CN116769051A - Topological structure star-shaped protein based on spy reaction and drug conjugate thereof - Google Patents

Topological structure star-shaped protein based on spy reaction and drug conjugate thereof Download PDF

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CN116769051A
CN116769051A CN202310405909.4A CN202310405909A CN116769051A CN 116769051 A CN116769051 A CN 116769051A CN 202310405909 A CN202310405909 A CN 202310405909A CN 116769051 A CN116769051 A CN 116769051A
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protein
antibody
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蒋锡群
蒋蕙忆
陈伟芝
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Nanjing University
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Abstract

The invention provides a topological structure star protein based on a spyware reaction, which is formed by connecting protein 1 and protein 2 through an isopeptide bond by the spyware reaction, wherein the protein 1 comprises an EGFR antibody or antigen binding fragment thereof, a polypeptide with an RGD motif and a spyware tag, and the protein 2 comprises an HER2 antibody or antigen binding fragment thereof and a spyware. The invention further discloses a multi-target drug coupling body prepared from the topological structure star-shaped protein. The drug conjugate has good targeting ability with different degrees, can be concentrated to tumor parts more, has longer residence time at the tumor parts, has excellent tumor inhibition and treatment ability, and effectively improves tumor inhibition and treatment efficiency compared with common targeting drugs.

Description

Topological structure star-shaped protein based on spy reaction and drug conjugate thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to star-shaped protein with a topological structure for tumor inhibition and treatment and a drug coupling body thereof.
Background
With the development of molecular biology research, strategies for developing novel antitumor drugs for cancer chemotherapy have changed significantly. Molecular targeted therapies represent an attempt to achieve antitumor effects by selectively altering the differences in biological characteristics between normal cells and cancer cells or between normal tissue and cancer tissue. Many targeted drugs can specifically inhibit cancer pathways in tumor cells or the environment or key molecules associated with tumor growth, progression, apoptosis, angiogenesis and metastasis, and also produce good therapeutic effects in patients. Recent technological advances in pharmacogenomics and proteomics have improved the identification of biomarkers for predicted responses, thereby identifying patients more likely to respond to such treatments. However, despite the great progress made by some of the new compounds and the new therapeutic approaches that have been put into clinical use for cancer patients, the cure rates remain low, especially in specific patient treatment cases, and the types of these new molecular targets still require constant confirmation and personalized customization. To address the differences in targets for different tumor types, as well as target failure issues, multi-targeting molecular targeted therapies are beginning to move into the field of view. Recently, a number of molecular targeted therapeutic agents have been developed that act on different targets. From the drug development process, these drugs are referred to as molecular targeted drugs, but do not have to act on any particular target alone, i.e. they selectively modify several targets. Since the signal transduction system consists of a complex network, multi-targeted molecules may be involved in inhibiting tumor growth by simultaneously blocking multiple pathways involved.
The EGFR family has important significance in the growth and transfer process of tumors and is also an important target point of anti-tumor drugs. Antibody drugs, antibody drug conjugates and small molecule inhibitors targeting human epidermal growth factor receptors 1 and 2 have been marketed in a number of products, such as cetuximab, trastuzumab, T-DM-1, gefitinib, afatinib, dacatinib, pyrroltinib, etc., with great success in the treatment of tumors, particularly breast cancer. Drug resistance of targeted drugs remains a challenge to be addressed. There are a number of mechanisms of drug resistance of targeted drugs, of which activation of the bypass signal is an important aspect. Numerous studies have shown that there is an intricate interaction between the signaling pathways of epidermal growth factor receptors 1 and 2 and the integrin pathway. After one receptor is inhibited, the other receptor may be activated, thereby inducing continued activation of downstream signals. Therefore, simultaneous targeted inhibition of multiple receptor targets with interactions can maximally inhibit the associated signaling pathway, ultimately maximizing the anti-tumor effect. How to effectively fuse multiple targeting motifs into the same molecule is a great challenge, especially for large-sized antibody drugs. More importantly, there are currently no multispecific antibody drug conjugate products on the market.
The preparation of the traditional multi-specific antibody requires higher technical barriers and complex process conditions, and the formation of the recombinant protein by connecting the targeting motifs in series may affect the biological activity of the targeting motifs, so the invention proposes that the multi-specific recombinant protein constructing a star topology can be combined with a plurality of tumor targets simultaneously. In space, the influence of steric hindrance on each targeting element is reduced to the greatest extent, more opportunities are created for the binding of targets, and the better binding of targets achieves the effect of inhibiting related signal paths. The multi-specificity recombinant protein drug conjugate body with a star topology structure is further constructed, and more drugs are delivered into tumor tissues through simultaneous combination of multiple targets.
Disclosure of Invention
The spy reaction is a reaction process in which an isopeptide bond spontaneously forms between a spy tag (SpyTag) and a SpyCatcher (SpyCatcher). The two components of the reaction are completely based on natural amino acid units, and chemical reactivity can be directly written into the sequence of the protein in a gene coding mode, so that the method has wide prospects in the aspects of protein engineering, biological materials, chemical biology, synthetic biology and the like.
The invention is based on the technical conception of the spy reaction, respectively designs two polypeptides with multiple targets containing the spy tag and the spy catcher, and forms an isopeptide bond (a peptide bond formed by two amino acids through side chain carboxyl or side chain amino) through the amino acid residues in the spy tag (spyTag) and the spy catcher (SpyCatcher) sequences, so that a topological star protein (spy tag-spy catcher complex) is obtained, the topological star protein can identify multiple targets, has strong targeting property, can be concentrated to tumor sites more, has longer stay time at the tumor sites, has excellent tumor inhibition and treatment capability, and effectively improves tumor inhibition and treatment efficiency compared with common targeting drugs.
The specific technical scheme of the invention is as follows:
a topologically star protein linked by an isopeptide bond by a spy reaction from protein 1 and protein 2, said protein 1 comprising an EGFR antibody or antigen binding fragment thereof, a polypeptide having an RGD motif and a spy tag, said protein 2 comprising a HER2 antibody or antigen binding fragment thereof and a spy trap.
In the technical scheme of the invention, EGFR antibody or antigen binding fragment thereof, protein with RGD motif and HER2 antibody or antigen binding fragment thereof are tumor targeting functional fragments, so that star protein can target three targets of EGFR, HER2 and integrin simultaneously. The spyware tag and spyware capture sequences are functional fragments that enable the attachment of protein 1 and protein 2.
The EGFR antibody or antigen binding fragment thereof, the polypeptide with the RGD motif and the spy tag in protein 1 may be arranged in any order, either by a linker peptide or directly.
The HER2 antibody or antigen binding fragment thereof and the spyware in protein 2 may be arranged in any order, either by a linker peptide or directly.
The invention adopts a specific mode that: protein 1 forms peptide chains by sequentially connecting EGFR antibody antigen binding fragments, connecting peptides, spy labels, connecting peptides and iRGD peptide chains.
Protein 2 was linked in sequence with HER2 antibody antigen binding fragment, linker peptide, spyware to form a peptide chain.
Mixing the protein 1 and the protein 2 according to a certain proportion (preferably, the molar ratio is 1:1) to obtain the topological structure star protein.
The EGFR antibody or antigen binding fragment thereof, the polypeptide having an RGD motif and the HER2 antibody or antigen binding fragment thereof may be all functional fragments capable of achieving tumor targeting disclosed in the art, the antibodies may be in the form of IgG1, igG2, igG3 or IgG4, and the antigen binding fragment may be one or more of Fab, fab ', (Fab') 2, fv, scFv, VH. Preferably a human antibody or antigen binding fragment.
The EGFR antibody of the present invention may be any EGFR antibody obtained by the prior art, including but not limited to anti-EGFR monoclonal antibodies, anti-EGFR single chain antibodies, anti-EGFR nanobodies, and the like.
The HER2 antibody of the present invention may be any HER2 antibody obtained by the prior art, including but not limited to anti-HER 2 mab, anti-HER 2 single chain antibodies, anti-HER 2 nanobodies, etc.
In a specific example of the invention, the amino acid sequence of the EGFR antibody is shown in SEQ ID No. 3, and the amino acid sequence of the HER2 antibody is shown in SEQ ID No. 4.
Polypeptides having RGD motifs of the present invention include, but are not limited to RGD, cRGD, iRGD, phenylalanine-linked RGD peptides (F-RGD), serine-linked RGD peptides (RGD-S), glutamate-linked RGD peptides (G-RGD). According to a preferred technical scheme, the polypeptide containing the RGD motif is iRGD, and the amino acid sequence is shown in SEQ ID No: shown at 7.
The connecting peptide (GGGGS) of the invention n N represents an integer of 1 to 50, preferably n is 2 or 3.
The protein 1 and/or protein 2 of the present invention may further comprise a linker peptide or elastin-like, which modulates the distance between functional fragments or introduces a modifiable site. The elastin has repeating units consisting of VPGXG, wherein X in each repeating unit is the same or different, X is any amino acid except proline, at least one of the repeating units is lysine, and the number of the repeating units of VPGXG in the elastin is 2-50. Preferably, X is selected from V, P, G or K, preferably the number of repeating units of VPGXG is preferably 20.
The spy tag and spy predator of the present invention were first disclosed in 2012 by Bijan zakri et al (Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin, PANS) to find that the streptococcus pyogenes fibronectin binding protein FbaB comprises a domain with a spontaneous isopeptidic bond between lysine and aspartic acid. By splitting this domain and fragment, a peptide (SpyTag) is obtained which forms an amide bond with its protein partner (SpyCatcher) within a few minutes, while both peptide chains do not cause any toxicity to the human body. The original spy-tag and spy-catcher sequence are therefore used in the present invention for the construction of the star topology.
In a specific example of the present invention, the amino acid sequence of the SPYTAG (SPYTAG) is as shown in SEQ ID No:1, wherein the amino acid sequence of the Spyware (SPYCATCHER) is shown as SEQ ID No: 2.
In a specific example of the present invention, the amino acid sequence of the protein 1 (EHI) is shown in SEQ ID No:5, the amino acid sequence of the protein 2 (HSC) is shown in SEQ ID No: shown at 6.
The invention also aims at providing a multi-target drug coupling body, and the star-shaped protein is coupled with a drug. The coupling mode is selected from one or more of lysine residue coupling, disulfide bond reduction cysteine coupling, non-amino acid coupling, catalytic coupling, coupling through transpeptidation mediated transpeptidation, coupling through MTG mediated transpeptidation, and coupling mode through modification of N-glycan on an aspartic acid residue of an antibody.
The medicine provided by the invention has or is modified to have one or more groups of sulfhydryl, hydroxyl, carbonyl, carboxyl and amino, and is coupled with star-shaped protein through one or more of disulfide bonds, hydrazone bonds, carbonic ester bonds, amide bonds and hydrogen bonds.
In a specific example of the present invention, the drug is an anticancer drug such as camptothecin, hydroxycamptothecin, topotecan or irinotecan.
The invention also aims to provide the application of the star-shaped protein or the multi-targeting drug conjugate in preparing the drug for treating the cancers. Preferably, the cancer is selected from colorectal cancer, lung cancer, liver cancer, breast cancer or pancreatic cancer.
In one specific example of the present invention, 7-ethyl-10-hydroxycamptothecin (SN 38) multi-targeted drug conjugates are provided.
The invention firstly constructs recombinant DNA molecules of protein 1 and protein 2 by PCR, carries out escherichia coli transformation, expresses protein purification, and mutually reacts the protein 1 and the protein 2 to obtain the multi-target antibody protein (S) with a star-shaped topological structure.
The SN38 end groups were then protected with di-tert-butyl dicarbonate to give Boc-SN38. Boc-SN38 and carboxy-PEG-azide (N 3 -PEG-COOH) to give N 3 PEG-SN38-Boc, N after deprotection of Boc 3 The PEG-SN38 reacts with propargyl maleimide to obtain MAL-PEG-SN38 with maleimide terminal group, and the structural formula of the final product is as follows:
the MAL-PEG-SN38 is coupled with sulfhydryl groups on amino acid residues of a multi-target antibody protein (S) with a star topology structure through an addition reaction, so that a target conjugate (SD) can be obtained, and the structural diagram is as follows:
the specific steps for preparing the multi-targeted star protein drug conjugate are as follows:
a. designing DNA sequences for encoding protein 1 and protein 2, and fusing several functional polypeptide fragments and protein at gene level by utilizing PCR technology to make DNA fragments pass through gene recombination so as to change original gene encoding of plasmid.
b. The DNA sequences encoding protein 1 and protein 2 (SEQ ID No:10, SEQ ID
No. 11) was introduced into the expression host Ecoli.BL21 (DE 3), and after single colony selection, the monoclonal strain was stored in 15% glycerol in a freezer at-80 ℃.
c. After the amplification culture, the escherichia coli is collected, broken by ultrasonic waves, the supernatant is centrifugally taken, the supernatant is purified by a nickel column, the nano antibody is eluted into 300mM imidazole, and the solution is dialyzed into PBS for standby.
d. Protein 1 and protein 2 obtained by the method are mixed according to the equivalent weight of 1:1, and stirred for 2 hours at 4 ℃ to finally obtain the multi-targeted star protein S.
e. Adding 7-ethyl-10-hydroxycamptothecin and di-tert-butyl dicarbonate into a round bottom flask, adding a small amount of dichloromethane and pyridine for dissolution, stirring for 3 hours at 25 ℃, and then washing, drying, filtering and rotary steaming to obtain a yellow solid compound Boc-SN38, wherein the structure is as follows:
f. the compound Boc-SN38 was reacted with carboxy-PEG-azide (N 3 -PEG-COOH) was dissolved in anhydrous dichloromethane and N, N' -diisopropylcarbodiimide and 4-dimethylaminopyridine were added thereto and stirred at 25 ℃ for more than 12h. Separating by size exclusion column to obtain transparent glassy solid compound N 3 -PEG-SN38-Boc having the structural formula:
g. compound N 3 Adding a small amount of trifluoroacetic acid into PEG-SN38-Boc, stirring for more than 4 hours at room temperature, and regulating pH after rotary evaporation to obtain PEG-SN38, wherein the structural formula is as follows:
h. adding PEG-SN38 and propargyl maleimide into N-formyldimethylamine for dissolution, then adding a small amount of cuprous iodide and pentamethyldiethylenetriamine, repeatedly vacuumizing, introducing nitrogen, injecting a small amount of ascorbic acid, stirring for 18h at 40 ℃, after post-treatment, purifying by a size exclusion column, and finally obtaining MAL-PEG with maleimide group
SN38, having the structural formula:
i. MAL-PEG-SN38 and multi-targeting protein S are mixed in PBS with PH=7.4, a small amount of TCEP and EDTA are added, the mixture is slowly stirred at 4 ℃ overnight, and the mixture is purified and separated by a dextran column, so that the target conjugate is finally obtained.
The star-shaped multi-targeting antibody drug conjugate body is a trispecific antibody fusion protein with EGFR, HER2 and integrin multi-targeting function, and can target a plurality of high-expression target tumor lesions such as tumor cell surfaces, tumor vascular tissues and the like.
The invention has the advantages that:
(1) Compared with linear targeting proteins, star-shaped targeting proteins have better specific targeting ability and stronger uptake ability on various cancer cells such as MCF-7, heLa, A549 and the like.
(2) The two components of the star protein have better expression efficiency in protein expression efficiency.
(3) The biocompatibility of the SN38 anticancer drug is improved.
(4) The star-shaped structure avoids the problem of steric hindrance of a linear result, and can keep the binding activities of different targets to the greatest extent.
The invention solves the problems of target point difference of different tumor types and target point failure existing in the prior art. By adding targets and changing the topological structure of protein peptide chains, the antibody protein can infiltrate tumor cells better and target focus.
Drawings
Fig. 1: SDS-PAGE electrophoresis of multi-targeted proteins of different topologies.
Fig. 2: high performance liquid chromatograms of SN38 and PEG-SN38.
Fig. 3: UV absorbance profile of protein drug conjugates.
Fig. 4: confocal microscopy imaging of star-type and linear multi-targeting proteins co-cultured with a549 cells.
Fig. 5: cytotoxicity of multi-targeted protein drug conjugates of different topologies.
Fig. 6: antitumor effect of multi-targeting protein drug conjugates of different topologies.
Detailed Description
The following examples further illustrate the invention but do not limit the scope of the invention.
Example 1: preparation of multi-targeted star protein.
a. The expression plasmids of protein 1 and protein 2 of star-shaped protein which form a topological structure are constructed, the amino acid sequence of protein 1EGFR-Spy tag-iRGD (ETI) is shown as SEQ ID No. 5, the amino acid sequence of protein 2HER2-Spy catcher (HSC) is shown as SEQ ID No. 6, and the gene vector pET28a+ and the expression host Ecoli.BL21 (DE 3) are shown.
b.2 mu L of the constructed plasmid is respectively taken and dissolved in an elision Buffer to blow 50 mu L of BL21 competent cells, the competent cells are placed on ice for incubation for more than 5min, and then the competent cells are taken out and placed in a water bath at 42 ℃ for 90s. Competent cells were then again incubated in ice for more than 5 min. Sucking out the well-incubated competent cells, pumping the cells into an SOC culture medium, and culturing the cells in a 200rmp shaking incubator at 37 ℃ for 1h. The cultured competent cells were then plated on a plate with a resistance to Carna, and single colonies were picked up in 5mL LB medium after shaking at 37℃for 12 hours, and amplified by overnight culture. Finally, the strain is preserved as protein expression bacterial liquid.
c. The stored bacterial liquid was picked up, and the bacterial liquid was inoculated into a shaking tube of 5mL of LB medium at an inoculum size of 0.1%, and cultured overnight. The bacteria in the rocker tube were inoculated into 400mL of TB medium at an inoculum size of 0.1%, and 0.001M of a Kana antibiotic was added thereto for 3 hours at 37℃and 200 rpm. After 3h, the OD600 value of the bacterial liquid is measured, and when the OD600 is 0.8, IPTG with the final concentration of 0.5M is added for induction expression. Then, the culture was continued at 18℃and 200rpm for 12 hours.
d. Centrifuging at 8500rmp and 4deg.C for 10min, collecting precipitate, discarding supernatant, washing with ultrapure water for one time, and collecting precipitate at 800rmp and 4deg.C for 10min. The submerged bacteria were resuspended with 20mL binding buffer (500mM NaCl,20mM PB,20mM imidazole,pH =7.4). The cells are broken up and intracellular proteins are released by ultrasonic breaking with ice bath at 400W for 2s under intermittent conditions of 5s for 30 min. Centrifuging at 13000rmp and 4deg.C for 30min, removing cell residue, and collecting supernatant.
e. The ability of the protein to bind with His tag and nickel column was purified using AKTA purification system. The sonicated supernatant was passed through a HisTrp 5mL column at a flow rate of 3mL/min, after washing with a bind buffer of 3 column volumes, a linear gradient elution was performed with a wash buffer, each peak was collected, SDS-PAGE was performed, and the product position collection was confirmed. The proteins removed to 300mM binding buffer were dialyzed into PBS for use.
EI-tag and H-C proteins were mixed at a molar ratio of 1:1, stirred at low speed overnight at 4℃and SDS-PAGE was performed to confirm successful synthesis of star protein S.
The amino acid sequences of the linear multi-targeting fusion proteins AntiHER2-anti EGFR-iRGD (HEI) and the anti EGFR-anti HER2-iRGD (EHI) prepared by the method are shown as SEQ ID No. 8 and 9.
The SDS electrophoresis results of the multi-targeted star protein and the linear protein are shown in FIG. 1. The results show that the molecular weights of the multi-targeted and linear proteins are consistent with the theoretical molecular weight, indicating successful preparation and binding.
Example 2: the preparation of maleimide end group PEG-SN38 prodrug molecules can be used for coupling proteins.
a. 1g of hydroxycamptothecin and 1g of di-tert-butyl dicarbonate were each dissolved in 100mL of anhydrous methylene chloride, and 5mL of anhydrous pyridine was added thereto. Stir at room temperature for 3h. Then, the mixture was washed three times with 0.5N diluted hydrochloric acid, washed with saturated sodium bicarbonate solution, extracted with dichloromethane, dried by adding a small amount of anhydrous sodium sulfate powder, and filtered to obtain Boc-SN38 as a yellow powder by rotary evaporation. The synthesis of Boc-SN38 was confirmed by thin layer chromatography.
b. 80mg of Boc-SN38 and 267mg of polyethyleneglycol with one end being azide and one end being carboxyl are added into anhydrous dichloromethane respectively, 48 mu L N, N' -diisopropylcarbodiimide and 30mg of 4-dimethylaminopyridine are added, and stirring is carried out at normal temperature for more than 12 hours. After the reaction, evaporating and drying the solvent in a rotary way, redissolving the solvent by using methanol, purifying the solvent by using a size exclusion column to obtain glassy transparent solid, and confirming that the reaction is successful to synthesize N by using mass spectrum 3 -PEG-BocSN38。
c. The obtained PEG(s)BocSN38 is dissolved in 1mL of trifluoroacetic acid, if the solution is turbid, a small amount of dichloromethane can be added to assist dissolution, and after complete dissolution is confirmed, the reaction is stirred at normal temperature for more than 4 hours. Then spin-evaporating the solvent, adjusting pH to neutrality, and spin-evaporating again to obtain N 3 PEG-SN38. Confirmation of N by thin layer chromatography 3 -PEG-SN38 generation.
d. 80mg N 3 PEG-SN38 and 4.32mg propargyl maleimide are added into 5 mLN-formyldimethylamine for dissolution, then 1.1mg of cuprous iodide and 4.7mg of pentamethyldiethylenetriamine are added into the mixture, the mixture is put into a Schlenk bottle, the mixture is repeatedly vacuumized and introduced with nitrogen for three times, then a small amount of ascorbic acid is injected, the mixture is stirred for 18 hours at 40 ℃, the mixture is evaporated and dried in a rotary manner after the reaction is finished, methanol is added for redissolution, the mixture is added into diethyl ether solution in a dropwise manner, and the mixture is centrifuged for 10 minutes at 4 ℃ by a floor centrifuge 8000r, and the supernatant is discarded. The methanol redissolved product was added and dried by rotary evaporation. Adding methanol again for redissolution, filtering, purifying by a size exclusion column, and finally obtaining MAL-PEG-SN38 with maleimide group. The completion of MAL-PEG-SN38 synthesis with maleimide group was confirmed by nuclear magnetic resonance spectroscopy.
High performance liquid chromatograms of SN38 and PEG-SN38 are shown in FIG. 2 (Wondasil C18,5um,4.6 x 150mm; mobile phase A0.1% TFA-H) 2 O, mobile phase B MeCN; elution gradient: 0min,95% A+5% B;15min 5% A+95% B; detection wavelength: SN 38-365/373 nm; PEG 214/220 nm), the result shows that the PEG was successfully linked to SN38, forming a peak at 365nm ultraviolet different from monomer SN38. As can be seen from FIG. 2, SN38 under HPLC analysis has a different retention time than PEG-SN38, indicating the formation of new species, i.e., successful synthesis.
Example 3: preparation of star-shaped multi-target antibody drug conjugate and linear multi-target antibody drug conjugate.
20mg of MAL-PEG-SN38 is added into 200 mu L of phosphate buffer solution which is pH7.8 and contains 25mM EDTA and 5mM TCEP, 10mL of phosphate buffer solution which contains 2mg/mL of star-type multi-target antibody protein S, linear double-target protein HEI or EHI is added into buffer solution with MAL-PEG-SN38 respectively, reaction is carried out at 4 ℃ overnight, ultrafiltration is carried out for five times by an ultrafiltration tube with a molecular weight cutoff 10000, and then the star-type multi-target antibody drug conjugate is obtained by dialysis for two days by using the phosphate buffer solution. The preparation of the linear multi-targeting antibody drug conjugate is as above.
The ultraviolet spectra of HEI-PEG-SN38, EHI-PEG-SN38, S-PEG-SN38 and PEG-SN38 are shown in figure 3, and the antibody drug conjugate has ultraviolet absorption peak at 368nm as the PEG-SN38, which proves that the protein and the drug are successfully conjugated.
Example 4: star-type multi-targeting protein and cell uptake condition research of linear multi-targeting protein.
a. Cells were 1X 10 per well 5 Density of individuals were seeded in 6-well plates, each well was covered with a coverslip at 5% CO 2 Is incubated overnight at 37 ℃.
200. Mu.L of rhodamine b labeled star protein S, HEI, EHI was added to the wells and subsequently incubated at 37℃for 24h.
c. The medium was aspirated, rinsed three times with PBS and fixed with 4% paraformaldehyde for 8-10min at room temperature.
After three washes with PBS, nuclei were stained with Hoechst 33258 for 8-10min at room temperature, washed with PBS, and coverslips were placed in PBS.
e. Protein cell entry was observed under a confocal laser microscope using a 63-fold oil microscope.
As shown in FIG. 4, the confocal laser fluorescence microscopy image shows that the cellular uptake of the star-shaped multi-target protein is obviously higher than that of other two linear multi-target proteins, and the cells adopt human alveolar adenocarcinoma basal epithelial cells A549.
Example 5:
in vitro cytotoxicity of the three protein drug conjugates was assessed using an MTT assay.
In this example, human lung cancer cell A549, human cervical cancer cell HeLa and human breast cancer cell MCF-7 were used. The cell culture was performed using DMEM plus 10% fetal bovine serum. Cells were seeded at a density of 5000 per well in 96-well plates and incubated overnight at 37 ℃ in a humid environment with 5% CO 2. The medium of the cells was replaced with serum-free medium and starved for 4h. The serum-free medium was then removed and replaced with medium containing different concentrations of protein drug conjugate (10,20,40,80,160,320. Mu.g/ml). A background well and a positive control well containing no protein and only cells were set simultaneously. After incubation in a humid environment of 5% CO2 for 24h at 37℃the medium was aspirated, fresh medium was added, while 20. Mu.L of MTT solution (5 mg/mL in PBS) was added and incubated for 4h at 37 ℃. The liquid in each well was aspirated and 150. Mu.L of DMSO was added. After 10min, absorbance at 490nm was measured for each well with a microplate reader.
Cell viability was calculated using the following formula:
cell viability (%) = a490 of test well cells/a 490 x 100% of control well cells
Wherein A490 of the test well cells and A490 of the control well cells are the values obtained by subtracting the absorbance of the background well, respectively.
It can be seen from FIG. 5 that tumor cells are generally more sensitive to protein drug conjugates with star topology (S-PEG-SN 38) than to protein drug conjugates with linear topology (EHI-PEG-SN 38). And this sensitivity is exhibited in all three tumor cells selected in this example.
Example 6:
constructing a mouse subcutaneous HeLa tumor model to study the anti-tumor effect of the star-shaped multi-target antibody drug conjugate and the linear multi-target antibody drug conjugate. The number of injections was 1X 10 by subcutaneous injection in nude mice 6 And (3) constructing a mouse tumor model. When the subcutaneous tumor of the mice is about 80mm after one week of injection 3 In size, antibody drug conjugates were injected into mice at 0.8mg/kg (as drug dosimeter) via the tail vein, once every 2 days, and the change in tumor volume of mice was studied after 6 consecutive injections. As shown in FIG. 6, the results show that the star-type multi-targeting antibody drug conjugate and the linear multi-targeting antibody drug conjugate both inhibit tumor growth to different degrees, wherein the inhibition effect of the star-type multi-targeting antibody drug conjugate is particularly obvious, even almost blocks tumors, particularly smaller tumors (100 mm) 3 The following). The star-shaped multi-target antibody drug conjugate body can obviously inhibit the growth of tumors and can be used for tumor treatment and blocking.

Claims (10)

1. A topologically star protein characterized by being connected by an isopeptide bond by a spiking reaction of protein 1 and protein 2, said protein 1 comprising an EGFR antibody or antigen binding fragment thereof, a polypeptide having an RGD motif, and a spiking tag, said protein 2 comprising a HER2 antibody or antigen binding fragment thereof, and a spiking catch.
2. The star protein of claim 1, wherein the spy tag has an amino acid sequence as set forth in SEQ ID No:1, wherein the amino acid sequence of the spyware is shown as SEQ ID No: 2.
3. The star protein according to claim 1, characterized in that the antibody is an antibody in the form of IgG1, igG2, igG3 or IgG4 and the antigen binding fragment is one or more of Fab, fab ', (Fab') 2, fv, scFv, VH.
4. The star protein of claim 1, wherein the EGFR antibody has the amino acid sequence set forth in SEQ ID No:3, the amino acid sequence of the HER2 antibody is shown as SEQ ID No: 4.
5. The star protein according to claim 1, wherein the protein 1 has the amino acid sequence shown in SEQ ID No:5, the amino acid sequence of the protein 2 is shown as SEQ ID No: shown at 6.
6. A multi-targeting antibody-drug conjugate, characterized in that the topologically star protein of any one of claims 1-5 is conjugated to a drug.
7. The multi-targeting antibody drug conjugate according to claim 6, wherein the conjugation means is selected from one or more of lysine residue conjugation, disulfide bond-changing reduced cysteine conjugation, non-amino acid conjugation, catalytic conjugation, conjugation through transpeptidation mediated transpeptidation, conjugation through MTG mediated transpeptidation, conjugation means through modification of N-glycan on an aspartic acid residue of an antibody, and drug conjugation to star protein through one or more of disulfide bond, hydrazone bond, carbonate, ester bond, amide bond, hydrogen bond.
8. The multi-targeted antibody drug conjugate of claim 7, characterized in that the drug is camptothecine, hydroxycamptothecin, topotecan or irinotecan.
9. Use of a star protein according to any one of claims 1-5 or a multi-targeting antibody drug conjugate according to claims 6-8 for the preparation of a medicament for the treatment of cancer.
10. Use according to claim 9, characterized in that the cancer is selected from colorectal cancer, lung cancer, breast cancer or pancreatic cancer.
CN202310405909.4A 2023-04-17 2023-04-17 Topological structure star-shaped protein based on spy reaction and drug conjugate thereof Pending CN116769051A (en)

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