by intramolecular addition elimination of tandem chemistry as contemplated by the inventors. In addition, the toxicity test of the HER2 negative MCF7 cell shows that the cell survival rate is not obviously reduced under the illumination (figure 5b), so that the antibody conjugated drug can realize the targeted light-controlled release of the drug.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims (20)

1. A photo-responsive autocatalytic cleavage type linker having a structure represented by formula (I1),
Figure FDA0002813974970000011
wherein, in the formula (I1),
R1is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl;
n is 2 or 3.
2. The linker of claim 1, wherein in formula (I1),
R1is 4, 5-dimethoxy-2-nitrobenzyl, and n is 3.
3. The linker of claim 1, wherein the linker has a structure represented by formula (I2), and in formula (I2), R is1Is 4, 5-dimethoxy-2-nitrobenzyl;
Figure FDA0002813974970000012
4. a drug-carrying linker of a photoresponsive autocatalytic cleavage formula, characterized in that the drug-carrying linker has a structure represented by formula (II1) wherein, in formula (II1), DOX is a group provided by doxorubicin,
Figure FDA0002813974970000021
wherein R is1Is as defined in any one of claims 1 to 3.
5. A method of preparing a drug-loaded linker of formula (II1), comprising:
Figure FDA0002813974970000022
(1) reacting a compound shown in a formula (1) with triphosgene in the presence of diisopropylethylamine and a solvent to obtain a compound shown in a formula (2);
(2) reacting the compound shown in the formula (2) with adriamycin in the presence of diisopropylethylamine and a solvent to obtain a compound shown in a formula (3);
(3) reacting the compound shown in the formula (3) with azido polyethylene glycol maleimide shown in the formula (4) and ascorbate in the presence of copper sulfate, dimethylformamide and water to obtain the drug-loaded linker;
Figure FDA0002813974970000023
Figure FDA0002813974970000031
wherein, in the formula (II1), the formula (1), the formula (2) and the formula (3), R1Is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl.
6. The method according to claim 5, wherein in the step (1), the compound represented by the formula (1) is used in a molar ratio of 1: (2-3): (1-1.5).
7. The process according to claim 5, wherein, in step (1), the solvent is tetrahydrofuran.
8. The method of claim 5, wherein, in step (1), the reaction conditions comprise: the temperature is 10-40 ℃, the time is 4-24h, and the stirring speed is 50-1000 rpm.
9. The method according to claim 5, wherein in the step (2), the compound represented by the formula (2) and the diisopropylethylamine and the adriamycin are used in a molar ratio of 1: (2-3): (1-1.5).
10. The process of claim 5, wherein in step (2), the solvent is dichloromethane.
11. The method of claim 5, wherein in step (2), the reaction conditions comprise: the temperature is 10-40 ℃, the time is 1-15h, and the stirring speed is 50-1000 rpm.
12. The method according to claim 5, wherein in the step (3), the compound represented by the formula (3) and the azido polyethylene glycol maleimide, ascorbate and copper sulfate represented by the formula (4) are used in a molar ratio of 1: (1-1.5): (0.8-1.2): (0.4-0.6).
13. The method of claim 5, wherein, in step (3), the conditions of the reaction comprise: the temperature is 10-40 ℃, the time is 2-30h, and the stirring speed is 50-1000 rpm.
14. A method of preparing a cell-penetrating peptide-conjugated drug, the method comprising:
(1) dissolving the drug-loaded linker of claim 4 in a solvent to obtain a drug-loaded linker stock;
(2) and carrying out addition reaction on the drug-loaded linker storage solution and cell penetrating peptide H3-V35C to obtain the cell penetrating peptide coupling drug.
15. The method of claim 14, wherein, in step (2), the drug-loaded linker stock solution and the cell penetrating peptide H3-V35C are used in a molar ratio of (1-1.5): 1.
16. the method of claim 14, wherein in step (2), the conditions of the addition reaction comprise: the reaction temperature is 20-60 ℃, and the reaction time is 0.5-8 h.
17. The method according to claim 14, wherein, in step (2), the addition reaction is carried out in the presence of a HEPES buffer.
18. A cell penetrating peptide conjugate prepared by the method of any one of claims 14 to 17.
19. A method of preparing an antibody-conjugated drug, the method comprising:
(1) dissolving the drug-loaded linker of claim 4 in a solvent to obtain a drug-loaded linker stock; and carrying out reduction reaction on the monoclonal antibody and a reducing agent solution to obtain a solution after the antibody is reduced;
(2) carrying out addition reaction on the drug-loaded linker storage solution and the solution obtained after the reduction of the antibody to obtain the antibody conjugate drug;
wherein, in the step (1), the monoclonal antibody is a trastuzumab monoclonal antibody.
20. An antibody-conjugated drug prepared by the method of claim 19.
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