Preparation method and application of antibody-polymer-drug conjugate

Abstract

The present invention relates to the field of pharmaceutical preparations, and specifically discloses a compound, an antibody-polymer-drug conjugate and a preparation method thereof, wherein the antibody-polymer-drug conjugate is formed by polymerizing a polymer monomer and a drug molecule monomer on the surface of an antibody-trigger conjugate. Compared with existing antibody-drug conjugates, the present invention can greatly increase the drug / antibody conjugation ratio of antibody-drug conjugates, expand the types of conjugated drugs, and simultaneously improve the solubility, stability, pharmacokinetics and therapeutic efficacy of antibody-drug conjugates and reduce their immunogenicity. The preparation method provided by the present invention has high yield, easy purification, simple quality control, and versatility, and has great application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a preparation method and application of antibody-polymer-drug conjugates. Background Art

[0002] Antibody-drug conjugates (ADCs) are an emerging form of targeted drugs that can well combine the high specificity of antibodies with the high activity of drugs. Currently, the FDA has approved more than ten new antibody-drug conjugates for clinical treatment, and they have shown great clinical advantages in the treatment of major diseases such as cancer. However, the drug / antibody ratio (DAR) of existing antibody-drug conjugates is generally four. This is because a smaller number of drug conjugates will reduce the efficacy of the drug, while a larger number of drug conjugates will affect the protein structure and stability of the antibody, resulting in faster clearance in the body, affecting pharmacokinetic indicators, and further affecting the final efficacy. Therefore, after years of research and optimization by scientific researchers, it is a consensus in the current design and development of antibody-drug conjugates to conjugate an average of 4 drugs to each antibody.

[0003] However, the fact that each antibody can only have four conjugated drugs on average also brings some problems. For example, the conjugated small molecule drug must be a highly toxic toxin molecule, and its half-inhibitory concentration (IC50) value is generally in the nM to pM range, so as to ensure that the overall therapeutic effect of the antibody-drug conjugate is significant enough. However, if these highly toxic toxin small molecules are released in the circulatory system, they will cause serious toxic side effects; at the same time, the invention, synthesis and process development of highly toxic toxin small molecules are very complicated, which brings great difficulty and risk to the design of drugs and linkers of antibody-drug conjugates and subsequent clinical development. Therefore, in the antibody-drug conjugates approved so far, only four types of toxin molecules are used, including maytansinoids, auristatins, calicheamicin, and irinotecan derivatives (SN-38 and DXd): (U.S. Patent Nos. 5208020; 5416064; 6333410; 6441163; PCT Publication No. WO2019219891).

[0004] On the other hand, there are currently 45 small molecule anti-tumor chemotherapy drugs in clinical use. The IC50 values ​​of these chemotherapy drugs are generally in the μM range. If they are directly coupled with monoclonal antibodies to form antibody-drug conjugates, the efficacy is not significant enough. Therefore, among the currently approved antibody-drug conjugate products, no clinically used chemotherapy drugs are directly coupled to antibodies. Therefore, there is an urgent need to design a universal method that can effectively solve the problem of low drug / antibody ratio and further apply chemotherapy small molecule drugs to antibody-drug conjugates to expand the development scope of traditional antibody-drug conjugates. Summary of the invention

[0005] The first aspect of the present invention provides a compound having a structure as shown in Formula I:

[0006]

[0007] wherein R1 is selected from C1-C4 alkyl, -OH or -H; R2 is selected from C1-C4 alkyl, -OCH3, -OH or -H; R3 is selected from -H, -OH or The chiral configuration of the carbon atom to which R3 is connected can be R or S; R4 can be selected from C1-C4 alkyl or -H; R5 can be selected from -H, -NH2 or -OH; R6 can be selected from -H, -NH2 or -OH.

[0008] Preferably, R1 is selected from -OH or -H; R2 is selected from -OCH3 or -H; R3 is selected from -OH or The chiral configuration of the carbon atom to which R3 is connected is R or S; R4 is selected from -CH3 or -H; R5 is selected from -NH2 or -OH, and R6 is -OH.

[0009] The compound of the present invention is more preferably a compound represented by formula II-VII:

[0010]

[0011] The present invention also provides a method for preparing the above compound, specifically: using doxorubicin and methacryloyl hydrazide hydrochloride as raw materials to prepare the compound through an imidization reaction (with acid as catalyst). The specific conditions of the imidization reaction are known to those skilled in the art, and the present invention does not impose any particular limitation on this.

[0012] The present invention also provides the use of the above-mentioned compounds in the preparation of antibody-polymer-drug conjugates, specifically, as drug molecule monomers to prepare antibody-polymer-drug conjugates, which can not only greatly increase the drug / antibody conjugation ratio of the antibody-drug conjugate, thereby improving its in vitro and in vivo anti-tumor efficacy, while reducing side effects; but also greatly expand the types of drugs used for antibody conjugation, so that several low-toxic chemotherapy drugs that cannot be used for conjugation in the prior art can be used and further developed, avoiding the use of highly toxic cytotoxins, and also making multi-drug combination possible.

[0013] The second aspect of the present invention provides an antibody-polymer-drug conjugate, which is formed by polymerizing polymer monomers and drug molecule monomers on the surface of an antibody-initiator conjugate;

[0014] The antibody-initiator conjugate comprises an antibody, and at least one initiator coupled to a disulfide bond or a free thiol group of the antibody;

[0015] The polymer monomer is selected from at least one of acrylate, methacrylate, acrylamide and methacrylamide;

[0016] The drug molecule monomer is selected from drug molecules modified by at least one of acrylate functional groups, methacrylate functional groups, acrylamide functional groups and methacrylamide functional groups.

[0017] Wherein, the disulfide bond is selected from at least one of an intrachain disulfide bond and an interchain disulfide bond.

[0018] In the antibodies provided by the present invention, disulfide bonds or free thiol groups can be introduced by a chemical or biological modification method selected from gene mutation, introduction of non-natural amino acids, oxidation-reduction, and enzyme catalysis. The specific introduction method is known to those skilled in the art and is not particularly limited in the present invention.

[0019] Preferably, the antibody is selected from monoclonal antibodies and polyclonal antibodies related to medicine, agriculture, scientific research and other industrial fields.

[0020] More specifically, the antibody is selected from abavotumab, adelimumab, alaximab, atumomab, mammotumomab, acitumomab, baviximab, bevacizumab, bivacizumab, bonatumomab, brevituzumab, cantuzumab, catumaxomab, caproumab, cetuximab, situtuzumab, clivanizumab, cannatumumab, daclizumab, edrecolomab, epratuzumab, ertuinomab, irrituzumab, farletuzumab, fijitumumab, gemtuzumab, golimumab, ibritumomab, tiuxetan, igovomab, intozumab, inotuzumab, labetuzumab, lexalimumab , lintuzumab, rukatumomab, matuzumab, mitumomab, nartumomab, nesimumab, oplitumumab, orgovotumab, panitumumab, pertuzumab, pertuzumab, protumumab, rituximab, rituximab, robatumumab, satumomab, xipotuzumab, tapulimab, tetumomab, tetumomab, tecitumomab, tegatuzumab, trastuzumab, tositumomab, tremetumomab, turocizumab, voroxizumab and zalutumumab and one or more of an antibody nanobody derived from Fab, Fab2, scFv or a camel antibody heavy chain fragment.

[0021] In particular, when the antibody selects a humanized IgG1 monoclonal antibody, especially a humanized IgG1 monoclonal antibody such as trastuzumab (preferably Herceptin), pertuzumab (preferably Perjeta), bevacizumab, etc., it can be more conducive to the drug coupling to the interchain disulfide bonds in the hinge region of this type of monoclonal antibody, and is not likely to affect the spatial structure and biological activity of the monoclonal antibody.

[0022] The drug molecule monomer of the present invention preferably has a structure represented by any one of Chemical Formulas 6 to 9:

[0023]

[0024] Among them, the R2 group in chemical formulas 6 to 9 is selected from the pharmacophoric group of the drug molecule; the R1 group is selected from the chemical functional group that responds to external chemical, physical, and biological stimuli (such as pH, temperature, redox potential, and biological enzymes).

[0025] Preferably, the R1 group is selected from pH-responsive acylhydrazone bonds, carbonate amide bonds, phenylboronic acid ester bonds, imine bonds, redox-responsive disulfide bonds, thioether bonds, diselenide bonds, selenoether bonds, thioketal bonds, aminoacrylate groups, oligoproline groups, peroxalate groups, and cathepsin B-responsive chemical groups, asparagine endopeptidase-responsive chemical groups, β-glucuronidase-responsive chemical groups, and matrix metalloproteinase-responsive chemical groups;

[0026] The pharmacophore of the drug molecule refers to the core group in the drug molecule that can maintain the drug activity, including anthracycline groups, tricyclic diterpenoid groups, quinoline groups, etc.

[0027] Specifically, the drug molecule is selected from one or more of small molecule chemotherapeutic drugs, cytotoxins, enzyme inhibitors, and immune agonists, preferably one, two or three;

[0028] More preferably, the drug molecule is selected from doxorubicin, epirubicin, pyrarubicin, paclitaxel, methotrexate, docetaxel, gemcitabine, cyclophosphamide, fluorouracil, nimustine, carmustine, irinotecan, hydroxycamptothecin, vincristine, vindesine, teniposide, etoposide, cisplatin, carboplatin, oxaliplatin, dacarbazine, procarbazine, maytansinoids, auristatins, calicheamicin, irinotecan derivatives, imatinib, sorafenib, lapatinib, dasatinib, larotrectinib, entrectinib, erlotinib, gefitinib, afatinib, At least one of the following: nivolumab, dacomitinib, osimertinib, crizotinib, alectinib, ceritinib, brigatinib, lorlatinib, trametinib, dabrafenib, regorafenib, lenvatinib, cabozantinib, everolimus, palbociclib, ribociclib, bomazenib, olaparib, rucaparib, niraparib, sunitinib, axitinib, pazopanib, vemurafenib, conciertinib, cobimetinib, binimetinib, vandetanib, bosutinib, ibrutinib, ponatinib, bortezomib, carfilzomib, panobinostat, ixezomib, and ruxolitinib.

[0029] The drug molecule monomer of the present invention is preferably a compound represented by Formula I; more preferably a compound represented by Formula II-VII:

[0030]

[0031]

[0032] The polymer described in the present invention is at least one selected from homopolymers, heteropolymers, block polymers, copolymers, and terpolymers, preferably homopolymers and / or copolymers.

[0033] Preferably, the polymer monomer used for polymerization reactions such as ATRP, RAFT and ROMP may have a structure represented by any one of Chemical Formulas 2 to 5:

[0034]

[0035] The R groups in Chemical Formulae 2 to 5 are each independently selected from an oligoethylene glycol group, an ethylphosphocholine group, and the like.

[0036] The polymer monomer of the present invention may be water-soluble or biodegradable.

[0037] In some embodiments of the invention:

[0038] The polymer monomer is selected from at least one of lactic acid, epichlorohydrin, acrylate, methacrylate, acrylamide, methacrylamide, norbornene and oxa-norbornene, especially methacrylate.

[0039] Preferably, the high molecular weight monomer is a PEG-type monomer, which helps to increase the water solubility and biocompatibility of the product, increase the drug loading, maintain the spatial structure and biological activity of the antibody, and reduce immunogenicity and the production of anti-drug antibodies.

[0040] Preferably, in the antibody-polymer-drug conjugate, the molar ratio of the antibody to the polymer monomer unit is 1:10 to 1:1000, and the molar ratio of the antibody to the drug monomer unit is 1:1 to 1:100.

[0041] And / or, the molar ratio of the drug monomer unit to the polymer monomer unit is 1:1 to 1:1000.

[0042] The antibody-polymer-drug conjugate provided by the present invention, each of which has at least one polymer chain obtained by polymerizing a polymer monomer and a drug molecule monomer on the antibody surface.

[0043] The third aspect of the present invention provides a method for preparing a site-specific antibody-polymer-drug conjugate, the method comprising:

[0044] Preparation of antibody-initiator conjugate: The initiator is coupled to two adjacent free thiol groups formed after the disulfide bonds in the hinge region of the antibody are reduced;

[0045] The antibody-trigger conjugate is polymerized with a polymer monomer and a drug molecule monomer in a buffer to prepare the antibody-polymer-drug conjugate.

[0046] The method provided by the present invention can synthesize site-specific responsive antibody-polymer-drug conjugates with high efficiency and high yield, improve the drug / antibody ratio of the antibody-drug conjugates, expand the types of conjugated drugs, and improve the overall quality of the antibody-drug conjugates.

[0047] Specifically, the method comprises:

[0048] a1) preparing an antibody-initiator conjugate: the initiator is coupled to two adjacent free thiol groups formed after the disulfide bonds in the hinge region of the antibody are reduced;

[0049] b1) The antibody-initiator conjugate is mixed with a polymer monomer and a drug molecule monomer in a buffer solution, and the polymer monomer and the drug molecule monomer are polymerized under the action of a catalyst to prepare the antibody-polymer-drug conjugate.

[0050] Preferably, in step a1), the hinge region disulfide bond of the antibody is reduced to two adjacent free thiols, and then the initiator is coupled to the two adjacent free thiols at the same time, thereby achieving re-bridging after the reduction and cleavage of the original disulfide bond of the antibody. The in-situ polymerization method is characterized by high efficiency, high yield, and high activity retention rate by modifying specific sites of the antibody and growing drug-containing polymers in situ.

[0051] The polymerization may be selected from atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer polymerization (RAFT), ring-opening metasitu polymerization (ROMP) and combinations thereof, preferably atom transfer radical polymerization (ATRP).

[0052] The preparation method provided by the present invention can regulate polymerization by adding a catalyst.

[0053] For example:

[0054] The catalyst used to initiate the ATRP polymerization reaction includes, but is not limited to, copper ions and their ligands (ligands include: bipyridine and its derivative ligands, π receptor derivative ligands, nitrogen atom chelate ligands and some fatty polyamine ligands).

[0055] Catalysts for initiating RAFT polymerization reactions include, but are not limited to, water-soluble free radical initiators, such as 4,4'-azo(4-cyanovaleric acid), 2,2'-azo[2-(2-imidazolin-2-yl)propane] dihydrochloride, 2,2'-azo[2-(2-imidazolin-2-yl)propane]-anhydrous pyrosulfate, 2,2'-azo(2-ethylpropionamidine) dihydrochloride, and the like.

[0056] The catalyst for initiating the ROMP polymerization reaction includes, but is not limited to, a water-soluble Grubbs catalyst and the like.

[0057] The preparation method provided by the present invention, the ATRP or RAFT polymerization reaction, preferably uses methacrylate or methacrylamide monomers, a composite catalyst composed of cupric ions / cuprous ions / fatty polyamine ligands (such as CuCl2 / CuCl / HMTETA, wherein the molar ratio of CuCl to CuCl2 is 1:0-5, and the molar ratio of CuCl to HMTETA is 1:1-10), the reaction conditions are that the polymerization is carried out in a low oxygen or inert gas atmosphere, the reaction time is 0.5 to 48 hours (for example, 12 hours, 24 hours or 48 hours), and the reaction temperature is 0 to 50°C (for example, 20°C, 30°C, 40°C, 50°C).

[0058] Furthermore, the preferred reaction conditions are as follows: the ATRP polymerization reaction uses methacrylate or methacrylamide monomers (such as methacrylate oligoethylene glycol ester) as raw materials, uses a composite catalyst composed of cupric chloride / cuprous chloride / 1,1,4,7,10,10-hexamethyltriethylenetetramine, and the reaction conditions are carried out under a high-purity nitrogen atmosphere. The reaction time is 2 to 24 hours and the reaction temperature is 0 to 37°C.

[0059] The initiator of the present invention can be selected from ATRP initiator, RAFT initiator or ROMP initiator.

[0060] More specifically, the initiator molecule is of Chemical Formula 1:

[0061]

[0062]

[0063] In the chemical formula 1, R 1 It is a functional group and its functional group derivatives that can initiate polymerization reactions such as ATRP, RAFT or ROMP.

[0064] For example:

[0065] Functional groups capable of initiating ATRP reactions include, but are not limited to, N-(2-aminoethyl)-2-bromo-2-methylpropionamide, N-(2-aminoethyl)-2-chloro-2-methylpropionamide, 2-bromo-N-(2-(2-hydrazinoacetylamino)ethyl)-2-methylpropionamide, 2-chloro-N-(2-(2-hydrazinoacetylamino)ethyl)-2-methylpropionamide, and the like.

[0066] Functional groups capable of initiating RAFT reactions include, but are not limited to: R'C(=S)SR, wherein the R group may be a cysteine ​​group, a hydrazine group, or a hydroxylamine group; the R' group may be a phenyl group, a long-chain alkyl group, or a phthalimidomethyl group;

[0067] The functional groups that can simultaneously initiate ROMP reaction include but are not limited to AB type functional groups, A can be a cysteine ​​group, a hydrazine group, a hydroxylamine group, and B can be a Grubbs initiator of a ruthenium-carbene complex.

[0068] R 2 and R 3 It may be, but is not limited to, H, I, Br, Cl, C6H5S, CH3C6H5S, Ts and other functional groups that are easily substituted by nucleophilic groups and their functional group derivatives; R 2 and R 3can be the same or different; X and Y can be but not limited to NH, O, S, Se and other atoms (or strong electron-withdrawing functional groups and their functional group derivatives), X and Y can be the same or different, and Z includes but is not limited to N or CH.

[0069] Further, the ATRP initiator may be 2-(2-(2-(3,4-dibromomaleimide-N-ethoxy)ethoxy)ethoxy)ethyl 2-bromo-2-methylpropionate (DBEB), the RAFT initiator may be 2-(dodecylthiocarbonylthiothio)-2-methylpropionic acid (3,4-dibromomaleimide-N-ethoxy) ester; the ROMP initiator may be a Grubbs initiator having a ruthenium-carbene complex structure.

[0070] Furthermore, if desired, biodegradable materials (polymer monomers, oligomers or polymers) can also be introduced into the structure of the antibody-polymer-drug conjugate.

[0071] In some embodiments of the present invention, the antibody-polymer-drug conjugate is prepared by in situ polymerization, and the specific technical scheme is as follows:

[0072] Using methacrylate oligoethylene glycol ester and methacryloylhydrazone doxorubicin monomers and antibody macromolecular initiator Herceptin-Br as raw materials to form a reactant (preferably in an oxygen-free buffer);

[0073] Using cuprous chloride and hexamethyltriethylenetetramine as a catalyst system (in a dosage ratio of 1:1 to 10) to form a catalyst system solution;

[0074] After the reactants and the catalyst system solution are mixed, the mixture is reacted at 0-50° C. (preferably 4° C.) in an oxygen-free atmosphere for 2-48 hours (preferably 24 hours), the reaction is terminated, and the antibody-polymer-drug conjugate is obtained through purification.

[0075] The fourth aspect of the present invention provides the above-mentioned antibody-polymer-drug conjugate, the above-mentioned preparation method, and the use of the antibody-polymer-drug conjugate prepared by the preparation method in the preparation of anti-tumor drugs.

[0076] The preparation method of the present invention has the following advantages:

[0077] First, compared with existing antibody-drug conjugates, the present invention can greatly increase the drug / antibody conjugation ratio of the antibody-drug conjugate, thereby improving its in vitro and in vivo anti-tumor efficacy while reducing side effects.

[0078] Secondly, the present invention not only does not affect the structure, biological activity (antibody-antigen binding ability) and stability of the antibody while increasing the drug / antibody coupling ratio, but also can improve the activity and stability of the coupled antibody due to site-specific coupling and polymer modification.

[0079] Thirdly, the present invention greatly expands the types of drugs used for antibody conjugation, allowing dozens of low-toxic chemotherapy drugs that could not be used for conjugation before to be used and further developed, avoiding the use of highly toxic cytotoxins, and also making multi-drug combination possible. In addition, the present invention introduces a responsive linker into the antibody-polymer-drug conjugate, and utilizes the acidic environment after cell endocytosis to achieve the effect of controlled release, thereby effectively reducing side effects.

[0080] Finally, the preparation method of the present invention has high yield, easy purification, simple quality control and versatility. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 The steps and process of synthesizing antibody-polymer-drug conjugate (Herceptin-P-DOX) by in situ polymerization are shown.

[0082] Figure 2 The steps and process of synthesizing antibody drug conjugates (Herceptin-DOX) by traditional conjugation method are shown.

[0083] Figure 3 Non-reduced SDS-PAGE is shown to characterize different Herceptin-P-DOX. Lane 1: pre-stained protein standards of different molecular weights; Lane 2: Herceptin-P-DOX with a DAR of 15; Lane 3: Herceptin-P-DOX with a DAR of 32; Lane 4: Herceptin-P-DOX with a DAR of 55; Lane 5: Herceptin-Br; Lane 6: Herceptin

[0084] Figure 4 The molecular weight of the polymer POEGMA in Herceptin-P-DOX after enzymatic and acid hydrolysis as determined by gel permeation chromatography (GPC) is shown to be 66.11 kDa.

[0085] Figure 5 The yields of Herceptin-P-DOX with different DAR values ​​are shown (the DARs of samples 1 to 4 are 15, 32, 55, and 78, respectively).

[0086] Figure 6 Different drug / antibody ratios of Herceptin-P-DOX and Herceptin-DOX are shown, as well as a comparison of the two at the same antibody concentration (Herceptin-DOX on the left, Herceptin-P-DOX on the right).

[0087] Figure 7 Dynamic light scattering (DLS) determination of the hydrated diameters of Herceptin-P-DOX and Herceptin-DOX is shown.

[0088] Figure 8 Shown are the hydrated diameters of Herceptin-P-DOX and Herceptin-DOX measured by dynamic light scattering (DLS) before and after one week of storage at 4°C, respectively.

[0089] Fig. 9 The binding activity of Herceptin-P-DOX and Herceptin-DOX to HER2 antigen measured by immunofluorescence is shown.

[0090] Fig.10 The release kinetics of DOX from Herceptin-P-DOX and Herceptin-DOX in pH=7.4 and pH=5.0 buffers are shown.

[0091] Fig.11 The internalization effects of doxorubicin (DOX), Herceptin-DOX and Herceptin-P-DOX in ovarian cancer cells SKOV-3 are shown.

[0092] Fig.12 The cytotoxicity of DOX, Herceptin-DOX and Herceptin-P-DOX in breast cancer cells BT-474 and ovarian cancer cells SKOV-3, respectively, is shown, with DOX concentration used as a quantitative comparison.

[0093] Fig.13 The cytotoxicity of Herceptin-DOX and Herceptin-P-DOX in breast cancer cells BT-474 and ovarian cancer cells SKOV-3, respectively, is shown, with Herceptin concentration used as a quantitative comparison.

[0094] Fig.14 The figure shows the changes in body weight of mice after receiving different gradient doses of Herceptin-P-DOX, and the results show that 30 mg / kg is the maximum tolerated dose in mice.

[0095] Fig.15 The graph shows the inhibition of tumor growth curves of phosphate buffered saline (PBS), DOX (dose 5 mg / kg), Herceptin, Herceptin-DOX, and Herceptin-P-DOX in the SKOV-3 nude mouse tumor model (6-8 mice in each group) after a single administration of Herceptin at a dose of 136 mg / kg.

[0096] Fig.16The results show the different effects of single administration of phosphate buffered saline (PBS), DOX (dose 5 mg / kg), Herceptin, Herceptin-DOX, and Herceptin-P-DOX on the survival time of SKOV-3 tumor-bearing mice (6-8 mice in each group) when the corresponding dose of Herceptin was 136 mg / kg. DETAILED DESCRIPTION

[0097] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0098] The detection of raw materials, reagents, products, etc. involved in the following embodiments all adopts known detection means, and the applicable equipment and reagents are also known, and the present invention does not make any special limitation on this.

[0099] Example 1 Synthesis of drug molecule monomer - methacryloylhydrazone doxorubicin (mDOX)

[0100]

[0101] The specific steps are as follows:

[0102] 1. Weigh 1g (10mmol) of Boc hydrazine, dissolve it in 50mL of anhydrous dichloromethane, and add 1.2 equivalents of triethylamine (1.2g, 12mmol) as an acid-binding agent and catalyst. Place the flask in an ice-water bath and stir for 20 minutes. Slowly drop an equivalent amount of methacryloyl chloride (1.3g, 10mmol) into the flask in an ice bath. After the addition is complete, remove the ice-water bath and slowly warm the system to room temperature. Continue stirring the reaction for 10 hours, and monitor the progress of the reaction by thin-layer chromatography. After the reaction is completed, add an appropriate amount of silica gel powder, concentrate and load the sample, and separate the product Boc methacryloyl hydrazide (colorless oil, 1.3g, yield 65%) by silica gel rapid column chromatography. 1 H-NMR (400MHz, CDCl3) δ1.42 (s, 9H), 1.98 (s, 1H), 5.72 (s, 1H), 5.79 (s, 1H), MS (ESI) m / z: 201 [M+H] + .

[0103] 2. Weigh 200 mg of the obtained Boc methacryloyl hydrazide, add 10 mL of 3M hydrogen chloride ethyl acetate solution, react at room temperature for 15 minutes, rotary evaporate ethyl acetate and hydrogen chloride in an ice water bath, add a small amount of dichloromethane and dissolve it in an ice water bath and rotary evaporate it, precipitate it in 200 mL of ice ether, centrifuge to obtain a white powder solid, dry the ether in a vacuum oven to obtain the final product methacryloyl hydrazide hydrochloride (white powder solid, 120 mg, yield 88%). 1H-NMR (400MHz, CD3OD): δ1.98(s,1H),5.72(s,1H),5.79(s,1H), MS(ESI)m / z:123[M+Na] + .

[0104] 3. Weigh 58 mg (0.1 mmol) of doxorubicin and dissolve it in 20 mL of anhydrous methanol, add 68 mg (0.5 mmol) of methacryloyl hydrazine hydrochloride, stir until dissolved, then add 100 μL of trifluoroacetic acid (CF3COOH), and react at room temperature in the dark for 96 hours. After the reaction, concentrate the solution to 5 mL, add anhydrous acetonitrile until the solution begins to become turbid, recrystallize twice at -20 ° C, filter and dry to obtain methacryloyl hydrazone doxorubicin mDOX (dark red solid, 40 mg, yield 64%). 1 H-NMR (400MHz, CD3OD): δ1.98(s,1H),5.72(s,1H),5.79(s,1H),3.90(s,3H),7.3-7.9(m,3H),1 .1(d,3H),1.8-2.2(m,4H),2.7-3.7(m,5H),4.4(s,2H),4.5-4.7(s,2H),MS(ESI)m / z:626[M+H] + .

[0105] Example 2 Synthesis of Antibody Macromolecular Initiator - Herceptin-Br

[0106] The specific steps are as follows:

[0107] ATRP initiator DBEB was inserted into the disulfide bonds between antibody chains: monoclonal antibody Herceptin (7.5 mg, 0.05 μmol) was dissolved in 1 mL Tris·HCl (50 mM, 150 mM, NaCl, pH = 7.4), and an aqueous solution of reducing agent tris(2-carboxyethyl)phosphine (TCEP, 50 μL, 5 mM) was added at 37°C and incubated for 3 hours. Desalting columns (AKTA, GE Healthcare, HiTrap TM Desalting Column 5mL, mobile phase is 50mM Tris·HCl, 150mM NaCl, pH=7.4) to remove small molecules to obtain purified reduced Herceptin (Herceptin-SH). Add the DMF solution of ATRP initiator DBEB (5μL, 50mM) to the Tris·HCl buffer of the above Herceptin-SH (the solution turns light yellow), incubate at 37°C for 14 hours, and remove excess ATRP initiator through the desalting column to obtain the antibody-initiator conjugate (Herceptin-Br). After the above two-step reaction, the ATRP initiator is successfully inserted into the interchain disulfide bond of the antibody. The chemical structure of DBEB is as follows:

[0108]

[0109] Example 3 Preparation of Antibody-Polymer-Drug Conjugate (Herceptin-Polymer-Adriamycin Conjugate) by In-Situ Polymerization

[0110] The specific steps are as follows (see Figure 1 ):

[0111] Preparation of acid-sensitive Herceptin-P-DOX: In situ ATRP polymerization was initiated at the disulfide bond position between antibody chains: CuCl (0.4 mg) and hexamethyltriethylenetetramine (8 mg) were dissolved in 1 mL of deionized water.

[0112] 250 mg of oligoethylene glycol methacrylate (OEGMA, MW about 500 Da) and a certain amount of mDOX were dissolved in a buffer solution of Herceptin-Br (10 μM, 5 mL) in Tris·HCl (50 mM, 150 mM NaCl, pH=7.4), and the molar ratio of Herceptin-Br: OEGMA: mDOX was 1:1000:100. Argon was bubbled into the two solutions for 30 minutes (deoxygenation), and the first solution was introduced into the second solution at room temperature (through a two-way needle). After reacting at 25°C for 8 hours under an argon atmosphere, air was bubbled into the reaction solution (to terminate the polymerization reaction). The reaction solution was dialyzed in PBS for three days with a 10 kDa regenerated cellulose membrane to remove unreacted small molecules and catalysts to obtain Herceptin-P-DOX.

[0113] Example 4 Preparation of Antibody-Polymer-Drug Conjugate (Herceptin-Polymer-Epirubicin Conjugate) by In-Situ Polymerization

[0114] Preparation of Methacryloylhydrazone Epirubicin mEPI The preparation method of methacryloylhydrazone Epirubicin mDOX in Reference Example 1 was used.

[0115] Preparation of acid-sensitive Herceptin-P-EPI: In situ initiation of ATRP polymerization at the disulfide bond position between antibody chains: CuCl (0.4 mg) and hexamethyltriethylenetetramine (8 mg) were dissolved in 1 mL of deionized water. 147 mg of 2-methacryloyloxyethyl phosphorylcholine and a certain amount of mEPI were dissolved in a buffer solution of Herceptin-Br (10 μM, 5 mL) in Tris·HCl (50 mM, 150 mM NaCl, pH = 7.4), Herceptin-Br: MPC: mEPI was 1: 2000: 50. Argon was bubbled into the above two solutions for 30 minutes (deoxygenation), and the first solution was introduced into the second solution at room temperature (through a two-way needle). After reacting at 4°C under argon atmosphere for 24 hours, air was bubbled into the reaction solution (to terminate the polymerization reaction). The reaction solution was dialyzed in PBS for three days using a 10 kDa regenerated cellulose membrane to remove unreacted small molecules and catalysts to obtain Herceptin-P-EPI.

[0116] Example 5 Preparation of Antibody-Polymer-Drug Conjugate (Perjeta-Polymer-Daunorubicin Conjugate) by In-Situ Polymerization

[0117] Preparation of methacryloylhydrazone daunorubicin mDNR Refer to the preparation method of methacryloylhydrazone doxorubicin mDOX in Example 1. Synthesis of antibody macromolecular initiator Perjeta-Br Refer to the synthesis of Herceptin-Br in Example 2.

[0118] Preparation of acid-sensitive Perjeta-P-DNR: In situ initiation of ATRP polymerization at the disulfide bond position between antibody chains: CuCl (0.4 mg) and hexamethyltriethylenetetramine (4 mg) were dissolved in 1 mL of deionized water. 250 mg of oligoethylene glycol methacrylate (OEGMA, MW about 500 Da) and a certain amount of mDNR were dissolved in Perjeta-Br (10 μM, 5 mL) in Tris·HCl (50 mM, 150 mM NaCl, pH=7.4) buffer solution, Perjeta-Br: OEGMA: mDNR was 1: 500: 20. Argon was bubbled into the above two solutions for 30 minutes (deoxygenation), and the first solution was introduced into the second solution at room temperature (through a two-way needle). After reacting at 37°C for 2 hours under argon atmosphere, air was bubbled into the reaction solution (to terminate the polymerization reaction). The reaction solution was dialyzed against PBS for three days using a 10 kDa regenerated cellulose membrane to remove unreacted small molecules and catalysts to obtain Perjeta-P-DNR.

[0119] Comparative Example 1 Synthesis of Antibody Drug Conjugate (Herceptin-DOX) in Control Group

[0120] The specific steps are as follows (see Figure 2): Herceptin-SH was prepared in the same manner as in Example 2. The linker 4-maleimidobutyric acid hydrazide hydrochloride MBH (58.4 μg, 250 nmol) was added to the above-mentioned Herceptin-SH (10 μM, 1 mL) in Tris·HCl buffer (pH=7.4), incubated at 37°C for 14 hours, and the excess MBH was removed through a desalting column to obtain the Herceptin linker conjugate (Herceptin-MBH). DOX (0.25 mg, 0.4 μmol) was added to the acetic acid / sodium acetate (50 mM, 150 mM NaCl, pH=5.0) buffer solution of Herceptin-MBH (10 μM, 1 mL), and 100 mM aniline was added as a catalyst. After reacting at room temperature for 16 hours, the reaction solution was dialyzed in PBS (pH=7.4) with a 10 kDa regenerated cellulose membrane for three days to remove unreacted DOX and aniline to obtain Herceptin-DOX.

[0121] Experimental Example 1 Characterization of the physicochemical properties and biological activity of Herceptin-P-DOX

[0122] The specific steps are as follows:

[0123] 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): Prepare 80 μL of sample with a concentration of 1 mg / ml, add 20 μL of loading buffer (×5, without β-mercaptoethanol). Load the above 10 μL sample onto a 7% polyacrylamide gel and run electrophoresis for 2 hours at 80-120 V and 40 mA (electrophoresis buffer: 25 mM Tris, 250 mM Glycine, 0.1% SDS). Figure 3 It showed that the polymer was successfully coupled to Herceptin through ATRP polymerization initiated in situ by Herceptin, and there was no obvious difference in the molecular weight of the conjugates with different DAR values.

[0124] 2. Molecular weight determination: First, a certain amount of Herceptin-P-DOX was hydrolyzed with proteinase K (0.1-1 mg / mL) at 45°C. The hydrolysis reaction was carried out. The obtained hydrolyzate was treated with a pH 5 buffer for 72 hours, and then the small molecule drug impurities and amino acid fragments were removed by dialysis (MWCO3-5 kDa), and pure high molecular weight poly(ethylene glycol methacrylate) (POEGMA) was purified. Pure high molecular weight POEGMA was analyzed on a Waters gel permeation chromatography (GPC) system equipped with a UV-visible detector (Waters 2489) and a differential refractive index detector (Waters 2414). The analysis conditions were a flow rate of 0.5 mL / min, with Tris·HCl buffer (50 mM Tris·HCl, 150 mM NaCl, pH 7.4) as the mobile phase, and the analysis was carried out on an Asahpak GS-520HQ column connected after the guard column. Using PEG standards with different molecular weights, a calibration curve for determining relative molecular mass was established. The molecular weight of the sample was calculated to be 66.11 kDa based on the calibration curve using Breeze software (see Figure 4 ), the molar ratio of antibody to polymer monomer unit is calculated to be 1:529, and the molar ratio of drug monomer unit to polymer monomer unit is 1:9.6.

[0125] 3. Drug / antibody ratio measurement: First, determine the Herceptin concentration by the Bradford method, prepare a bovine serum albumin (BSA) standard at 0.5 mg / mL (PBS), add the standard to the standard wells of a 96-well plate at 0, 1, 2, 4, 8, 12, 16 and 20 μL, and add PBS to make up to 20 μL. Add an appropriate volume (2-20 μL) of sample to the sample wells of the 96-well plate, and add PBS to make up to 20 μL. Add 200 μL of Bradford working solution to each well and leave at room temperature for 5 minutes. Determine the OD value at 595 nm, and calculate the protein concentration c (antibody) of the sample based on the standard curve. Secondly, measure the OD value at 495 nm using a UV / Vis spectrophotometer, and based on the 1.00×10 4 M -1 cm -1 The DOX concentration c(drug) of the sample is calculated based on the molar extinction coefficient, and the drug / antibody ratio DAR = c(drug) / c(antibody). Figure 5The yields of Herceptin-P-DOX with different DAR values ​​are shown (the DARs of samples 1 to 4 are 15, 32, 55, and 78, respectively). As the DAR value increases from 15 to 55, the yield of Herceptin-P-DOX remains basically unchanged. When the DAR value increases to 78, the yield of Herceptin-P-DOX decreases significantly. This is because too many drug molecules affect the stability of the antibody, causing it to aggregate and precipitate. Figure 6 The DAR value of Herceptin-P-DOX is 55, while that of Herceptin-DOX is 3.2, which means that in Herceptin-P-DOX, on average, 55 doxorubicin molecules are coupled to each Herceptin monoclonal antibody molecule, while the traditional antibody-drug conjugate Herceptin-DOX can only couple 3.2 drug molecules, and the coupling ratio is increased by 17.2 times. At the same time, it can be intuitively observed that the color of the Herceptin-P-DOX solution is darker red at the same protein concentration, indicating that the amount of DOX coupled to it is greater.

[0126] 4. Dynamic light scattering (DLS): DLS test uses Malverm Zetasizer Nano-zs90. The sample must pass through a 0.22μm filter membrane before testing, and the sample concentration is 15μM. Data processing uses Zetasizer software6.32. Figure 7 The hydrated diameters of Herceptin-P-DOX and Herceptin-DOX determined by dynamic light scattering (DLS) are shown. Figure 7 It was shown that the purified Herceptin-DOX and Herceptin-P-DOX both had only one smooth characteristic peak of nanoparticle distribution (the hydrated diameters were 9.03 nm and 26.5 nm, respectively, and the PDIs were 0.16 and 0.19, respectively).

[0127] 5. Storage stability test: The PBS solutions of Herceptin-P-DOX and Herceptin-DOX (the concentration of the sample was 15 μM) were stored in a refrigerator at 4° C., taken out after one week, and directly measured by DLS without filtering the membrane to observe the light intensity distribution diagram. Figure 8 The results show that after one week of storage, Herceptin-DOX produced new peaks at 18, 100, 820, and 5500 nm in the light intensity distribution, indicating that protein aggregation and precipitation occurred during the storage process. However, after one week of storage, Herceptin-P-DOX did not produce new peaks, indicating that it stably maintained a single-molecule dispersion state, showing that the Herceptin-P-DOX prepared by this method has better stability than traditional Herceptin-DOX.

[0128] 6. Fluorescence immunoassay of in vitro antigen binding activity of Herceptin-P-DOX and Herceptin-DOX: 100 μL of antigen (HER2, 1.3 μg / mL) was added to a 96-well plate and incubated at 4°C overnight. After removing the antigen (HER2), the cells were washed three times with PBS (200 μL), and then 200 μL of bovine serum albumin (BSA, 1%) was added, and the cells were shaken at room temperature for 3 hours. After removing BSA, the cells were washed three times with PBS (200 μL), and 100 μL of serially diluted Herceptin, Herceptin-P-DOX, and Herceptin-DOX (27 μg / mL, 9 μg / mL, 3 μg / mL, 1 μg / mL, 0.333 μg / mL, 0.111 μg / mL, 0.037 μg / mL, 0.012 μg / mL) were added, and the cells were shaken at room temperature for 1 hour. The cells were washed twice with PBS-T (0.1% Tween-20, 200 μL), and once with PBS (200 μL), and fluorescent secondary antibodies (goat anti-human Herceptin-PE, Santa Cruz Biotechnology Co., Ltd.) were added. Biotechnology) 100 μL, shake at room temperature for 1 hour, wash twice with PBS (200 μL) and read on a microplate reader (MolecμLar Devices, SpectraMax M3). Repeat three times for each sample. Fig. 9 The results showed that the in vitro antigen binding activity of Herceptin itself was 0.4603 μg / mL, that of Herceptin-P-DOX was 0.5372 μg / mL, and the activity retention rate was 85.7%, while that of Herceptin-DOX was 0.7080 μg / mL, and the activity retention rate was only 65.0%. It can be seen that after polymer modification, not only the coupling ratio of the drug can be increased, but also the antigen binding activity retention rate of the antibody can be improved.

[0129] 7. Responsive release of drugs: DOX was released from Herceptin-P-DOX and Herceptin-DOX by dialysis method. 100 μM equivalent of DOX conjugate solution samples were placed in two dialysis bags and immersed in 20 mL of PBS solution with pH of 5.0 and 7.4, respectively. Shake at 180 rpm at 37 °C, and measure the concentration of DOX in the external buffer by UV-visible spectrophotometry at 495 nm at different time points of 0.5, 1, 2, 5, 8, 16, 24 and 48 h to compare the release behavior of DOX at different pH values. Fig. 9 The results showed that at pH 7.4, Herceptin-P-DOX released less than 5% of DOX within 24 hours. In contrast, at pH 5.0, free drug was released rapidly, and 44% of DOX was released within 24 hours. These data suggest that the acylhydrazone bond is stable at blood pH but breaks at lower pH values, such as in lysosomes.

[0130] 8. Cell endocytosis and intracellular drug release: SKOV-3 cells were incubated with 20 μM DOX equivalent concentrations of Herceptin-P-DOX, Herceptin-DOX and DOX at 37°C for 24 hours. The cells were then fixed with 4% (wt / vol) cold paraformaldehyde. The cell membrane and nucleus were stained with 2 μg / mL wheat germ agglutinin (WGA) Alexa Fluor 594 (Invitrogen) and 1 μg / mL Hochest 33342 (Sigma). The cells were then imaged using a laser scanning confocal microscope (LSM710 Carl Zeiss). The excitation / emission wavelengths of Hochest, DOX and WGA were 346 / 460 nm, 485 / 590 nm and 590 / 617 nm. The images were analyzed by Zen_2012 software. Fig.11 It was observed that the polymer-modified Herceptin-P-DOX was able to deliver DOX into ovarian cancer cells SKOV-3.

[0131] 9. Anti-tumor cell in vitro proliferation activity test: BT-474 and SKOV-3 cells were cultured with RPMI1640 medium (GIBCO). Both cells overexpress HER2 receptors. In vitro cytotoxicity was determined using an MTS assay kit. Cells seeded in 100 μL of culture medium on a 96-well plate were incubated for one day before adding DOX, Herceptin-DOX or Herceptin-P-DOX. Wells filled with culture medium (n=6) were defined as 0% viability, and wells filled with only PBS-treated cells (n=6) were defined as 100% viability. Serial dilutions of DOX, Herceptin-DOX or Herceptin-P-DOX were added to other wells to obtain final DOX equivalent concentrations of 75, 25, 8.33, 2.78, 0.926, 0.309, 0.103, 0.0343, 0.0114 and 0.00381 μM. After 48 hours of incubation, 20 μL of MTS reagent was added to the cells and incubated for another 4 hours at 37°C. The absorbance was measured at 490 nm using a SpectraMax M3 microplate reader (Molecular Devices). The viability percentage and 50% inhibitory concentration (IC50) were determined using GraphPad Prism 5.0 software. Fig.12The results showed that, based on the concentration of doxorubicin, the IC50 of DOX itself was 5.49 and 0.31 μM in BT-474 and SKOV-3 cells, respectively, the IC50 of Herceptin-DOX was 26.5 μM (BT-474) and 1.34 μM (SKOV-3), and the in vitro anti-tumor cell proliferation activity retention rate was 20.7% and 23.1%, while the IC50 of Herceptin-P-DOX was 22.9 μM (BT-474) and 0.99 μM (SKOV-3), with a higher activity retention rate of 24.0% and 31.3%, respectively. More importantly, as Fig.13 The results showed that based on the quantification of Herceptin concentration, the IC50 of Herceptin-DOX was 8.28μM (BT-474) and 0.42μM (SKOV-3), while the IC50 of Herceptin-P-DOX was 0.42μM (BT-474) and 0.018μM (SKOV-3), with the activity increased by 19.7 and 23.3 times.

[0132] 10. Six-week-old healthy female SPF BALB / c mice were injected with Herceptin-P-DOX via the tail vein at doses of 30 and 50 mg DOX equivalents per kg body weight (n=3). Changes in mouse body weight and survival rate were monitored daily for two weeks. Mice that lost more than 10% of their pre-treatment body weight were euthanized. The highest dose at which no animal death was observed and weight loss did not exceed 10% was defined as the maximum tolerated dose. Fig.14 It can be seen that under the 50 mg DOX equivalent, the body weight of one mouse decreased by more than 10%, so 30 mg / kg DOX equivalent (or 136 mg / kg Herceptin equivalent) was determined to be the maximum tolerated dose.

[0133] In vivo antitumor activity test: BALB / c nude mice (female, 16-18 g) were acclimatized one week before treatment. SKOV-3 tumor cells (1×10 7 ) were implanted subcutaneously on the back of mice. 40 tumor-bearing mice were divided into 5 groups, with 6-8 mice in each group: PBS group, DOX group (5 mg / kg), Herceptin group (136 mg / kg), Herceptin-DOX group (136 mg / kg Herceptin equivalent) and Herceptin-P-DOX group (136 mg / kg Herceptin equivalent). When the tumor volume reached 50-70 mm 3 Each group of mice received 200 μL of intravenous drug at 1:10 pm. The tumor volume of mice was assessed twice a week with a caliper and determined by the following formula: tumor volume = 1 / 2 × LW 2 , where L is the longest diameter of the tumor and W is the smallest diameter of the tumor. Fig.15 It can be seen that at 49 days after injection, the average tumor volume of the PBS group and the DOX group was 1189 mm 3 and 1029mm3 The mean tumor volume in the Herceptin group and the Herceptin-DOX group was 436 mm 3 and 358mm 3 , while the Herceptin-P-DOX group was 245 mm 3 During treatment, if the mice lost >15% of their body weight or the tumor volume reached 500 mm 3 , the mice were recorded as dead to calculate the survival time. Fig.16 It can be seen that the median survival time of the PBS group and the DOX group was 35 days, indicating that a single DOX administration could not prolong the survival time. The median survival time of the Herceptin group was 56 days, indicating that Herceptin monoclonal antibody can effectively prolong the survival time of HER2-high-expressing ovarian cancer cell SKOV-3 tumor-bearing mice, while the median survival time of the Herceptin-DOX group was 57.5 days, indicating that the DAR value of 3.2 could not significantly improve the efficacy of the drug. The median survival time of the Herceptin-P-DOX group was 63 days, which significantly prolonged the survival time of mice compared with the Herceptin group and the Herceptin-DOX group.

[0134] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing an antibody-polymer-drug conjugate, characterized in that: Preparation of antibody-polymer-drug conjugates by in situ polymerization; include: preparing antibody-elicitor conjugates; The antibody-initiator conjugate is polymerized with a polymer monomer and a drug molecule monomer in a buffer to prepare the antibody-polymer-drug conjugate; wherein the antibody-polymer-drug conjugate is formed by polymerizing the polymer monomer and the drug molecule monomer on the surface of the antibody-initiator conjugate; the antibody-initiator conjugate comprises an antibody and at least one initiator connected to a disulfide bond or a free thiol group of the antibody; The polymer monomer is selected from at least one of acrylate, methacrylate, acrylamide and methacrylamide; The drug molecule monomer is selected from drug molecules modified by at least one of acrylate functional groups, methacrylate functional groups, acrylamide functional groups, and methacrylamide functional groups; The drug molecule is at least one of doxorubicin, epirubicin, and daunorubicin; The antibody is at least one of trastuzumab and pertuzumab; The initiator has the following structural formula: ； Among them, R 1 is a functional group or a functional group derivative thereof capable of initiating an atom transfer radical polymerization reaction; Wherein, the functional group capable of initiating atom transfer radical polymerization is selected from 2-bromo-2-methylpropionamide, 2-chloro-2-methylpropionamide, 2-bromopropionamide, 2-chloropropionamide, 2-bromo-2-methylpropionate, 2-chloro-2-methylpropionate, 2-bromopropionate, 2-chloropropionate; R 2 and R 3 Same or different, R 2 and R 3 Each is independently selected from a functional group that is easily substituted by nucleophilicity and a derivative of the functional group, the functional group that is easily substituted by nucleophilicity is selected from at least one of H, I, Br, Cl, C6H5S, CH3C6H5S, and p-toluene ring sulfonyl; X and Y are the same or different, X and Y are independently selected from NH, O, S, and Se atoms; Z is selected from N or CH.

2. The method for preparing an antibody-polymer-drug conjugate according to claim 1, characterized in that: The drug molecule monomer has a structure represented by any one of Chemical Formulas 6 to 9: ； Among them, the R2 group in chemical formulas 6 to 9 is selected from the pharmacophoric group of the drug molecule; the R1 group is selected from pH-responsive acylhydrazone bonds, carbonate amide bonds, phenylboronic ester bonds, imine bonds, oxime bonds, ester bonds, redox-responsive disulfide bonds, thioether bonds, diselenide bonds, selenoether bonds, thioketal bonds, aminoacrylates, oligoproline and peroxalate.

3. The method for preparing the antibody-polymer-drug conjugate according to any one of claims 1 to 2, characterized in that: The polymer monomer has a structure represented by any one of Chemical Formulas 2 to 5: ； Wherein, the R groups in Chemical Formulas 2 to 5 are each independently selected from an oligoethylene glycol group or an ethylphosphocholine group.

4. The method for preparing an antibody-polymer-drug conjugate according to any one of claims 1 to 2, characterized in that: In the antibody-polymer-drug conjugate, the molar ratio of the antibody to the polymer monomer unit is 1:10 to 1:1000, and the molar ratio of the antibody to the drug monomer unit is 1:1 to 1:

100.

5. The method for preparing the antibody-polymer-drug conjugate according to claim 1 or 2, characterized in that: The initiator is coupled to two adjacent free sulfhydryl groups formed after the disulfide bonds of the hinge region of the antibody are reduced.

6. The method for preparing the antibody-polymer-drug conjugate according to any one of claims 1 to 2, characterized in that: The polymerization is carried out in a low oxygen or inert gas atmosphere, the reaction time is 0.5 to 48 hours, and the reaction temperature is 0 to 50°C.

7. The antibody-polymer-drug conjugate prepared by the preparation method according to any one of claims 1 to 6.

8. A method for preparing the antibody-polymer-drug conjugate according to any one of claims 1 to 6 and / or use of the antibody-polymer-drug conjugate according to claim 7 in the preparation of anti-tumor drugs.

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