CN109439665B

CN109439665B - Aptamer drug conjugate capable of being combined with CD133 protein in targeted mode and application thereof

Info

Publication number: CN109439665B
Application number: CN201811484364.6A
Authority: CN
Inventors: 葛明华; 朱栩杭; 那仁满都拉; 黄萍; 杨畅; 张轶雯; 李清林; 朱欣; 张启弘; 钱杨洋
Original assignee: Zhejiang Cancer Hospital
Current assignee: Zhejiang Cancer Hospital
Priority date: 2018-12-06
Filing date: 2018-12-06
Publication date: 2021-04-02
Anticipated expiration: 2038-12-06
Also published as: CN109439665A

Abstract

The invention provides an aptamer drug conjugate combined with CD133 protein in a targeted manner and application thereof, wherein the sequence of the aptamer is shown as SEQ ID NO.1, the aptamer combined with the CD133 protein in the targeted manner is screened from a ssDNA library by adopting a cell screening method, and the aptamer AP-1-M is selected. In one scheme of the invention, the classical chemotherapy drug adriamycin (DOX) and AP-1-M are connected by using a physical mosaic mode to obtain the aptamer-cytotoxic conjugate (AP-1-M-Dox) capable of being combined with ATC in a targeted manner and explore the cytotoxic effect of the conjugate, so that a better target is provided for clinically screening and researching novel targeted drugs, and a new method and thought are provided for clinically treating ATC.

Description

Aptamer drug conjugate capable of being combined with CD133 protein in targeted mode and application thereof

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to a nucleic acid aptamer-cytotoxic conjugate in targeted combination with CD133 protein.

Background

The CD133 protein is a member of cell membrane protein superfamily, contains 865 amino acids, glycoprotein with the molecular weight of about 120kDa, and the molecular structure thereof comprises: an extracellular N-terminus, 2 extracellular Loop loops, a 5-transmembrane domain, a 59 amino acid cellular C-terminus and 8N-glycosylated termini. The predicted size of the CD133 protein is 97kDa, but the actual molecular weight of glycosylated CD133 is 120 kDa. In recent years, researchers find that the CD133 protein exists on the surfaces of various stem cell-like tumor cells, including liver cancer, colon cancer and ovarian cancer, and provide a good specific target for clinically screening and researching novel drugs, thereby providing a thought for developing safer and more effective molecular targeted drugs. In addition, the expression of the CD133 protein in undifferentiated thyroid carcinoma (ATC) has also been reported, which suggests that the membrane protein CD133 is expected to be one of the binding targets of aptamers for ATC targeted therapy.

The aptamer, also called chemical antibody, has precise targeting property and is composed of single-chain ribonucleotide or deoxyribonucleotide. Although most of the aptamers have no treatment effect, the aptamers can be connected with toxic drugs to form a targeted aptamer conjugate, and the drugs can be accurately delivered to target cells, so that the aptamer conjugate drugs are endocytosed by the target cells, and the drugs are released inside the cells to induce apoptosis, thereby effectively improving the drug concentration of local tumors, greatly reducing the toxicity of other tissues and organs in vivo, and achieving the effect of real targeted synergism and attenuation. In addition, aptamers also have the following advantages: 1. in vitro screening, rapid artificial synthesis can be realized; 2. the target types are wide, and proteins or complete cells and the like can be used as screening targets; 3. the molecular weight is not large, no immunoreaction occurs, the molecular weight is easy to be specifically combined with target molecules, the affinity is strong, and the dissociation constant can reach pmol to nmol; 4. good chemical stability, reversible denaturation and renaturation, easy room temperature transportation, long-term storage and the like.

Thyroid cancer can be classified into papillary carcinoma, follicular carcinoma, Hurthle Cell carcinoma, medullary carcinoma and undifferentiated carcinoma according to pathological typing, wherein undifferentiated thyroid cancer (ATC) is a highly malignant tumor with extremely poor prognosis, median survival of about 3-7 months and annual survival rate of about 10%. Although ATC accounts for only 2% of thyroid malignant tumors, the absolute number of ATC patients is not low due to the huge number of thyroid cancer patients in China. In addition, the number of patients who die due to ATC accounts for about 14% -39% of the total number of thyroid tumor deaths, and is one of the most fatal malignant tumors in humans, and conventional treatment methods such as surgery, radiotherapy and 131I are almost ineffective against ATC. Therefore, how to improve and improve the treatment effect of such patients and prolong the median survival time of patients has been a difficult point and a hotspot in the ATC research.

At present, aptamer-conjugated drugs are one of the main research directions for macromolecular targeted therapy of malignant tumors, and are gradually the hot spots of research in recent years. However, related researches on aptamers and cytotoxic conjugates thereof against thyroid cancer are few, and especially, researches on aptamers and cytotoxic conjugates related to ATC have not been reported.

Disclosure of Invention

The invention utilizes the methods of RT-PCR, immunoblotting, laser Confocal microscope (Confocal microscope) and the like to confirm that the CD133 protein is expressed in undifferentiated thyroid cancer cells and is expressed on cell membranes, while normal thyroid cells do not express the CD133 protein, which indicates that the CD133 protein can be used as one of the binding targets of ATC.

Then, the inventors screen an aptamer which is targeted to bind to the CD133 protein from a ssDNA library by using a cell screening method, detect the screened ssDNA by using a high-throughput sequencing method, and finally select the aptamer AP-1-M, namely the aptamer of the invention, by analyzing the stability and affinity of the obtained sequence by using methods such as flow cytometry and the like. Then, an immunofluorescence method proves that the aptamer AP-1-M can be specifically targeted and combined with the CD133 protein and is combined with ATC cells expressing the CD133 protein to play a role in targeted delivery.

In one scheme of the invention, the classical chemotherapy drug adriamycin (DOX) and AP-1-M are connected by using a physical mosaic mode to obtain the aptamer-cytotoxic conjugate (AP-1-M-Dox) capable of being combined with ATC in a targeted manner and explore the cytotoxic effect of the conjugate, so that a better target is provided for clinically screening and researching novel targeted drugs, and a new method and thought are provided for clinically treating ATC.

According to one aspect of the present invention, there is provided an aptamer targeted to bind to CD133 protein, wherein the sequence of the aptamer targeted to bind to CD133 protein is as shown in SEQ ID No.1 in the sequence table, and the sequence is: 5'-TACCAGCCGTTTCCCCGGAGGGTCACCCCTGACGCATTCGGTTGAC-3' are provided.

According to one aspect of the invention, the application of the aptamer which is targeted and combined with the CD133 protein is provided, and the aptamer is used as a CD133 positive tumor chemotherapy targeting drug carrier.

According to one aspect of the invention, an aptamer drug conjugate which is targeted to bind to a CD133 protein is provided, and the aptamer drug conjugate which is targeted to bind to the CD133 protein is a conjugate of an aptamer with a sequence shown as SEQ ID NO.1 and a cytotoxic drug.

According to one aspect of the present invention, there is provided an aptamer drug conjugate which is targeted to bind to a CD133 protein, wherein the aptamer and a cytotoxic drug are conjugated by attaching a GC-rich base pair to the 5' -end of the aptamer to thereby conjugate the cytotoxic drug.

According to one aspect of the invention, an aptamer drug conjugate which is targeted to bind to a CD133 protein is provided, wherein the 5' end of an aptamer of the conjugate is connected with 8 pairs of GC base sequences.

According to one aspect of the present invention, there is provided an aptamer drug conjugate that binds to a CD133 protein in a targeted manner, wherein the cytotoxic drug is doxorubicin.

According to one aspect of the invention, the invention provides a use of an aptamer drug conjugate which is targeted to bind to a CD133 protein, and an application of any one of the aptamer drug conjugates which is targeted to bind to the CD133 protein in preparation of a preparation for treating CD133 positive tumor.

According to one aspect of the invention, a pharmaceutical preparation for targeted therapy of CD133 positive tumors is provided, which comprises any one of the aptamer drug conjugates for targeted binding to CD133 protein and a pharmaceutically acceptable carrier or excipient.

According to one aspect of the invention, a preparation method of an aptamer drug conjugate which is targeted to bind to CD133 protein is provided, wherein a long GC base sequence is connected to the 5' end of an aptamer, then the aptamer and a cytotoxic drug are incubated, the molar concentration ratio is 1:10, the optimal concentration ratio is adopted, and the aptamer drug conjugate (drug conjugate) -AP-1-M-Dox is obtained.

The aptamer which is targeted and combined with the CD133 protein is screened from the ssDNA library by adopting a cell screening method, the screened ssDNA is detected by a high-throughput sequencing method, and finally, the aptamer is selected by analyzing the stability and the affinity of the obtained sequence by methods such as flow cytometry and the like. Then, the aptamer is proved to be capable of specifically targeting and binding to the CD133 protein through an immunofluorescence method, and the aptamer is combined with ATC cells expressing the CD133 protein to play a role in targeted delivery. This provides new ideas and methods for the targeted therapy of ATC.

The aptamer targeted to bind to the CD133 protein has the following advantages: 1. the screening in vitro can be carried out quickly and artificially; 2. the molecular weight is not large, no immunoreaction occurs, the molecular weight is easy to be specifically combined with target molecules, the affinity is strong, and the dissociation constant can reach pmol to nmol; 3. good chemical stability, reversible denaturation and renaturation, easy room temperature transportation, long-term storage and the like.

One of the objects of the present invention is to provide an aptamer-cytotoxic conjugate AP-1-M-Dox that binds to the CD133 protein by targeting. The DNA sequence rich in GC base pairs can polymerize by itself to form a pocket structure, so that the adriamycin is physically embedded.

Based on the above, the AP-1-M-Dox provided by the invention is formed by connecting 8 pairs of GC base sequences to the 5' end of the AP-1-M, and the AP-1-M can directly carry cytotoxic drugs (such as adriamycin) and selectively kill ATC cells positive for CD 133. Thus, in a further aspect of the invention, there is provided the use of the AP-1-M-Dox of the invention in the preparation of a formulation for the treatment of a CD133 positive tumour (e.g. ATC).

One of the beneficial effects of the invention is that: the CD133 aptamers of the invention have: (1) selectively bind to tumor cells that are CD133 positive, but bind weakly to cells that are CD133 negative; (2) preferably, the cytotoxic drug doxorubicin can be directly carried, and selectively binds and kills CD133 positive tumor cells.

In one embodiment of the invention, aptamer AP-1-M is coupled with classical chemotherapy drug adriamycin to obtain AP-1-M-Dox and confirm that the AP-1-M has better cytotoxicity, and because aptamer AP-1-M can selectively bind to CD 133-positive tumor cells and has weaker binding to CD 133-negative cells, in another aspect of the invention, the aptamer is provided as a carrier of CD 133-positive tumor chemotherapy targeting drugs, the chemotherapy drugs are combined on the aptamer and can selectively kill CD 133-positive tumor cells without toxicity to normal cells when tumor radiotherapy and chemotherapy are carried out, the above-mentioned chemotherapy drugs (cytotoxic drugs) can be selected from metabolic drugs such as methotrexate, fluorouracil, floxuridine, gemcitabine and raltitrexed, anticancer antibiotic drugs such as mitomycin C, bleomycin, doxorubicin, epirubicin, pirarubicin, plant alkaloids such as vinblastine, paclitaxel, hydroxycamptothecin, antitumor hormones such as tamoxifen, letrozole, prednisone, heteroclasses such as cisplatin, carboplatin, mirtuxantrone, antitumor small molecule targeted drugs such as gefitinib, imatinib or lapatinib, more preferably, the above chemotherapeutic drugs are selected from fluorouracil, floxuridine, methotrexate, cisplatin, gemcitabine, raltitrexed, mitomycin, bleomycin, vinblastine, paclitaxel, hydroxycamptothecin, carboplatin, tamoxifen, letrozole, prednisone, gefitinib, imatinib or lapatinib.

Drawings

FIG. 1 is a graph showing the expression and localization of CD133 protein in different cell lines according to one embodiment of the present invention;

FIG. 2 is a specific detection map of aptamer AP-1-M according to one embodiment of the present invention;

FIG. 3 is a graph showing the detection of the binding of aptamer AP-1-M to FRO cells according to one embodiment of the present invention.

FIG. 4 is a schematic diagram of the coupling synthesis of aptamers AP-1-M and Dox according to one embodiment of the present invention;

FIG. 5 is a specific assay for the aptamer AP-1-M according to one embodiment of the present invention.

Detailed Description

The invention will be further described with reference to the accompanying drawings.

Example 1: expression and localization of CD133 protein in different cell lines

As shown in FIG. 1, Caco-2: colon adenocarcinoma cells; FRO: undifferentiated thyroid cancer cells; NTH: normal thyroid cells; 293T: human embryonic kidney cell line.

The expression of CD133 protein in different cell lines was observed by Confocal Microscopy (Confocal Microscopy) (FIG. 1), red fluorescence (i.e., bright spots in the four lower left micrographs in FIG. 1) represents the expression distribution of the membrane protein CD133 protein, and blue fluorescence (i.e., bright spots in the nuclei of each micrograph in FIG. 1) represents DAPI, indicating the cell nucleus. The results showed that CD133 protein was expressed on the cell membrane in FRO cells of undifferentiated thyroid cancer and Caco-2 cells of colon adenocarcinoma cells as a positive control group, while HEK-293T cells and normal thyroid cells Nthy-ori3-1(NTH) as a negative control group did not express CD133 protein.

Example 2: aptamer AP-1-M binds to CD133 positive and negative cells.

As shown in FIG. 2, AP-1-M aptamer was used to label AP-1-M aptamer by FITC, HEK-293T cells over-expressing CD133 protein were used as positive cells, wild-type HEK-293T cells were used as negative cells, AP-1-M aptamer, positive cells and negative cells were incubated at 4 ℃ and observed under confocal laser microscopy to find that AP-1-M and positive cells were combined and localized on the cell membrane (FIG. 2-A) and not combined with negative cells (FIG. 2-B), which also indicates that AP-1-M is targeted aptamer for CD133 protein.

The results showed that AP-1-M is an aptamer that specifically binds to CD133 protein, and that AP-1-M has a Kd value of 101.4nM as determined by flow cytometry for the affinity curve of the aptamer.

Example 3: binding of aptamer CD133 and undifferentiated thyroid cancer cell FRO.

As shown in FIG. 3, AP-1-M and FRO cells were incubated at 4 ℃ and 37 ℃ for 30 minutes, respectively, and it was found that AP-1-M and FRO cells were bound and localized on the cell membrane due to the inhibition of endocytosis of the cells at 4 ℃ (FIG. 3-A); whereas AP-1-M was engulfed intracellularly by FRO cells due to endocytosis of the cells at 37 deg.C (FIG. 3-B).

The result shows that the AP-1-M can be combined with the FRO cell, is positioned on the cell membrane and can be endocytosed into the cell so as to achieve the aim of targeted delivery.

Example 4: method for coupling aptamer AP-1-M with drug

As shown in FIG. 4, the DNA sequence rich in GC base pairs is preferably studied, and because the DNA sequence rich in GC base pairs can polymerize by itself to form a pocket structure, drugs containing loops, such as adriamycin, erythromycin and the like, are physically embedded, and the drug loading of the Aptamer is detected and calculated by utilizing the characteristic that the adriamycin has a fluorescence spectrum of itself and is quenched in the GC base pairs.

Firstly connecting 8 pairs of GC base sequences to the 5' end of AP-1-M, then incubating AP-1-M and Dox in different concentration ratios, after incubating for 2 hours, comparing the absorbance at 590nM by using a spectrophotometer, finally determining 1:10 as the optimal concentration ratio, and obtaining the aptamer drug couplet-AP-1-M-Dox.

Based on this, the AP-1-M-Dox of the invention can be prepared into preparations for treating CD133 positive tumors (such as ATC).

Example 5: drug toxicity effect of AP-1-M-Dox on normal thyroid cells and undifferentiated thyroid cancer cells

FIG. 5 shows a specific assay diagram of aptamer AP-1-M (A: normal thyroid cell Nthy-ori 3-1; B: undifferentiated thyroid cancer cell: FRO)

Respectively taking a normal thyroid cell strain Nthy-ori3-1(NTH) and an undifferentiated thyroid (ATC) cell strain FRO to pave in a 96-well plate with 5000 per well (90 ul of RPMI-1640 culture solution containing 10% fetal bovine serum); NTH cell strain is control group A, FRO cell strain is experimental group B, and is divided into group A1 (AP-1-M-Dox treatment group) and group A2 (Dox treatment group), group B1 (AP-1-M-Dox treatment group) and group B2 (Dox treatment group) according to different cell treatment modes; then adding adriamycin (Dox) or AP-1-M-Dox (10 uL/well) with different concentrations into cells in different wells of each group, wherein 9 groups are provided, the first group of cells are not treated and are used as negative control, and the concentration of the rest 8 groups is sequentially increased by 2 times, the minimum concentration is about 0.016umol/mL, and the maximum concentration is 50 umol/mL; culturing in a saturated water vapor carbon dioxide incubator with 5% CO2 at 37 deg.C for 3 hr, and changing the culture solution of all the cells in the wells after 3 hr (90 ul of RPMI-1640 culture solution containing 10% fetal calf serum); and finally culturing in an incubator for 48 hours, adding 10 mu L of CCK into each hole, culturing in the incubator for 3 hours, and measuring the OD value of 450nm by using an enzyme-labeling instrument.

The result shows that AP-1-M-Dox has good cytotoxicity on the FRO of ATC cells, but the toxicity is slightly weaker than Dox, and the toxicity is obviously weaker than Dox on NTH of normal thyroid cells which do not express CD 133.

Sequence listing

<110> Zhejiang province tumor hospital

<120> aptamer drug conjugate for targeted binding to CD133 protein and application thereof

<130> CN-CN-2018-1031-1

<141> 2018-12-06

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 1

taccagccgt ttccccggag ggtcacccct gacgcattcg gttgac 46

Claims

1. a nucleic acid aptamer drug conjugate for targeting and binding to CD133 protein, characterized in that the nucleic acid aptamer drug conjugate for targeting and binding to CD133 protein is the sequence of SEQ ID NO.1 Conjugates of nucleic acid aptamers and cytotoxic drugs.

2. A nucleic acid aptamer drug conjugate targeting and binding to CD133 protein according to claim 1, wherein the coupling mode of the nucleic acid aptamer and the cytotoxic drug is by adding the nucleic acid The 5' end of the aptamer is linked to a GC-rich base pair for conjugation of cytotoxic drugs.

3. the nucleic acid aptamer drug conjugate of a kind of targeting binding CD133 protein according to claim 1, is characterized in that, described conjugate nucleic acid aptamer 5 ' end is connected with 8 pairs of GC bases sequence.

4 . The nucleic acid aptamer drug conjugate targeting and binding to CD133 protein according to claim 1 , wherein the cytotoxic drug is doxorubicin. 5 .

5. Use of a nucleic acid aptamer-drug conjugate for targeting and binding to CD133 protein, wherein the nucleic acid aptamer-drug conjugate for targeting and binding to CD133 protein according to any one of claims 2 to 4 Application in the preparation of tumor preparations for the treatment of CD133 positive.

6. A tumor drug preparation for targeted therapy CD133-positive, characterized in that, the preparation contains the nucleic acid aptamer drug conjugate that targets and binds to CD133 protein according to any one of claims 2 to 4 and a pharmaceutically acceptable drug conjugate. acceptable carrier or excipient.

7. a preparation method of the nucleic acid aptamer drug conjugate of targeting binding CD133 protein, it is characterized in that, at first the 5 ' end of nucleic acid aptamer is connected with a long GC base sequence, and then all The nucleic acid aptamer and the cytotoxic drug were incubated with a concentration ratio of 1:10 to obtain the aptamer drug conjugate, namely AP-1-M-Dox. The nucleic acid aptamer sequence is shown in the sequence table. SEQ ID NO. 1.