CN106497931B - A kind of ssDNA aptamers for Her3ECD and its application in diagnoses and treatment HER3 related disease - Google Patents

Abstract

Fas lignand system evolution technology (the systematic evolution of ligands by exponential enrichment that the invention discloses a kind of by index concentration, what SELEX) screening obtained is directed to HER3 extracellular region protein (extracellular domain, ECD) high-affinity, highly selective DNA aptamers.The present invention further proves that the aptamers either still all have certain specificity and compatibility to the breast cancer cell of high expression Her3ECD albumen to free Her3ECD by ELISA, cell streaming, laser co-focusing experiment, and the diagnoses and treatment for the highly expressed related disease of HER3 provides new thinking.

Description

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

technical field

The invention relates to the field of biotechnology, in particular to a ssDNA aptamer that specifically binds to the extracellular domain (Her3ECD) of epidermal growth factor HER3 and its application in the 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 for epidermal growth A member of the factor family with a size of 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, namely, the ligand-binding domain of the extracellular domain, the transmembrane domain, and the intracellular kinase domain. The extracellular domain of HER3 has a neuregulin binding site but no intracellular kinase activity. Therefore, it is necessary to form heterodimers with other members of this family to activate the MAPK and PI3K-Akt pathways, which are involved in the occurrence and 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 anti-cancer drugs targeting HER3. HER3 inhibitors include seribantumab (MM-121) of Merrimack Pharmaceuticals, which is currently in Phase III clinical stage; GKS2849330 of GlaxoSmithKline, which is in Phase I and the HER3/EGFR bidirectional inhibitor MEHD7945A of Roche's Genentech, which is in the middle stage of development.

Inhibitor drugs targeting Her3ECD can be used in HER3-overexpressing disease models, including: 1) HER3 is highly expressed in HER2-mediated breast tumors. Knockdown of HER3 expression in HER2-overexpressing tumors can cause tumor shrinkage, which is inhibited by knockdown of HER2, while knockdown of EGFR in this tumor model does not cause tumor reduction. In cancer specimens, the high expression of HER3 is highly consistent with the high expression of HER2, while the expression of EGFR and HER2 has no such correlation. 2) HER3 is highly expressed in melanoma. HER3 may act as a synergistic molecule of EGFR and/or HER4, which is significantly overexpressed in melanoma, and is associated with the malignancy and poor prognosis of the disease. 3) The incidence of high expression of HER3 in lung cancer is 18%-100%, especially in non-small cells resistant to EGFR tyrosine kinase inhibitor (TKI) 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 HER3 expression is significantly associated with brain metastasis and short survival. sex. 4) HER3 is significantly highly expressed in childhood osteosarcoma; 5) HER3 is highly expressed in certain types of prostate, colon and ovarian cancers (expression rates between 10-90%), although the mechanism of its involvement has not been elucidated, It is clear that the high expression of HER3 is closely related to its high metastasis and poor prognosis.

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

At present, there are few studies on aptamers targeting Her3ECD. Currently, only RNA aptamers with high affinity targeting Her3ECD have been reported in the literature in 2003, but no subsequent reports have been reported. Since 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.

SUMMARY 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 enriching ligands, and selects Her3ECD as the target protein. The Her3ECD aptamer-drug conjugates (ApDCs) delivery system was constructed, and its targeting, tumor-inhibiting effect and related mechanisms were studied to provide reference for the development of similar drugs.

The object 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 three days later, the culture medium is collected and purified by nickel column.

The aptamer in the solution of the present invention can undergo adaptive folding in the presence of the target molecule, and will form special three-dimensional spatial structures such as hairpins, stem loops, pseudoknots, convex loops, G-tetramers, etc. Hydrogen bonding, hydrophobic stacking, van der Waals force, etc. are closely combined with the target molecule, reflecting a 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 described in the present invention can also be RNA, and it is reported in the literature 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 development.

The aptamer for 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 and laser confocal experiments confirmed that the aptamer of the present invention has good affinity and specificity for the breast cancer cell line with high expression of Her3ECD.

Therefore, in another aspect, the present invention provides a reagent for diagnosing breast cancer with high expression of Her3ECD, which contains the ssDNA aptamer of the present invention.

In another aspect, 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.

In another aspect, 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 expression of Her3ECD.

In another aspect, the present invention provides the use of the ssDNA aptamer in the preparation of a targeted therapy drug 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. Decrease renal clearance and improve its pharmacokinetics. After stability modification, aptamers can also be linked to nanoparticles to achieve targeted delivery of chemotherapeutic drugs, nucleic acids and other drugs. It can be seen that aptamers are widely used in 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 chemically modified and applied to the diagnosis and treatment of related diseases with high HER3 expression, and can be constructed by constructing a targeted delivery system on the basis of improving drug efficacy and reducing toxic and side effects Achieve targeted delivery of chemotherapeutic drugs and nucleic acid drugs. The HER3-related diseases shown include EGFR antibody therapy-failed lung cancer, HER2-positive antibody drug-resistant breast cancer, and cervical cancer. The target aptamer can also be used in scientific research related to HER3, or applied research in medicine or pharmacy such as tumor.

The sequence described in the present invention is as follows:

7#GGGAGCTCAGAATAAAACGCTCAAAGGGTCAAGCTGATTACACTTTGTCCACTATTGGGTCCTTCGACATGAGGCCCGGATC

13#GGGAGCTCAGAATAAACGCTCAAGGCTAACAGCACGCAACGGGGGGGAGTAATCGTGTCTGTTCGACATGAGGCCCGGATC

Description of drawings

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

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

Fig. 3 Binding experiment of aptamer to BT474 cells by flow cytometry;

definition

Reference works, patents, patent applications and scientific documents mentioned in the present invention constitute the prior knowledge of those skilled in the art and are incorporated herein by reference as a whole to the same extent as when these documents are specifically incorporated by reference individually. In the event of any conflict between a document incorporated in this application and the specific meaning of this specification, the latter shall control. In addition, if there is any conflict between the existing understanding of the definitions of words and phrases in the art and the definitions based on the specific interpretation of the words or phrases in this specification, the latter shall prevail.

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

It should also be noted that, as used in this specification, the singular includes the plural of its referent unless explicitly and explicitly 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 an 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 less.

Detailed ways

The invention discloses an aptamer for Her3ECD, and its related affinity verification experiments prove that the aptamer has good affinity and specificity, which will provide new ideas for the diagnosis and treatment of related diseases with high HER3 expression . Those skilled in the art can learn from the content of this document and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The application of the present invention has been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.

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

Example

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

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

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

1.3 Use 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 the corresponding PBST (K ₂ HPO ₄ 50mM, sodium chloride 150mM, Tween- 20 0.05%, PH=7.5) and stored in a refrigerator at 4°C.

1.4 Binding of ssDNA library to target protein: Dissolve 1 nmol ssDNA library with 100 μL PBST, denature it at 95°C for 5 min, ice bath for 10 min, add it to 10 mL of magnetic beads linked to the target protein in the above step (3), rotate and bind at room temperature for 30 min , then use the magnetic stand to discard the supernatant, wash the magnetic beads three times with PBST buffer, discard the supernatant, and use Elution buffer (sodium phosphate 20 mM, sodium chloride 50 mM, imidazole 500 mM, pH=7.4). Elution, the ssDNA with a certain affinity will be bound to the protein, the eluate is used as a QPCR template for amplification, the primers are selected upI and downII, program: pre-denaturation at 95°C for 15min, PCR reaction: denaturation at 95°C for 10s; 58 Annealing at ℃ for 20s; extension at 72℃ for 30s. (Note: Corresponding experiments have been carried out to compare the protein nucleic acid mixture for qPCR or the combined nucleic acid precipitation recovery and then qPCR. Through the more sensitive silver staining, the difference between the two is very small, and considering that there are a large number of nucleic acid recovery processes. After the loss, the magnetic bead eluted product is directly used as a qPCR template.)

1.5 Streptavidin-biotin magnetic beads to separate dsDNA into ssDNA: take streptavidin-biotin magnetic beads and use corresponding Buffer (Tris-HCl (PH=7.5) 10mM, EDTA 1mM, NaCl 1M, Tween-20 0.01%-0.1%) wash appropriate amount of magnetic beads twice, add qPCR product at room temperature and mix for 30-60 min, wash three times with Buffer, discard the supernatant, add 50 μl of 0.1 mol/L freshly prepared NaOH, and incubate at 37 °C for 15 min to make dsDNA melts. 50 μl of NaOH solution containing ssDNA was directly used as the next round of screening library, or added to 1 ml of PBST, and the pH was adjusted to 7.5 with 10 mmol/ml dihydrogen phosphate buffer before storage.

1.6 Reverse screening of irrelevant proteins: Take irrelevant proteins for screening. The difference from positive screening is that we reserve 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) (standing for 5 min, centrifuging at 12000 rpm for 5 min), adding an equal volume of cold ice ethanol, mixing and placing on ice Let stand for 30 min, centrifuge at 12000 rpm for 10 min, discard the supernatant, rinse once with 70% ethanol, discard the supernatant, and air dry the pellet. Add ddH ₂ O to dissolve as template for qPCR.

1.7 qPCR melting curve combined with silver-stained band alignment to confirm the accuracy of the library after multiple screening.

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

Example 2. Her3ECD ssDNA aptamer affinity identification

Coat the ELISA plate with Her3ECD, set different concentration gradients, add 1.25, 2.5, 5, 10, 50, 100 and 200 pmol protein in sequence, dilute with 100 μl of coating solution, coat overnight at 4°C; block with 1% BSA at 37°C for 2h; Add 10 pmol 5'-end 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 values were consistent with Blank, which further proved that the aptamer has high specificity and affinity for Her3ECD. The KD value of the aptamer was obtained by fitting the affinity curve. The KD values of 7# and 13# were 116nM and 98nM respectively, and their sequences were SEQE ID NO:1 and SEQE ID NO:2 respectively. The secondary structure is shown in the figure. 1.

Example 3. Identification of Her3ECD ssDNA Cell Binding Specificity

Cell flow: Select the breast cancer cell line MCF-7 stimulated by NRG1 4h in advance (MCF7 cells belong to NRG1 stimulation positive cells, and the expression of HER3 on the membrane surface increases after stimulation) and the unstimulated breast cancer cell line BT474 for aptamer To verify the affinity and specificity of HER3, a commercial HER3 antibody was selected as the positive control, and the human embryonic cell line 293T with low expression of HER3 was selected as the negative control. The depleted cells were washed twice with PBS, resuspended and counted, and 1×10 ⁶ cells were taken and resuspended in 100 μl PBS. 100 pmol of the aptamer labeled with FAM at the 5' end and the commercial HER3 antibody labeled with APC were taken with cell avoidance. The cells were incubated with light for 1 h, washed twice with PBS, fixed with 2% paraformaldehyde, and the positive rate of cells was detected by flow cytometry respectively.

The experimental results show that the affinity of commercial HER3 antibody to cells is 95%, and the aptamer binds to the breast cancer cell line MCF-7 (Figure 2 Flow Detector: BD, American biosciences) and BT474 (Figure 3 Flow Detector: The positive rates of Guava, Merck Millipore, Germany) were all about 75%, and both showed extremely low affinity for 293T (about 5%), reflecting the good affinity and specificity of the aptamer itself.

Example 4. Her3ECD ssDNA targeting into cells

Laser confocal: Both commercial HER3 antibodies and aptamers theoretically bind to endogenously expressed HER3 ECD antigens in cells, and the two co-incubate to form target competition, but co-localization can also be used to demonstrate their binding properties with endogenous cell antigens. Breast cancer cell line MCF-7 was seeded on 24-well plate size circular glass slides in advance, NRG1 was added to stimulate MCF-7 cells 4 hours before the experiment, washed twice with PBS to remove residual medium, added 2% paraformaldehyde to fix cells, PBS Wash twice, add FAM-labeled aptamer and commercial rabbit-derived HER3 antibody for 1 h in the dark, wash twice with PBS, add PE-labeled goat anti-rabbit IgG secondary antibody against the antibody, incubate for 30 min, and wash twice with PBS , add 300nM DAPI to stain the nucleus for 3min, wash twice with PBS, add anti-quenching agent and place it on the cover glass upside down, seal with nail polish, and observe by laser confocal microscope. The results showed that FITC-aptamer was distributed in both the cytoplasm and nucleus, and the commercialized PE-HER3 antibody was mainly distributed in the cytoplasm, followed by the nucleolus, but less in the nucleus other than the nucleolus.

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

sequence listing

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

<120> A ssDNA aptamer for Her3ECD and its application in diagnosis and treatment of HER3-related diseases

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<170> PatentIn version 3.5

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gggagctcag aataaacgct caaggctaac agcacgcaac gggggggagt aatcgtgtct 60

gttcgacatg aggcccggat c 81

Claims (7)

1. A ssDNA aptamer that specifically recognizes the extracellular domain of HER3, the nucleotide sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2. 2. The modified product of the ssDNA aptamer of claim 1, the modification being selected from the group consisting of adding fluorine or oxymethyl group at the 2' position of the glycoside, linking high molecular weight polyethylene glycol or cholesterol, or linking nanoparticles. 3 . A breast cancer diagnostic reagent with high expression of HER3 extracellular domain, comprising the ssDNA aptamer of claim 1 . 4. A DNA chip for breast cancer diagnosis with high expression of HER3 extracellular domain, on which the ssDNA aptamer of claim 1 is immobilized. 5 . A targeted therapy drug for breast cancer with high HER3 extracellular domain expression, comprising the ssDNA aptamer of claim 1 . 6. Use of the ssDNA aptamer of claim 1 in the preparation of a breast cancer diagnostic reagent with high expression of HER3 extracellular domain. 7. Use of the ssDNA aptamer of claim 1 in the preparation of a targeted therapy drug for breast cancer with high HER3 extracellular domain expression.