CN106497931A - A kind of ssDNA aptamers and its application in diagnoses and treatment HER3 relevant diseases for Her3ECD - Google Patents

Abstract

The present invention discloses a DNA with high affinity and high selectivity for HER3 extracellular domain protein (extracellular domain, ECD) screened by systematic evolution of ligands by exponential enrichment (SELEX) aptamer. The present invention further proves that the aptamer has certain specificity and affinity for both free Her3ECD and breast cancer cells with high expression of Her3ECD protein through ELISA, cell flow cytometry and laser confocal experiments, and is related to the high expression of HER3. The diagnosis and treatment of diseases provides new ideas.

Description

A ssDNA aptamer targeting Her3ECD and its application in the diagnosis and treatment of HER3-related diseases disease application

technical field

The invention relates to the field of biotechnology, in particular to an ssDNA aptamer specifically combined with the extracellular domain of epidermal growth factor HER3 (Her3 ECD) and its application in diagnosis and treatment of HER3-related diseases.

Background technique

HER3, also known as ErbB3, LCCS2, ErbB-3, c-erbB3, erbB3-S, MDA-BF-1, c-erbB-3, p180-ErbB3, p45-sErbB3, p85-sErbB3, is a tyrosine kinase epidermal growth One of the factor family members, the size is 161kDa. The human HER3 gene is located on chromosome 12q13. The family consists of 4 members, in addition to HER3, there are EGFR, HER2, HER4. Each member is composed of three parts, which are the ligand-binding region of the extracellular region, the transmembrane region, and the intracellular kinase region. The extracellular domain of HER3 has a neuregulin binding site but no intracellular kinase activity. Therefore, it must form a heterodimer with other members of this family to activate the MAPK and PI3K-Akt pathways and participate in the development of various tumors including breast cancer, lung cancer, and head and neck cancer.

At present, many companies at home and abroad have started to develop anticancer drugs targeting HER3. HER3 inhibitors include seribantumab (MM-121) of Merrimack Pharmaceuticals, which is currently in Phase III clinical stage; GlaxoSmithKline’s GKS2849330 is in Phase I and the HER3/EGFR bidirectional inhibitor MEHD7945A of Roche's Genentech, which is in the mid-stage of development.

Inhibitor drugs targeting Her3ECD can be used in HER3 overexpression disease models, including: 1) HER3 is highly expressed in HER2-mediated breast tumors. Knocking down HER3 expression in HER2 high-expressing tumors will cause tumor shrinkage, and the effect of knocking down HER2 will be inhibited, while knocking down EGFR in this tumor model will not cause tumor reduction. In cancer samples, the high expression of HER3 is highly consistent with the high expression of HER2, but there is no such correlation between the expression of EGFR and HER2. The above studies have emphasized the unique and key role of HER3 in the mechanism of HER2 carcinogenesis. 2) HER3 is highly expressed in melanoma. HER3 may be a synergistic molecule of EGFR and/or HER4, which is significantly highly expressed in melanoma and is associated with the degree of malignancy and poor prognosis of the disease. 3) The incidence of high expression of HER3 in lung cancer is 18%-100%, especially for non-small cells resistant to EGFR tyrosine kinase inhibitor (tyrosine kinase inhibitor, TKI) gefitinib (gefitinib) treatment In non-small lung cell lung carcinomas (NSCLC), compared with treatment-sensitive NSCLC, the high expression rate of HER3 is 100%. In addition, in NSCLC patients, high expression of HER3 is significantly related to brain metastasis and short survival time sex. 4) HER3 is significantly highly expressed in childhood osteosarcoma; 5) HER3 is highly expressed in some types of prostate cancer, colon cancer and ovarian cancer (expression rate between 10-90%), although its involvement mechanism has not yet been elucidated, but It is clear that the high expression of HER3 is closely related to its high metastasis and poor prognosis.

In recent years, oligonucleotide aptamers, as promising alternative molecules for antibody molecules, have attracted much attention. Aptamers are ligands that can bind to targets with high specificity and high affinity, and are screened from single-stranded random oligonucleotide libraries by using systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have a wide range of target molecules; high specificity and affinity; binding conditions with target molecules can be adjusted, easy to artificially synthesize; good stability, easy to modify; small molecules, small steric hindrance and other advantages. And as "antibody analogs" with low immunogenicity, aptamer targeted therapy drugs have been used in the treatment of various cancers, macular degeneration, von Willebrand's disease, diabetes and other diseases, and can also be used as drug targets. For ligands, aptamer-drug conjugates (aptamer-drug conjugates, ApDCs) are developed to assist drug release at specific tissue sites. Judging from current research and clinical trials, aptamers provide a good platform for targeted therapy. And some drugs have been approved by the FDA for marketing, showing great application prospects in the field of biomedicine.

At present, there are few studies on aptamers targeting Her3ECD. At present, there are only reports on RNA aptamers with high affinity for Her3ECD in 2003, but there is no subsequent report. Based on the fact that HER3 plays a very important role in the occurrence, development, drug resistance and prognosis of various cancers, the present invention may be applied in the diagnosis, treatment and related research of HER3 related cancers.

Contents of the invention

The purpose of the present invention is to provide a ssDNA aptamer with high affinity and high selectivity for Her3ECD.

The method of the present invention utilizes the systematic evolution technology (SELEX technology) of exponentially enriched ligands, and selects Her3ECD as the target protein. Construct the delivery system of Her3ECD aptamer-drug conjugates (ApDCs), and study its targeting, anti-tumor effect and related mechanisms, so as to provide reference for the development of similar drugs.

The purpose of the present invention can be achieved by the following measures: the target protein Her3ECD antigen of the present invention is expressed by transiently transfecting the Her3ECD plasmid into 293E cells, the protein size is 100kDa, and the culture medium is collected three days later and purified by a nickel column.

The aptamer in the solution of the present invention can undergo adaptive folding in the presence of target molecules, and will form special three-dimensional spatial structures such as hairpins, stem loops, pseudoknots, convex rings, and G-tetramers. Hydrogen bonds, hydrophobic stacking interactions, van der Waals forces, etc. are closely combined with target molecules, showing high affinity.

In one aspect, the present invention provides ssDNA aptamers that can specifically recognize Her3ECD.

In a preferred embodiment, the ssDNA aptamer of the present invention has a nucleotide sequence selected from SEQ ID NO:1 or SEQ ID NO:2.

The aptamer of the present invention can also be RNA, and the literature reports that RNA aptamers generally show higher affinity, but RNA is not as stable as ssDNA, and there are great difficulties in the process of screening and drug formation.

The aptamer against Her3ECD of the present invention is verified by free Her3ECD protein and two breast cancer cell lines MCF7 and BT474 with high expression of HER3, and has good specificity and affinity. ELISA, cell flow cytometry, and laser confocal experiments have confirmed that the aptamer of the present invention has good affinity and specificity for breast cancer cell lines with high expression of Her3ECD.

Therefore, on the other hand, the present invention provides a reagent for diagnosing breast cancer with high expression of Her3ECD, which contains the ssDNA aptamer of the present invention.

On the other hand, the present invention provides a DNA chip for diagnosing breast cancer with high expression of Her3ECD, on which the ssDNA aptamer of the present invention is immobilized.

On the other hand, the present invention provides a pharmaceutical composition for treating breast cancer with high expression of Her3ECD, which contains the ssDNA aptamer of the present invention.

In another aspect, the present invention provides the use of the ssDNA aptamer in preparing a diagnostic reagent for breast cancer with high Her3ECD expression.

In another aspect, the present invention provides the use of the ssDNA aptamer in the preparation of targeted therapeutic drugs for breast cancer with high expression of Her3ECD.

The aptamer of the present invention is easy to obtain, can be synthesized in large quantities, is easy to carry out chemical modification and transformation, and has good stability. It can be modified by fluorine or oxygen methyl at the 2' position of the glycoside, or connected with high molecular weight polyethylene glycol or cholesterol. Reduce renal clearance and improve its pharmacokinetics. After stability modification, aptamers can also be linked to nanoparticles to achieve targeted delivery of drugs such as chemotherapeutics and nucleic acids. It can be seen that aptamers are widely used in both clinical diagnosis and drug research.

Beneficial effects of the present invention:

The Her3ECD high-affinity and high-specificity aptamer of the present invention can be modified by chemical modification and applied to the diagnosis and treatment of diseases related to high expression of HER3, and can improve drug efficacy and reduce side effects by constructing a targeted delivery system Realize the targeted delivery of chemotherapy drugs and nucleic acid drugs. The HER3-related diseases shown include EGFR antibody therapy-failed lung cancer, HER2-positive antibody drug-resistant breast cancer, cervical cancer, etc. The target aptamer can also be used in scientific research related to HER3, or in medical or pharmaceutical application research such as tumors.

The sequence described in the present invention is as follows:

7#GGGAGCTCAGAATAAACGCTCAAAGGGTCAAGCTGATTACACTTTGTCCACTATTGGGTCCTTCGACATGAGGCCCGGATC

13#GGGAGCTCAGAATAAACGCTCAAGGCTAACAGCACGCAACGGGGGGGAGTAATCGTGTCTGTTCGACATGAGGCCCGGATC

Description of drawings

Figure 1 illustrates the secondary structure diagram of the Her3ECD aptamer of the present invention;

Figure 2 illustrates the cell flow cytometric detection of the binding specificity of the aptamer of the present invention to MCF-7 cells;

Fig. 3 The binding experiment of cell flow cytometry detection aptamer and BT474 cell;

definition

References, patents, patent applications and scientific literature mentioned in this application constitute the prior knowledge of those skilled in the art and are hereby incorporated by reference in their entirety to the same extent as if these documents were specifically and individually incorporated by reference. In case of any conflict between the documents introduced in this application and the specific meanings of this specification, the latter shall prevail. In addition, if there is a conflict between the existing understanding of the definitions of words and phrases in the field and the definitions set by the special interpretation of the words or phrases in this specification, the latter shall prevail.

Before the present invention is further elucidated, it is important to realize that this invention is not limited to particular embodiments described, that is, variations may exist in particular forms. It should also be reminded that since the scope of the present invention is defined by the appended claims, the terminology used herein is for the purpose of describing particular embodiments only and not for the purpose of limiting the invention.

It should also be noted that, as used in this specification, a singular form includes a plural form of its referent unless clearly and clearly limited to one referent. The term "or" is used interchangeably with the term "and/or" unless the context clearly dictates otherwise.

In certain embodiments, the affinity between the aptamer and its target is characterized by KD (dissociation constant), which is lower than 10 ⁻⁷ M, 10 ⁻⁸ M, 10 ⁻⁹ M, 10 ⁻¹⁰ M, 10 ^-11 M, or about 10 ^-12 M or lower.

detailed description

The invention discloses an aptamer for Her3 ECD, and related affinity verification experiments prove that the aptamer has good affinity and specificity, which will provide a new idea for the diagnosis and treatment of related diseases with high expression of HER3 . Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The application of the present invention and the preferred embodiments have been described, and the relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention. Invent technology.

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific embodiments.

Example

Example 1. Screening of Her3ECD ssDNA aptamers based on SELEX technology

1.1 Construct a random single-stranded DNA (ssDNA) oligonucleotide library 5'-GGGAGCTCAGAATAAACGCTCAA(N38)TTCGACATGAGGCCCGGATC-3', wherein N38 is a random sequence of 38 bases, and the two ends are primer sequences with fixed sequences, and the primer upI is 5'-GGGAGCTCAGAATAAACGCTCAA-3', primer down I is 5'-GATCCGGGCCTCATGTCGAA-3', primer down II is 5'-biotin-GATCCGGGCCTCATGTCGAA-3', library capacity is more than 10 ¹⁴ , synthetic random single-stranded DNA oligonucleotide The library is divided into 1OD/tube, and the lyophilized powder is stored at -20°C for future use.

1.2 The Her3ECD plasmid was transiently transfected into 293E cells with PEI reagent (Invitrogen). Three days later, the cell culture supernatant was collected and purified by nickel column to obtain the target protein with His tag. The expression of the antigen band was identified by Coomassie brilliant blue staining and Western Blotting If it is correct and has good purity, it can be used as the target protein for subsequent screening.

1.3 Select magnetic beads with His tag to immobilize the protein through Binding Buffer (sodium phosphate 20mM, sodium chloride 50mM, imidazole 5mM, pH=7.4), and add corresponding PBST (K ₂ HPO ₄ 50mM, sodium chloride 150mM, Tween- 20 0.05%, PH=7.5) Store in a refrigerator at 4°C.

1.4 Binding of ssDNA library to target protein: Dissolve 1nmol of ssDNA library in 100μL PBST, denature at 95°C for 5min, and ice-bath for 10min, add to 10mL magnetic beads linked to target protein in step (3) above, and rotate at room temperature for 30min , then use the magnetic stand to discard the supernatant, wash the magnetic beads with PBST buffer 3 times, discard the supernatant, and use Elution buffer (sodium phosphate 20mM, sodium chloride 50mM, imidazole 500mM, PH=7.4) to remove the protein on the magnetic beads Elution, ssDNA with a certain affinity will bind to the protein, use the eluate as a QPCR template for amplification, select primers upⅠ and downⅡ, program: pre-denaturation at 95°C for 15min, PCR reaction: denaturation at 95°C for 10s; 58 Anneal at ℃ for 20s; extend at 72℃ for 30s. (Note: After doing corresponding experiments and comparing the protein-nucleic acid mixture for qPCR or the combined nucleic acid precipitation and recovery for qPCR, the difference between the two is very small through the identification of the more sensitive silver staining pattern, and considering that there are a large number of nucleic acids in the recovery process loss, the eluted product of the magnetic beads is directly used as a qPCR template.)

1.5 Separation of dsDNA into ssDNA by streptavidin-biotin magnetic beads: take streptavidin-biotin magnetic beads with corresponding Buffer (Tris-Hcl (PH=7.5) 10mM, EDTA 1mM, sodium chloride 1M, Tween-20 0.01%-0.1%) to wash an appropriate amount of magnetic beads twice, add qPCR product and mix at room temperature for 30-60min, wash with Buffer three times, discard the supernatant, add 50μl 0.1mol/L freshly prepared NaOH, and incubate at 37℃ for 15min, so that The dsDNA melts. Use 50 μl of NaOH solution containing ssDNA directly as the next round of screening library, or add it to 1 ml of PBST, adjust the pH to 7.5 with 10 mmol/ml dihydrogen phosphate buffer, and store it.

1.6 Reverse screening of irrelevant proteins: take irrelevant proteins for screening, the difference from positive screening is that we retain the buffer system that is not bound to irrelevant proteins. The system was extracted twice by adding an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) (stand still for 5 min, centrifuge at 12000 rpm for 5 min), add an equal volume of cold ice ethanol, mix well and place on ice Let stand for 30 minutes, centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, rinse once with 70% ethanol, discard the supernatant, and dry the precipitate. Add ddH ₂ O to dissolve as a template for qPCR.

1.7 qPCR melting curve combined with comparison of silver-stained bands to confirm the accuracy of the library after multiple screenings.

1.8 The final sequence screened was amplified and purified by PCR, and multiple aptamers were obtained by enzyme-linked cloning and sequencing. The secondary structure, free energy, and homology were analyzed, and several candidate sequences were selected.

Example 2. Her3ECD ssDNA aptamer affinity identification

Coat the microtiter plate with Her3ECD, set up different concentration gradients, add 1.25, 2.5, 5, 10, 50, 100 and 200 pmol protein in sequence, dilute with 100 μl coating solution, coat overnight at 4°C; block with 1% BSA at 37°C for 2 hours; Add 10 pmol 5' biotin-modified Her3ECD aptamer, incubate at 37°C for 1h; wash 5 times with PBST, add SA-HRP antibody (1:8000), incubate at 37°C for 1h; wash 5 times with PBST, add TMB substrate for color development . Among them, GPC3 and BSA were used as negative controls. The detection results showed that the screened Her3ECD aptamer did not bind to the other two proteins, and the value was consistent with Blank, which further proved that the aptamer had high specificity and affinity for Her3ECD. The KD values of the aptamers were obtained by fitting the affinity curve, among which the KD values of 7# and 13# were 116nM and 98nM respectively, and their sequences were respectively SEQE ID NO:1 and SEQE ID NO:2, and their secondary structures are shown in Fig. 1.

Example 3. Identification of Her3ECD ssDNA cell binding specificity

Cell flow cytometry: The breast cancer cell line MCF-7 stimulated by NRG1 4 hours in advance (MCF7 cells are positive cells stimulated by NRG1, and the expression of HER3 on the membrane surface increases after stimulation) and the unstimulated breast cancer cell line BT474 were selected for aptamer For the verification of affinity and specificity, a commercially available HER3 antibody was selected as a positive control, and the human embryonic cell line 293T with low expression of HER3 was selected as a negative control. The deactivated cells were washed twice with PBS, resuspended and counted, and 1×106 cells ^were taken, resuspended in 100 μl PBS, and 100 pmol of FAM-labeled aptamer at the 5' end and APC-labeled commercial HER3 antibody were taken and the cells were kept in contact with each other. Incubate with light for 1 h, wash twice with PBS, fix with 2% paraformaldehyde, and detect the positive rate of cells by flow cytometry.

The experimental results show that the commercialized HER3 antibody has an affinity of 95% for the cells, and the aptamer binds to the breast cancer cell line MCF-7 (Figure 2 flow cytometry: BD, American biosciences) and BT474 (Figure 3 flow cytometry: The positive rate of Guava, Merck Millipore, Germany) is about 75%, and both of them show very low affinity (about 5%) to 293T, reflecting the good affinity and specificity of the aptamer itself.

Example 4. Her3ECD ssDNA targeted into cells

Laser confocal: Commercial HER3 antibodies and aptamers theoretically bind to endogenously expressed HER3ECD antigens in cells, and co-incubation of the two forms target competition, but co-localization can also be used to illustrate their binding properties to endogenous antigens in cells. The breast cancer cell line MCF-7 was planted on a 24-well plate-sized circular slide in advance, and NRG1 was added to stimulate the MCF-7 cells 4 hours before the experiment, washed twice with PBS to remove the residual medium, and 2% paraformaldehyde was added to fix the cells, PBS Wash twice, add FAM-labeled aptamer and commercial rabbit HER3 antibody and incubate for 1 hour in the dark, wash twice with PBS, add PE-labeled goat anti-rabbit IgG secondary antibody against the antibody and incubate for 30 minutes, wash twice with PBS , add 300nM DAPI to stain the nuclei for 3min, wash twice with PBS, add anti-fade agent and place it upside down on the coverslip, seal the slide with nail polish, and observe under the laser confocal microscope. The results showed that the FITC-aptamer was distributed in both the cytoplasm and the nucleus, and the PE-HER3 commercial antibody was mainly distributed in the cytoplasm, followed by the nucleolus, but less in the nucleus except the nucleolus.

The above results show that the aptamer of the present invention and the commercialized antibody have co-localization, suggesting that both of them enter the cell after binding to the endogenous HER3 antigen. It is worth noting that in addition to colocalizing with the antibody, the aptamer is highly expressed in the nucleolus of the cell, and its nucleolus localization mechanism may be different from the microscopic distribution mechanism of the antibody after entering the cell. The specific mechanism needs to be further elucidated.

sequence listing

<110> Institute of Radiation and Radiation Medicine, Academy of Military Medical Sciences of the Chinese People's Liberation Army, Junke Zhengyuan (Beijing) Drug Research Co., Ltd.

<120> An ssDNA aptamer targeting Her3ECD and its application in the diagnosis and treatment of HER3-related diseases

<130>

<160> 2

<170> PatentIn version 3.5

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gggagctcag aataaacgct caaagggtca agctgattac actttgtcca ctattgggtc 60

cttcgacatg aggcccggat c 81

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<211> 81

<212>DNA

<213> Artificial sequence

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gggagctcag aataaacgct caaggctaac agcacgcaac gggggggagt aatcgtgtct 60

gttcgacatg aggcccggat c 81

Claims (8)

1. a kind of ssDNA aptamers of specific recognition Her3ECD albumen, which has selected from SEQ ID NO:1 or SEQ ID NO:Nucleotide sequence shown in 2.

2. the corresponding RNA of ssDNA aptamers described in claim 1.

3. the derivant of the ssDNA aptamers described in claim 1 or the product through specific modification.

4. the breast cancer diagnosis reagent that a kind of Her3ECD height is expressed, wherein containing the ssDNA aptamers described in claim 1.

5. a kind of breast cancer diagnosis DNA chip of the high expression of Her3ECD, is fixed with the ssDNA described in claim 1 thereon and fits Part.

6. a kind of target therapeutic agent of the high expression breast carcinoma of Her3ECD, is wherein adapted to containing the ssDNA described in claim 1 Body.

7. purposes of the ssDNA aptamers described in claim 1 in the breast cancer diagnosis reagent for preparing the high expression of Her3ECD.

8. the ssDNA aptamers described in claim 1 are in the target therapeutic agent for the high expression breast carcinoma of Her3ECD is prepared Purposes.