A targeted drug based on antibody IgG1-Fc domain and its preparation method and application

Abstract

The present invention describes a protein-drug conjugate based on the IgG1-Fc domain of an antibody, a preparation method and an application. The conjugate comprises a small molecule targeting ligand, a toxic drug warhead and an Fc domain protein of IgG1, and a tumor-targeting protein-drug conjugate is formed by coupling the ligand and the drug warhead to the N-terminus of the Fc protein. The present invention discloses the structure, preparation method and therapeutic use of an Fc protein-drug conjugate. The present invention fuses SMDC and the Fc domain of an antibody to obtain an antibody-drug conjugate structure-eADC. The natural pharmacokinetic advantages of antibody molecules are utilized to make up for the short half-life of small molecule drug conjugates, and the effect of "1+1>2" is achieved by taking advantage of each other's strengths and weaknesses.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a targeted protein-drug conjugate based on an antibody IgG1-Fc domain and a preparation method and application thereof. Background Art

[0002] Cancer has become the second largest health threat to mankind, with approximately 10 million people dying from cancer in 2020. For decades, chemotherapy based on cytotoxic drugs has been the main method for treating many cancers. However, since chemotherapy drugs can act on all rapidly dividing cells, it is difficult to distinguish between normal cells and tumor cells, greatly reducing the therapeutic index. To solve this problem, researchers have been committed to developing new tumor treatment measures with targeted capabilities.

[0003] In recent years, with the development of tumor molecular and immunology, a new drug structure for targeted delivery of chemotherapy drugs to tumor sites - antibody-drug conjugates (ADCs) has become a cutting-edge direction in the field of cancer treatment. ADCs are mainly composed of monoclonal antibodies, linkers and cytotoxic drugs. Combining the advantages of strong specificity of monoclonal antibodies and high lethality of cytotoxic small molecule drugs, it has achieved great success in clinical practice through the combination of immunotherapy and chemotherapy. So far, 14 ADC drugs have been approved for marketing, and more than 100 ADCs of different structural types are in the clinical research stage, which is enough to show the popularity of its research field. However, with the deepening of clinical research, some problems that need to be solved are still found. First of all, because the complete monoclonal antibody is a large molecule biological drug with a molecular weight of about 150kDa, there are certain difficulties in the permeability of solid tumors. Moreover, ADC has a complex structure, high R&D costs, and a long cycle.

[0004] Small molecule drug conjugates (SMDC) are similar in structure to ADCs and are composed of small molecule targeting ligands, linkers and cytotoxic small molecule drugs. The targeted drug delivery model based on small molecule ligands has many advantages; lower molecular weight helps penetrate solid tumors, and they are not immunogenic, and can be flexibly designed and modified using simple organic synthesis, thereby greatly reducing research and development costs. It is well known that the approval of a new drug mainly depends on its efficacy and safety. In the SMDC structure, highly efficient cytotoxic small molecule drugs provide a guarantee for its efficacy. In terms of safety, small molecule targeting ligands enable it to achieve precise strikes, distinguish normal cells from tumor cells, and reduce the systemic toxicity of the entire conjugate. Therefore, SMDC has sufficient drug standards. The mechanism of action of SMDC is similar to that of ADC, mainly including three aspects. SMDC enters the blood circulation by intravenous injection. First, recognize and bind antigens: After reaching the tumor tissue through the blood circulation, it recognizes and binds to specific antigens on the surface of tumor cells and is internalized into the cell through the endosome / lysosome pathway. 2. Release drugs: Smart linkers respond to specific microenvironments in cells or organelles to release cytotoxic drugs. 3. Play a therapeutic role: Targeting specific targets in cells, such as tubulin, DNA, topoisomerase, etc., it plays a killing role through a series of cascade reactions, ultimately leading to tumor cell apoptosis. Compared with the success of ADC, limited target selection has restricted the development of SMDC. Currently, the main clinical target selections are folate receptor (FR), prostate-specific membrane antigen (PSMA), growth hormone inhibitor receptor, carbonic anhydrase IX receptor, etc. Although many SMDCs have entered the clinical trial stage, there are currently no SMDCs on the market. The reason is that although the smaller molecular weight gives it an obvious advantage in the permeability of solid tumors, it also greatly shortens its circulation half-life, making it difficult to achieve good aggregation at the site of solid tumors, and the therapeutic index is greatly reduced.

[0005] Therefore, innovative designs and modifications are urgently needed to enable SMDC to achieve similar success as ADC. Summary of the invention

[0006] In order to solve the technical problems, the present invention provides a targeted drug based on the antibody IgG1-Fc domain, and its preparation method and application, by fusing SMDC and the antibody Fc domain to obtain a similar antibody drug conjugate structure - eADC. The natural pharmacokinetic advantages of antibody molecules are used to make up for the short half-life of small molecule drug conjugates, and the effect of "1+1>2" is achieved by taking advantage of their strengths and making up for their weaknesses.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] In the first aspect of the present invention, a tumor-targeted protein-drug conjugate based on the antibody IgG1-Fc domain is characterized in that the structural formula of the tumor-targeted protein-drug conjugate is as follows:

[0009]

[0010] in:

[0011] R1 is selected from hydrogen or C1-C4 alkyl;

[0012] Z is methionine, glutamine or unsubstituted;

[0013] aa is an L-type amino acid, wherein aa includes at least one of leucine, proline, glutamic acid, threonine and glycine;

[0014] n is selected from 1, 2, 3, 4, 5 or 6;

[0015] k is 1 or 2;

[0016] Q1 represents a small molecule targeting ligand, and the ligand includes one of DUPA, folic acid and acetazolamide.

[0017] Furthermore, the tumor-targeting protein-drug conjugate comprises:

[0018]

[0019] Wherein, R 1 is hydrogen; Z is unsubstituted; (aa)n is leucine-proline-glutamic acid-threonine; and k is 2.

[0020] Furthermore, the tumor-targeting protein-drug conjugate comprises:

[0021]

[0022] Wherein R1 is hydrogen; Z is methionine; (aa)n is leucine-proline-glutamic acid-threonine; and k is 2.

[0023] Furthermore, the tumor-targeting protein-drug conjugate includes any of the following compounds:

[0024]

[0025]

[0026] In the above structural formula, the rightmost part (the part with N at the top and C at the bottom) is IgG1 Fc, and the amino acid sequence of the IgG1 Fc is shown in SEQ ID NO. 1. The chemical drug is coupled to the N-terminus of IgG1-Fc.

[0027] In the second aspect of the present invention, a pseudopeptide intermediate is provided, the structural formula of the intermediate is as follows:

[0028]

[0029] In the compound shown in Formula IA, the definitions of each letter are the same as described above; the intermediate is the structure of a Sortase A pseudopeptide substrate, including the drug MMAF and an enzyme recognition fragment, wherein the drug and the recognition fragment are connected by a peptide bond.

[0030] Furthermore, the intermediate includes any of the following compounds:

[0031]

[0032]

[0033] In the third aspect of the present invention, a method for preparing the tumor-targeting protein-drug conjugate is provided, the method comprising:

[0034] In PBS buffer, His-Fc was mixed and incubated with Sortase A enzyme at 1 mM di-Gly, to obtain IgG1Fc. The reaction equation is as follows:

[0035]

[0036] In TBS buffer solution, the pseudopeptide intermediate and the IgG1 Fc protein are subjected to transpeptide coupling reaction under the catalysis of Sortase A, and then a small molecule targeting ligand is added for click chemical grafting to obtain a tumor-targeted protein drug conjugate based on the antibody IgG1-Fc domain.

[0037] Furthermore, as a specific embodiment, the preparation method comprises:

[0038] Using pET-21-Cetuximab plasmid as a template and the primer pair shown in SEQ ID NO.3-NO.4 to perform PCR amplification to obtain an IgG1-Fc gene fragment; the IgG1-Fc gene fragment was connected to a pET-28 vector by double restriction digestion with BamHI / BBoⅠ, and a recombinant plasmid pET28-IgG1-Fc was obtained after transformation;

[0039] The recombinant plasmid pET28-IgG1-Fc was double-digested with NdeⅠ / BamHI, followed by electrophoresis and recovery to obtain a recovered DNA fragment; the positive chain and the antisense chain of SEQ ID NO.5-NO.6 of the substrate LPETG Linker recognized by the Sortase A enzyme were annealed to obtain an annealed fragment; the recovered DNA fragment and the annealed fragment were connected and transformed to obtain a recombinant plasmid pET28-IgG1-Fc / LPETG;

[0040] The recombinant plasmid pET28-IgG1-Fc / LPETG was induced to express and the protein was purified, and the His tag used for protein purification was removed by SortaseA to obtain the target protein IgG1-Fc;

[0041] Select 2-chlorotrityl resin, perform a condensation reaction on amino acids by solid phase synthesis to obtain a pseudopeptide with an amino group; add MMAF to DCM and DIPEA for ultrasonication, then add di-tert-butyl dicarbonate for reaction and purification to obtain Boc-MMAF; perform a condensation reaction on the pseudopeptide with an amino group and the Boc-MMAF to obtain a bifunctional pseudopeptide intermediate;

[0042] The target protein IgG1-Fc and the bifunctionalized pseudopeptide intermediate are mixed in a TBS solution, and Sortase A enzyme is added to perform a transpeptidation reaction to achieve drug loading, followed by purification to obtain an antibody loaded with MMAF small molecule toxin and azide functionalization;

[0043] The small molecule ligand is mixed, THPTA, copper sulfate and sodium ascorbate are added to react at room temperature, and then purified to obtain an alkynyl functionalized small molecule ligand;

[0044] The alkynyl-functionalized small molecule ligand, the loaded MMAF small molecule toxin and the azide-functionalized antibody are mixed, and then copper sulfate and sodium ascorbate are added to perform a click chemistry reaction to allow the alkynyl and azide groups to undergo cycloaddition to obtain a targeted drug based on the antibody IgG1-Fc domain.

[0045] In the fourth aspect of the present invention, provided is the use of the targeted protein-drug conjugate based on the antibody IgG1-Fc domain in the preparation of drugs for treating and preventing cancer.

[0046] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0047] The present invention provides a targeted protein-drug conjugate based on the antibody IgG1-Fc domain, and a preparation method and application thereof. First, the vector of the IgG1-Fc antibody protein is constructed and expressed in prokaryotes. Secondly, a bifunctionalized pseudopeptide of azide and MMAF is synthesized, and transpeptidation occurs under the mediation of the Sortase A enzyme to achieve controllable loading of the drug. Finally, the small molecule ligand library and the protein are grafted for different targets; that is, the present invention uses "Sortase A" as a tool enzyme for bridging the MMAF small molecule toxin and the bifunctionalized pseudopeptide of azide and the antibody Fc to achieve site-specific and controllable drug coupling and improve the homogeneity of eADC. "Click chemistry" is used as a grafting tool for grafting the small molecule ligand library and the antibody Fc. A series of drug structure molecules with uniform structure are obtained, and it is expected that one or more macromolecular biological drugs with drug development prospects can be screened to achieve clinical transformation.

[0048] This paper selected three small molecule targeting ligands that are widely used in clinical trials as model molecules to examine the universality and flexibility of our antibody drug delivery platform. Through click chemistry, a flexible target head replacement is achieved in one step, and finally a series of innovative drug candidate molecules with structures similar to antibody-drug conjugates are obtained, which are expected to achieve clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0050] Figure 1 Schematic diagram of the plasmid of pET28-IgG1-Fc / LPETG expression vector.

[0051] Figure 2 The SDS-PAGE analysis shows the protein purified by nickel column and the target protein after enzyme digestion. The first lane in the figure is the protein marker, and lanes 2-3 are the protein bands reduced by TCEP. Lane 2 is the Fc protein with His-tag, and lane 3 is the target protein band after enzyme digestion. The last two lanes in the figure are protein bands without reduction treatment, lane 4 is the Fc protein with His-tag, and lane 5 is the target protein band after enzyme digestion.

[0052] Figure 3 Schematic diagram of Sortase A enzyme-mediated transpeptidation and small molecule ligand library and Fc protein grafting.

[0053] Figure 4Figure 2 is the flow cytometry analysis results of SMDC and eADC.

[0054] Figure 5 Laser confocal imaging of SMDC-1070 and eADC-1070 in 22Rv1 cells.

[0055] Figure 6 Laser confocal imaging of SMDC-274 and eADC-274 in SKRC-52 cells.

[0056] Figure 7 Laser confocal imaging of SMDC-606 and eADC-606 in KB cells.

[0057] Figure 8 This is a diagram for analyzing the cytotoxicity of drug molecules to tumor cells. Figure 8 A shows that compared with MMAF, the cytotoxicity to target cells is significantly enhanced after small molecule ligand grafting. Figure 8 B shows that compared with PSMA receptor-negative PC 3 cells, eADC-1070 has a significant killing effect in receptor-positive 22Rv1 and LNCaP cells and has obvious selectivity.

[0058] Fig. 9 The anti-tumor effects of different drug molecules in the corresponding tumor cell xenograft animal models. DETAILED DESCRIPTION

[0059] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0060] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0061] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0062] The overall idea of ​​the technical solution of the present invention is as follows:

[0063] Fc fusion protein, combined with the natural pharmacokinetic advantages of antibody molecules, can expand the application range of antibody drugs by optimizing and modifying its structure. Because traditional protein drugs have a short half-life in plasma, long-term high-dose administration is required to achieve therapeutic effects, which brings serious side effects, while Fc fusion protein greatly prolongs the half-life of drugs, and can be further extended after modification.

[0064] This application develops a new type of innovative drug molecule that simulates the structure of ADC. That is, SMDC and the antibody Fc domain are fused to obtain a structure similar to an antibody-drug conjugate - eADC. The natural pharmacokinetic advantages of antibody molecules are used to make up for the short half-life of small molecule drug conjugates, and the "1+1>2" effect is achieved by taking advantage of their strengths. Specifically, "Sortase A" is used as a tool enzyme for MMAF small molecule toxins and azide bifunctionalized pseudopeptides and antibody Fc bridges to achieve site-specific and controllable drug conjugation and improve the homogeneity of eADC. "Click chemistry" is used as a grafting tool for small molecule ligand libraries and antibody Fc grafting. A series of drug-antibody ratios of 2 and uniformly structured drug structure molecules are obtained, and it is expected that one or more large molecule biological drugs with drug development prospects can be screened to achieve clinical transformation.

[0065] Through extensive and in-depth research, this application has optimized an efficient antibody drug delivery platform with controllable drug-antibody ratio and flexible modification. The experimental results show that by adjusting the ratio of bifunctional short peptides and IgG1-Fc, controllable drug loading can be achieved, and the uniformity of the drug structure is good. On the other hand, after grafting two small molecule ligands, the targeted delivery efficiency in vivo and in vitro has been significantly improved (such as Figure 8 ). Moreover, the introduction of the Fc domain significantly increases the circulation half-life of the drug, and therefore also exhibits efficient anti-tumor activity.

[0066] According to a typical embodiment of the present invention, a method for preparing a targeted drug based on the antibody IgG1-Fc domain is provided, the method comprising:

[0067] (1) Construction of pET28-IgG1-Fc / LPETG prokaryotic expression vector and protein purification and transformation, including steps S1 to S3:

[0068] Step S1, using pET-21-Cetuximab plasmid as a template and the primer pair shown in SEQ ID NO.3-NO.4 to perform PCR amplification to obtain an IgG1-Fc gene fragment; connecting the IgG1-Fc gene fragment to a pET-28 vector through BamHI / BBoⅠ double restriction digestion, and obtaining a recombinant plasmid pET28-IgG1-Fc after transformation;

[0069] Step S2, the recombinant plasmid pET28-IgG1-Fc is double-digested with NdeⅠ / BamHI, followed by electrophoresis and recovery to obtain a recovered DNA fragment; the positive chain and the antisense chain of SEQ ID NO.5-NO.6 of the substrate LPETG Linker recognized by the Sortase A enzyme are annealed to obtain an annealed fragment; the recovered DNA fragment and the annealed fragment are connected and transformed to obtain a recombinant plasmid pET28-IgG1-Fc / LPETG;

[0070] Step S3, inducing expression and protein purification of the recombinant plasmid pET28-IgG1-Fc / LPETG, and using Sortase A to cut off the His tag used for protein purification to obtain the target protein IgG1-Fc;

[0071] In the steps S1 to S3,

[0072] We selected the Fc fragment of IgG1 monoclonal antibody, which is widely used in ADC, as the core part of the entire antibody drug delivery platform. Because the natural favorable pharmacokinetic advantages of the Fc domain can prolong the circulation half-life and improve the efficiency of targeted therapy. At the same time, some immune effects can also be mediated, for example, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated phagocytosis (ADCP), etc. Therefore, in the present invention, we insert the base sequence of the Fc domain in cetuximab into the pET-28 plasmid, and introduce the substrate sequence recognized by the Sortase A enzyme at its front end. The prokaryotic expression vector includes three functional domains, (His tag, substrate sequence for Sortase A enzyme digestion, functional domain of IgG1-Fc) and its self-induced expression in the expression strain SBuffle T7E.coli.

[0073] (2) Design and synthesis of MMAF small molecule toxin C-terminal coupling and azide bifunctionalized pseudopeptide, including steps S4 to S6:

[0074] Step S4, selecting 2-chlorotrityl resin, and subjecting amino acids to a condensation reaction by solid phase synthesis to obtain a short peptide with a protecting group; as a specific embodiment, the step S4 specifically includes:

[0075] Step S41, weighing 2-chlorotrityl resin, adding anhydrous DMF solution to swell, and then draining the solvent to obtain treated 2-chlorotrityl resin;

[0076] Step S42, adding N,N-diisopropylethylamine DIPEA and DMF to dissolve the amino acid Fmoc-TBr(OtBu)-OH, then adding the treated 2-chlorotrityl resin, mixing and reacting for a period of time, then adding methanol to continue the reaction, and then washing with DMF, methanol and DCM in sequence to obtain Fmoc-TBr-resin;

[0077] Step S43, after adding the decapping reagent to the Fmoc-TBr-resin for reaction, the resin was washed with DMF and ethanol in sequence, dried, and Fmoc-Glu(OtBu)-OH dissolved with the peptide grafting reagent was added for reaction, and the resin was washed with DMF, methanol, and DCM respectively to obtain Fmoc-Glu-TBr-resin;

[0078] Step S44, adding the decapping reagent to the Fmoc-Glu-TBr-resin to react, then washing with DMF, methanol, and DCM in sequence, then adding Fmoc-Pro-OH dissolved with the peptide-attaching reagent to react, drying, and washing with DMF, methanol, and DCM in sequence to obtain Fmoc-Pro-Glu-TBr-resin;

[0079] Step S45, adding the decapping reagent to the Fmoc-Pro-Glu-TBr-resin to react, then washing with DMF, methanol, and DCM in sequence, then adding Fmoc-Leu-OH dissolved with the peptide-attaching reagent to react, drying, and washing with DMF, methanol, and DCM in sequence to obtain Fmoc-Leu-Pro-Glu-TBr-resin;

[0080] Step S46, adding the decapping reagent to the Fmoc-Leu-Pro-Glu-TBr-resin to react, then washing with DMF, methanol, and DCM in sequence, then adding Fmoc-Azl-OH dissolved with the peptide-attaching reagent to react, blowing dry, and washing with DMF, methanol, and DCM in sequence to obtain Fmoc-Azl-Leu-Pro-Glu-TBr-resin.

[0081] Step S47, using a cleavage reagent to cut the polypeptide Fmoc-Azl-Leu-Pro-Glu-TBr-OH from the resin, and precipitating the crude polypeptide with water.

[0082] Step S48, condense the crude product with tert-butyl bromoacetylglycine in THF solvent, purify on silica gel, and then remove the Fmoc protecting group with piperidine to obtain a pseudopeptide with an amino group.

[0083] Step S5, adding MMAF to DCM and DIPEA, sonicating, and then adding di-tert-butyl dicarbonate to react and purify to obtain Boc-MMAF;

[0084] The molar ratio of the MMAF to the di-tert-butyl dicarbonate is 1:3; such a ratio is conducive to complete reaction.

[0085] Step S6, subjecting the pseudopeptide with an amino group to a condensation reaction with the Boc-MMAF to obtain a bifunctional pseudopeptide;

[0086] In step S6, the method of introducing the small molecule toxin MMAF and the azide functional group in the synthesis of the bifunctional pseudopeptide is shown in the figure below.

[0087]

[0088] The step S6 specifically includes:

[0089] Step S61, mixing the polypeptide condensing agent HATU and Boc-MMAF, adding DMF and DIPEA to react; then adding the pseudo-peptide with amino group to continue the reaction; the molar ratio of HATU, Boc-MMAF and pseudo-peptide with amino group is (8-12):6:5; preferably 10:6:5, which is conducive to complete reaction;

[0090] Step S62, after the reaction is completed, water is added to quench the reaction, and DCM is used for extraction for multiple times. The organic phase is washed with water and saturated NaCl solution in turn to obtain a pseudopeptide skeleton with a protective group; then the protective group is removed with TFA, and the solvent is removed under reduced pressure to obtain a pseudopeptide with an amino group.

[0091] In the steps S4 to S6,

[0092] The design of the entire bifunctional short peptide mainly includes three effector parts. The first is a small molecule cytotoxin that exerts anti-tumor activity, MonometByl auristatin F (MMAF), which is an effective microtubule polymerization inhibitor and a small molecule toxin that is widely used in marketed drugs and clinical trials. However, it is basically modified, transformed and coupled at the N-terminus of MMAF. The present invention attempts to use the carboxylic acid at the C-terminus for direct drug coupling to provide a new drug connection mode. The second is the introduction of the azide functional group. We choose the specially functionalized Fmoc-Lys(N3)-OH and introduce it in the solid phase synthesis for subsequent grafting of the small molecule ligand library. The last is the synthesis of the pseudopeptide skeleton that plays a transpeptidation role, which is combined by solid phase synthesis and organic reaction. The obtained bifunctional pseudopeptide can achieve fixed-point and controllable drug loading under the action of enzymes.

[0093] (3) Sortase A enzyme-mediated site-specific, controllable drug loading; specifically comprising step S7:

[0094] Step S7, mixing the target protein IgG1-Fc and the bifunctionalized pseudopeptide in a TBS solution, adding Sortase A enzyme to perform a transpeptidation reaction to achieve drug loading, and then purifying to obtain an antibody loaded with MMAF small molecule toxin and azide functionalization;

[0095] In step S7,

[0096] The molar ratio of the target protein IgG1-Fc to the bifunctionalized pseudopeptide is 1: 10. This molar ratio range is conducive to the complete progress of the transpeptide coupling reaction.

[0097] During the coupling process of the target protein IgG1-Fc and the bifunctionalized pseudopeptide, the concentration of Sortase A is 10-1000 nM. This concentration is conducive to the rapid progress of the transpeptide coupling reaction, while too low or too high a concentration is not conducive to improving the yield.

[0098] (4) Synthesis of an alkynyl functionalized small molecule ligand library; specifically comprising step S8:

[0099] Step S8, according to the literature (Org. Biomol. CBem., 2014, 12, 3181; J. Med. CBem. 2020, 63, 10897-10907; J. Am. CBem. Soc. 2018, 140, 1617-1621), folic acid, DUPA and acetazolamide are condensed with valeric acid to obtain an alkynyl functionalized small molecule ligand;

[0100] (5) "One-step method" realizes the grafting of small molecule ligand libraries with different targets and antibody Fc, and constructs a variety of innovative drug molecules similar to antibody-drug conjugates. Specifically, it includes step S9:

[0101] Step S9, after mixing the alkynyl-functionalized small molecule ligand, the loaded MMAF small molecule toxin and the azide-functionalized antibody, copper sulfate and sodium ascorbate are added to perform a click chemistry reaction to allow the alkynyl and azide groups to undergo cycloaddition to obtain a targeted drug based on the antibody IgG1-Fc domain.

[0102] The molar ratio of the loaded MMAF small molecule toxin and the azide-functionalized antibody to the small molecule ligand is 1:3.

[0103] In the process of grafting small molecule ligand libraries, one-step click chemistry reaction achieves flexible ligand replacement.

[0104] The effects of the present application will be described in detail below in conjunction with embodiments and experimental data.

[0105] Unless otherwise specified, the molecular cloning methods, protein expression and purification methods, cell culture methods, and various detection methods mentioned in the following schemes are all traditional experimental methods and can be obtained by consulting the literature; the relevant reagents used can be purchased from the corresponding reagent suppliers.

[0106] Example 1: A method for preparing a targeted drug based on the IgG1-Fc domain of an antibody

[0107] 1. Vector construction and prokaryotic expression of pET28-IgG1-Fc / LPETG antibody protein

[0108] 1. Primer Design and Synthesis

[0109] We selected the Fc domain of Cetuximab as the protein component in our antibody drug delivery platform and expressed it in prokaryotic cells via pET-21 plasmid. We designed two upstream and downstream primers to amplify the IgG1-Fc target fragment, inserted it into the pET-28 plasmid via two restriction endonucleases, and then designed two gene fragments, directly annealed to produce sticky ends, and then inserted them into the N-terminus of IgG1-Fc by enzyme digestion and ligation, and used it as the substrate sequence recognized by Sortase A enzyme, and finally obtained a 6030bp target plasmid.

[0110] Each primer was synthesized by Shanghai Shenggong Bioengineering Technology Co., Ltd. The specific primer names and sequences are as follows:

[0111] Table 1

[0112]

[0113]

[0114] 2. PCR Amplification

[0115] Using the commercial pET-28 plasmid as a template, the cDNA gene of IgG1-Fc was synthesized, and then the PCR amplification reaction system and specific reaction conditions were as follows:

[0116] 1) IgG1-Fc gene amplification

[0117]

[0118] Mix the above components with a pipette and perform the following reactions on a PCR thermal cycler:

[0119] Denature at 95°C for 3 min, then perform 30 cycles of 95°C for 15 sec, 63°C for 15 sec, and 72°C for 60 sec, and 72°C for 5 min. Purify the obtained product using a DNA purification kit (product of Omiga).

[0120] 2) LPETG Linker Annealing

[0121] After obtaining the dry powder product, centrifuge the tube instantaneously and add annealing buffer to dissolve it to a concentration of 100 μM. Mix the sense strand and antisense strand at a molar ratio of 1:1, heat the mixed system to 100°C for 2 minutes, then slowly cool it down, and after it cools to room temperature, dilute it with annealing buffer to the required concentration and store it at -20°C for later use.

[0122] 3. Construction of pET28-IgG1-Fc recombinant plasmid

[0123] The pET-28 vector (product of Addgene) was double-digested with restriction endonucleases BamHⅠ and BBoⅠ. The IgG1-Fc product amplified by PCR in step 3.1 was also digested with the same double enzymes. The digested products were subjected to 1% agarose gel electrophoresis. DNA fragments were recovered using a DNA gel recovery kit (product of Omiga, hereinafter the same) (according to the instructions of the kit). The plasmid fragments and the amplified fragments were ligated at a ratio of 1:3. The ligated products were transformed into Escherichia coli DH5α. Positive clones were picked out and inoculated into 10 ml of LB medium containing kanamycin (50 μg / mL), and cultured at 37°C overnight. Plasmid DNA was extracted using a plasmid purification kit (product of Omiga, hereinafter the same) (according to the instructions of the kit). The recombinant plasmid pET28-IgG1-Fc was obtained after restriction analysis and nucleotide sequencing verification.

[0124] 4. Construction of pET28-IgG1-Fc / LPETG recombinant plasmid

[0125] The pET28-IgG1-Fc vector was double-digested with restriction endonucleases NdeⅠ and BamHⅠ, and agarose gel electrophoresis was performed. DNA fragments were recovered by a DNA recovery kit (product of Omiga) (according to the instructions of the kit), and the fragments annealed in the step were connected with the DNA fragments recovered above at a ratio of 5:1. The connection product was transformed into Escherichia coli DH5α, and the positive clones were picked out and inoculated into 10 ml of LB medium with kanamycin (50 μg / mL), and cultured at 37°C overnight. Plasmid DNA was extracted by a plasmid purification kit (product of Omiga, the same below) (according to the instructions of the kit), and the recombinant plasmid pET28-IgG1-Fc / LPETG was obtained after enzyme digestion analysis and nucleotide sequencing verification.

[0126] 5. Prokaryotic expression and protein purification of pET28-IgG1-Fc / LPETG

[0127] The recombinant plasmid pET28-IgG1-Fc / LPETG with correct sequencing was transformed into SBuffle T7 E.coli competent cells, and the self-inducing protein expression conditions were adopted, and the cells were cultured for 36 B at 25°C and 220 rpm. The cells were collected by centrifugation at 8000g for 10 min, resuspended in binding buffer, and crushed under high pressure. The resulting suspension was subjected to ultracentrifugation at 10000g / 25min. The target protein was purified using a nickel column to obtain the final target protein His-Fc.

[0128] Description of artificial protein sequence (SEQ ID NO.2):

[0129] MGSSHHHHHHSSGLVPRGSHMASLPETGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

[0130] 6. Enzymatic cleavage of His-Fc protein and preparation of IgG1-Fc

[0131] The purified His-Fc protein was dissolved in PBS, 1 mM triGly peptide was added, and then Sortase A enzyme (0.5 μM) was added, and incubated overnight at room temperature. The His tag used for protein purification was cut off. After the cutting was complete, the protein was precipitated with ammonium sulfate and purified with a G25 desalting column, using TBS as the mobile phase to obtain the protein IgG1-Fc.

[0132] Protein artificial sequence description (SEQ ID NO.1):

[0133] GGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0134] 2. Design and synthesis of MMAF small molecule toxin conjugation and azide bifunctionalized pseudopeptide

[0135]

[0136] Synthetic route, Z is methionine, glutamine or unsubstituted.

[0137] 1. Solid phase synthesis of peptides

[0138]

[0139] 1) Amino acid coupling

[0140] Select 2-chlorotrityl resin, and carry out condensation reaction of corresponding amino acids in sequence according to the standard operation method of solid phase synthesis based on Fmoc protection method. The specific amino acid sequence is Z-azido Lys-Leu-Pro-Glu-TBr (Z is methionine, glutamine or unsubstituted). Weigh 1.5g (1.8mmol) of 2-chlorotrityl resin (1.2mmol / g) and load it into the reactor. Add 10mL of anhydrous DMF solution to swell for 15min, and drain the solvent. Weigh 795mg (2mmol) of the first amino acid to be reacted into a 50mL centrifuge tube, add 1.5mL DIPEA (N,N-diisopropylethylamine) and 10mL DMF to fully dissolve it, add it to the reactor, react at room temperature for 2B, add 5mL methanol, and continue to react for 15min. Wash the resin three times with DMF, methanol, and DCM in turn. The reaction conditions for removing Fmoc are 20% piperidine and shaking at room temperature for 30min. After draining, replace new piperidine and repeat once. Then, the resin was tested with ninhydrin solution, and washed with DMF, methanol, and DCM three times each. HCTU / DIPEA or HATU / DIPEA was selected as the reagent for amino acid condensation. The dosage was 2-5 times of the amino acid. The removal of Fmoc protecting group, testing, amino acid condensation, and washing were repeated until the last amino acid was coupled.

[0141] 2) Cutting resin

[0142] Add cutting reagent (glacial acetic acid: 1 mL; trifluoroethanol: 2 mL; DCM: 7 mL) and react at room temperature for two hours, filter the reaction solution, continue to wash the resin twice with a mixed solution of DCM and trifluoroethanol, and combine the filtrate. Drain under reduced pressure until the solution volume is less than 5 mL, add 25 mL of deionized water, mix thoroughly, cool it on ice, allow it to fully precipitate, centrifuge, pour off the supernatant, collect the precipitate, and obtain crude peptide compound 1.

[0143] 2. Pseudopeptide Synthesis

[0144]

[0145] Z is methionine, glutamine or unsubstituted.

[0146] Compound 2 and the obtained crude peptide compound 1 were reacted at room temperature overnight in TBAI as a catalyst, DIPEA as a base, and THF solvent. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with DCM for multiple times. The organic phase was washed with water and saturated NaCl solution in turn to obtain the corresponding pseudopeptide skeleton compound 3. Finally, the Fmoc protecting group at the N-terminus was removed in a 10-20% piperidine solution to expose the amino group for subsequent condensation reactions. The reaction was monitored by TLC. When the reaction was complete, the solvent was drained, and the obtained solid compound was washed three times with n-hexane, and the n-hexane was removed by centrifugation to obtain the desired product compound 4.

[0147] 3.Boc-MMAF Synthesis

[0148]

[0149] MMAF is a derivative of dolastatin 10 (D10), which can inhibit cell mitosis and has strong anti-tumor activity. Its antibody-drug conjugate formed by conjugating with tumor-targeting antibodies has entered the clinical research stage. The chemical structure of MMAF is composed of 5 monomers: MeVal, Val, Dil, Dap, and PBe.

[0150] Weigh 146.4 mg (0.2 mmol) of MMAF into a 25 mL round-bottom flask, add 10 mL of DCM and 165 μL of DIPEA, sonicate for 1 minute, then add 175 mg of di-tert-butyl dicarbonate (0.8 mmol), react overnight at room temperature, and monitor by TLC. When the reaction is complete, drain the solvent and purify it with a silica gel column, the eluent is (DCM: MeOH: AcOH = 20: 1: 0.01), and obtain 158 mg, with a yield of 95%.

[0151] 4. Synthesis of bifunctionalized pseudopeptides

[0152]

[0153] Z is methionine, glutamine or unsubstituted.

[0154] The pseudopeptide compound 4 with amino group obtained above was subjected to condensation reaction with Boc-MMAF. 76 mg HATU (0.2 mmol) and 100 mg Boc-MMAF (0.12 mmol) were weighed into a 10 mL round-bottom flask, 5 mL DMF and 33 μL DIPEA were added, and the reaction was carried out at room temperature for 15 min. 90 mg pseudopeptide (0.1 mmol) was added thereto, and the reaction was continued at room temperature, and monitored by TLC. After the reaction was completed, water was added to quench, and DCM was extracted several times. The organic phase was washed with water and saturated NaCl solution in turn to obtain pseudopeptide skeleton compound 5 with protective group. Finally, 20% TFA was used to remove the protective group, and the solvent was removed under reduced pressure. The obtained mixture was separated and purified by RP-HPLC to obtain the target compound.

[0155] When Z is unsubstituted, that is, Pseudopeptide-1441;

[0156]

[0157] When Z is methionine, it is Pseudopeptide-1572;

[0158]

[0159] When Z is glutamine, it is Pseudopeptide-1569;

[0160]

[0161] 3. Drug loading and small molecule ligand grafting

[0162] 1. Drug-Fc protein conjugation

[0163] In order to achieve controllable and fixed-point drug loading, we mixed the purified target protein (5 mg / mL) and pseudopeptide-1441 (1-5 times or 10-100 times molar equivalent) in TBS solution, added Sortase A enzyme (500nM), under which a transpeptidation reaction occurred to achieve drug loading, and reacted at room temperature for 6B. The drug-antibody ratio was analyzed by hydrophobic interaction chromatography (HIC). After the reaction was complete, ammonium sulfate was used for precipitation, the supernatant was discarded, and the G25 desalting column was used for purification. PBS was used as the mobile phase to remove the residual small molecules, and it was characterized by ESI-TOF-MS and SDS-PAGE. Finally, an antibody drug platform with MMAF small molecule toxin loaded and azide functionalized was obtained.

[0164] 2. Grafting of small molecule ligands in protein-drug conjugates

[0165] In order to achieve precise targeted delivery and combine the natural and powerful pharmacokinetic advantages of antibody Fc, we functionalized the corresponding small molecule ligand with alkynyl groups, and the structure is as follows Figure 3 As shown, click chemistry reaction was performed. Antibody and small molecule ligand were added in a molar ratio of 1:3, THPTA, copper sulfate, and sodium ascorbate were added, and the reaction was carried out at room temperature for 2B. The protein was precipitated with ammonium sulfate and the supernatant was discarded. It was re-dissolved with PBS and purified by molecular exclusion chromatography, and PBS was used as the mobile phase to obtain the final protein-drug conjugate grafted with small molecule ligand.

[0166] eADC-1070 (prostate-specific membrane antigen) was obtained by grafting DUPA small molecule ligand;

[0167]

[0168] Grafting folic acid small molecule ligand to obtain eADC-606 (folate receptor);

[0169]

[0170] Grafting acetazolamide gave eADC-274 (carbonic anhydrase IX).

[0171]

[0172] 3. Preparation of SMDC Reference

[0173] We conducted a click chemistry reaction between the corresponding small molecule ligand and the pseudopeptide 1441 to prepare the corresponding SMDC reference substance. The main difference between this SMDC and the eADC protein drug conjugate is that it does not contain Fc protein. The pseudopeptide and the small molecule ligand were added in a 1:3 molar ratio, THPTA, copper sulfate, and sodium ascorbate were added, and the reaction was carried out at room temperature for 2B. The SMDC sample was purified by reverse phase preparative liquid chromatography:

[0174] SMDC-1070 (prostate-specific membrane antigen) was obtained by grafting DUPA small molecule ligand;

[0175]

[0176] Grafting folic acid small molecule ligand to obtain SMDC-606 (folate receptor);

[0177]

[0178] SMDC-274 (carbonic anhydrase IX) was obtained by grafting acetazolamide small molecule ligand.

[0179]

[0180] Example 2: Investigation of anti-tumor activity of targeted drug delivery platform

[0181] 1. Verification of drug structure and affinity with corresponding target receptors

[0182] To verify its binding specificity to PSMA receptor, folate receptor and carbonic anhydrase IB, eADC or SDMC were co-incubated with LNCaP, 22Rv1, KB, SKRC-52 and PC-3 cells at room temperature to reduce internalization.

[0183] The specific operation steps are as follows: eADC or SMDC (labeled with Cy5) and cancer cells (about 10,000) were incubated in serum-free RPMI 1640 medium supplemented with 1% BSA for 1 hour. Centrifuged at 3000 rpm for 3-10 min at 4°C, resuspended in 0.5-1 mL pre-cooled sterile PBS, and washed twice. Statistical analysis was performed using a BD LSR II flow cytometer (BecktonDickinson).

[0184] The grafted drug structure molecules [SMDC-1070 (prostate-specific membrane antigen), SMDC-606 (folate receptor), SMDC-274 (carbonic anhydrase IX)] can bind well to the corresponding cell surface receptors, and overall, the targeting of eADC is better than SMDC. In view of the different receptor expressions of different cells, our targeted-antibody drug delivery platform shows good universality.

[0185] 2. Cellular Uptake Experimental Analysis

[0186] In order to further explore the internalization and distribution of eADC in cells, we used laser confocal scanning microscopy to co-localize the distribution of eADC or SMDC in LNCaP, SKRC-52, and KB tumor cells. The specific steps are as follows: eADC or SMDC (labeled with Cy5) was co-incubated with the corresponding tumor cells (approximately 5000) at 37°C for 3B. Endosomes and lysosomes were labeled with lysotracker, cell membranes were labeled with WGA-AF 594, and cell nuclei were labeled with BoecBst 33342. At pre-set time points, the images were taken and analyzed using a laser confocal scanning microscope; the results are shown in Figure 2. Figure 5 , Figure 6 and Figure 7 shown.

[0187] Depend on Figure 5 , Figure 6 and Figure 7It can be seen that the grafted drug structure molecules SMDC-1070 (prostate-specific membrane antigen), SMDC-606 (folate receptor), and SMDC-274 (carbonic anhydrase IX) can bind well to the receptors on the cell membrane surface and internalize into the cell over time. Carbonic anhydrase IB is a non-internalizing receptor, and no obvious internalization process was observed.

[0188] 3. In vitro Antitumor Activity Analysis

[0189] Tumor cells in the logarithmic growth cycle were digested with trypsin to prepare a single-cell suspension and the cell density was adjusted to 1×10 5 / mL, inoculated into 96-well plates, set up 3 replicates, and placed in a cell culture incubator for overnight culture. After the cells adhered to the wall and stretched, the original culture medium was discarded. The blank control group: normal serum culture medium. The experimental group: the drug group diluted with normal serum culture medium. After acting on 72B, the cell viability was measured at a wavelength of 450nm according to the CCK 8 standard operating procedure; the results are shown in Figure 8 shown.

[0190] Figure 8 In A, compared with MMAF, the cytotoxicity to target cells was significantly enhanced after small molecule ligand grafting. Figure 8 In B, compared with PSMA receptor-negative PC 3 cells, eADC-1070 has a significant killing effect in receptor-positive 22Rv1 and LNcaP cells, and has obvious selectivity. Figure 8 It can be seen that the grafted drug structure molecules SMDC-1070 (prostate-specific membrane antigen), SMDC-606 (folate receptor), and SMDC-274 (carbonic anhydrase IX) can significantly inhibit the growth of tumor cells. This phenomenon was not observed in receptor-negative cells, indicating the anti-tumor potential in subsequent animal experiments.

[0191] 4. In vivo Antitumor Activity Analysis

[0192] Trypsinized 22Rv1 or SKRC-52 or KB cells were washed twice with RPMI 1640 serum-free medium, and then resuspended in a 1:1 mixture of RPMI 1640 and Matrigel. The resuspended cells were subcutaneously injected into 6-8 week old male BALB / c-nu mice (CBarles River). When the tumor size reached about 100 mm 3 When the tumor size exceeded 1000 mm, the drug was administered by tail vein according to the dose and frequency according to the experimental purpose. The weight and tumor size of the mice were monitored and recorded every 2 days. 3 When Fig. 9 shown.

[0193] Depend on Fig. 9 It can be seen that after a single dose, compared with the PBS control group and the SMDC group, the drug structure molecule grafted with the antibody Fc domain showed a significant anti-tumor effect and could inhibit the growth of solid tumors for a long time.

[0194] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0195] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0196] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations. Sequence Listing <110> Wuhan University <120> A targeted drug based on antibody IgG1-Fc domain and its preparation method and application <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 235 <212> PRT <213> Artificial Sequence <400> 1 Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 1 5 10 15 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 20 25 30 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 35 40 45 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 50 55 60 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 65 70 75 80 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 85 90 95 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 100 105 110 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 115 120 125 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 130 135 140 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 145 150 155 160 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 165 170 175 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 180 185 190 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 195 200 205 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 210 215 220 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 235 <210> 2 <211> 262 <212> PRT <213> Artificial Sequence <400> 2 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Leu Pro Glu Thr Gly Gly Ser Glu Pro 20 25 30 Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 35 40 45 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 50 55 60 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 65 70 75 80 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 85 90 95 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 100 105 110 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 115 120 125 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 130 135 140 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 145 150 155 160 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 165 170 175 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 180 185 190 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 195 200 205 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 210 215 220 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 225 230 235 240 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 245 250 255 Ser Leu Ser Pro Gly Lys 260 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <400> 3 aaaggatccg aaccgaaaag ctgcg 25 <210> 4 <211> twenty four <212> DNA <213> Artificial Sequence <400> 4 gtggtgctcg agttatttgc ccgg 24 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <400> 5 ctagcctgcc ggaaaccggt ggcggtggtg 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <400> 6 gatccaccac cgccaccggtttccggcagg 30

Claims

1. A tumor-targeting protein-drug conjugate based on the antibody IgG1-Fc domain, characterized in that: The structural formula of the tumor targeting drug-Fc protein conjugate is as follows: Wherein, the amino acid sequence of the IgG1 Fc is shown in SEQ ID NO.

1.

2. A method for preparing the tumor-targeting protein-drug conjugate according to claim 1, characterized in that: The method comprises: In PBS buffer, His-Fc was mixed and incubated with SortaseA enzyme at 1 mM di-Gly, to obtain IgG1 Fc. The reaction equation is as follows: In TBS buffer solution, the pseudopeptide intermediate and the IgG1 Fc protein are subjected to transpeptide coupling reaction under the catalysis of SortaseA, and then a small molecule targeting ligand is added for click chemical grafting to obtain a tumor-targeted protein drug conjugate based on the antibody IgG1-Fc domain, wherein the amino acid sequence of the His-Fc is shown in SEQ ID NO.2; the pseudopeptide intermediate includes any of the following compounds:

3. Use of the tumor-targeting protein-drug conjugate according to claim 1 in the preparation of a drug for treating prostate cancer with high expression of folate receptor or prostate-specific membrane antigen receptor.

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