RU2528870C2 - Aptamer specific to human tumour lung tissues - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, more specifically to detecting lung cancer by means of an aptamer and can be used in diagnostics. The aptamers are prepared by selection involving alternating rounds of positive selection of the aptamers to minced human tumour lung tissues sampled from oncological patients after the operation and of negative selection to healthy lung tissues and healthy whole blood to determine a pool of the highest-affinity aptamers to be cloned, sequenced and analysed for a binding specificity to tumour lung cells.

EFFECT: prepared aptamers possess high sensitivity to tumour debris and circulating tumour cells in peripheral blood of the patient suffering lung cancer that enables more effective diagnosis of human lung cancer.

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Description

The invention relates to the diagnosis of cancer, in particular to the detection of lung cancer using aptamers.

Among all types of oncological diseases, lung cancer is the most common form of malignant neoplasms. It affects more than a million people worldwide and accounts for about 25% of all cancer deaths (E.V. Artamonova. Major achievements in biology, screening, diagnosis and treatment of non-small cell lung cancer (NSCLC). Practical Oncology. - 2011. - T. 12. - No. 1. - P.26-35; Sunil K. Arya, Shekhar Bhansali. Lung Cancer and Its Early Detection Using Biomarker-Based Biosensors. Chem. Rev. - 2011. - V.111. - P.6783- 6809).

The relative five-year survival of patients with lung cancer is very low, it is 13-15% for developed countries (Francesco Paolo Fiorentino, Marcella Macaluso, Fabrizio Miranda, Micaela Montanari, Antonio Russo, Luigi Bagella, Antonio Giordano. CTCF and BORIS Regulate Rb2 / p130 Gene Transcription: A Novel Mechanism and a New Paradigm for Understanding the Biology of Lung Cancer. Molecular cancer research. - 2011. - V.9. - P.225-233; BE Shevchenko, HE Arnotskaya, O.P. Trifonova, AC Dashkevich , BA Yurchenko, DG Zaridze, Profiling of a low molecular weight plasma proteome for the detection of potential markers of lung cancer, Mass spectrometry. - 2007. - T.4. - No. 4. - S.245-253) and 9% for developing I (EV Levchenko. Screening for lung cancer. Practical oncology. - 2010. - T.11. - No. 2. - P.88-95).

However, despite the variety of existing diagnostic methods, the detection rate of patients with stages I-II of lung cancer is 26.5%, and patients with stages III-IV are about 66.4%. Moreover, 5-year survival after treatment of stage I of RL corresponds to 70%, and stage IV - less than 5% (Chissov V.I., Starinsky V.V., Petrova G.V. Oncological care for the population of Russia in 2009. M.: Federal State Institution “Moscow Scientific Research Institute named after PA Herzen of Rosmedtehnologii.” - 2010. - 196 p.).

The most common histological types of lung cancer according to the WHO classification are squamous cell carcinoma of the lung, adenocarcinoma and small cell carcinoma of the lung.

For the clinical diagnosis of cancer, aptamers, single-stranded DNA oligonucleotides can be used, which, due to their unique conformations and spatial arrangement of charges, have high specificity and affinity for given targets. In addition, aptamers have a picomolar level of sensitivity, and therefore can be used to diagnose the early stages of the development of diseases.

The potential use of aptamers for the detection of small cell lung cancer was demonstrated by Weihong Tan et at. (Hui William Chen, Colin D. Medley, Kwame Sefah, Dihua Shangguan, Zhiwen Tang, Ling Meng, Josh E. Smith, and Weihong Tan. Molecular Recognition of Small-Cell Lung Cancer Cells Using Aptamers. ChemMedChem. 2008). The described aptamers were obtained by cell-SELEX method for cell cultures of small cell lung cancer (MRL). To prove the possibility of using the selected aptamers in clinical practice, cells from the MRL culture were mixed with human whole blood samples, followed by examination of the sample using flow cytometry.

Known inventions that describe methods for detecting lung cancer using aptamers selected for recombinant surface proteins or cell cultures of lung cancer.

Thus, there is a method for the diagnosis of human lung cancer, as well as the prevention and treatment of lung cancer using immunogenic peptides (WO 2004015390 A2, C07K 14/47, G01N 33/574, 2004.02.19), in which five target proteins are expressed by different types of cancer tissues lung. Aptamers can be used as agents that bind to the described proteins.

A known method for the detection and differentiation of various subtypes of non-small cell lung cancer (CN 101538570, C12N 15/10, C12N 15/11, 2009.09.23), which consists in the selection of aptamers to surface markers of various types of non-small cell lung cancer (NSCLC). Aptamers, binding to markers of different histological types of non-small cell lung cancer, allow their differentiation. The method described in the invention allows early diagnosis of NSCLC, as well as more accurate genotyping.

The aptamers used in the diagnosis of cancer in the methods described above were matched to recombinant proteins. Since the conformations of recombinant (purified) and native (located on the cell membrane) proteins can vary significantly, the specificity of detection of lung cancer using such aptamers may not be high enough.

A known method for the diagnosis of small cell lung cancer (US 2009239762 A1, C07H 21/04, C12Q 1/68, 2009.09.24), according to which aptamers were selected for cell cultures of MRL by cell-SELEX. The obtained aptamers had the ability to bind to the tissues of MRL with high affinity. When cultures of various histological types of lung cancer were mixed, the cells of MRL cultures were extracted from the mixture using aptamers associated with magnetic and fluorescent particles.

The method for detecting lung cancer using aptamers selected for cell cultures is more sensitive than the methods in which aptamers were selected for recombinant proteins, since aptamers selected for whole cells are more specific for native cell receptors and, in addition, can contact with a large number of targets on the surface of the cell. However, cells growing in culture can transform and differ from tumor cells growing in the body due to different microenvironments, which can give false negative results in detecting circulating cells in blood samples. In addition, since the described aptamers are not specific to the decay products of the tumor and many intracellular targets, only circulating cancer cells can be detected with their help. As a result, the sensitivity of cancer detection methods using aptamers to whole cells will not be high enough.

The present invention is the selection of aptamers, which will allow with high sensitivity to identify the decay products of the tumor and circulating cancer cells in the peripheral blood of patients with lung cancer of the most common histological types (squamous lung cancer, glandular carcinoma, small cell lung cancer).

The problem is solved in that the aptamer specific for tumor tissue of the human lung, obtained as a result of selection of aptamers, according to the invention, was obtained in the selection process, including the alternation of rounds of positive selection of aptamers to the crushed tumor tissues of the human lung, which were taken from cancer patients after surgery and negative selection for healthy tissues of the lungs and whole blood of healthy people, with the identification of the pool of aptamers with the highest affinity, its cloning, sequencing, checking for the specificity of binding to lung tumor cells has an established nucleotide sequence selected from SEQ ID NO 1-SEQ ID NO 34.

The pool of aptamers with the highest affinity for given targets, obtained in one of the selection rounds, can be detected using flow cytometry.

The aptamer specific for human lung tumor tissues, having the highest affinity, is characterized by an established nucleotide sequence selected from SEQ ID NO 1-SEQ ID NO 5.

The technical result of the invention is expressed in the higher specificity of the obtained aptamers to the tumor tissues of the human lung due to the possibility of detecting oleum-specific targets in clinical samples of patients with lung cancer.

The invention is illustrated in graphic materials. Figure 1 shows a histogram comparing the affinity of the original library of single-stranded DNA oligonucleotides and obtained during the selection of the pool of aptamers to tumor lung cells. The number 1 in figure 1 shows the histogram of intact cells, the number 2 - the histogram of lung cancer cells with a library of aptamers, the number 3 - the histogram of lung cancer cells with a pool of aptamers of 10 round selection. Figure 2 presents samples of human peripheral blood, the analysis of which was carried out using aptamers having the highest affinity for lung tumor cells. In the form A. figure 2 presents a sample of the peripheral blood of a patient with lung cancer incubated with aptamers labeled with a fluorescent label; Figure B shows a sample of the peripheral blood of a healthy person incubated with aptamers labeled with a fluorescent label; Figure C shows a peripheral blood sample of a patient with kidney cancer incubated with aptamers labeled with a fluorescent label.

When selecting aptamers for tumor tissues of the most common histological types of lung cancer, SELEX technology was used with alternating positive and negative selection (Ellington. AD and Szostak. JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818- 822).

Positive selection of aptamers was carried out to the tumor tissues of the lung of a person who climbed after the operation of cancer patients. Negative selection was carried out to healthy tissues of the lungs and whole blood of healthy people.

The tissue was crushed with a scalpel in order to increase the number of aptamer binding sites. The targets for the selection of aptamers were whole cells, necrotic cells, tumor decay products, released proteins, and intracellular contents.

Aptamers were selected for 13 rounds of selection using a library of single-stranded DNA oligonucleotides. As a result of selection, aptamers specific to lung tumor tissues were obtained.

In the process of selection, the affinity of aptamers for targets increases to a certain round of selection, after which it remains constant or may decrease. Therefore, for further research, the aptamer pool with the highest affinity for the given targets was obtained during one of the selection rounds. To identify the most affinity aptamer pool obtained as a result of selection, the method of flow cytometry was used. The fluorescence of the studied aptamer pools bound to the target was determined using an FC-500 flow cytometer (Beckman Coulter Inc .. USA). The results of flow cytometry showed that the aptamers of the 10th round of selection have the highest affinity for the tumor tissue of a human lung.

From the histograms shown in FIG. 1, it is seen that the fluorescence level of aptamers incubated with lung tumor cells is higher than the fluorescence levels of a single-stranded DNA oligonucleotide library incubated with lung cancer cells and intact lung cancer cells, which indicates the specificity of binding of round 10 aptamers selection with lung tumor cells.

The 10-round selection aptamer pool was cloned using the M13mp18 Perfectly Blunt Cloning Kit (Novagen, Gemany) and sequenced to determine the desired aptamer sequences. Aptamers were chemically synthesized. As a result, 34 unique aptamers binding to human lung tumor tissues were obtained, represented as SEQ ID NO 1-SEQ ID NO 34. Each of the synthesized aptamers was tested for binding specificity to lung tumor cells.

The specificity of aptamers to tumor cells of a human lung was checked using a flow cytometer. For this, lung cancer cells were incubated for 15 minutes with masking RNA from yeast (final concentration 0.1 mg / ml) to reduce non-specific binding, after which aptamers with a fluorescent label FAM were added to the incubation mixture and incubated for another 30 minutes at room temperature. Aptamer binding analysis was performed using a flow cytometer. As a result, the 5 most specific sequences for lung tumor tissues were identified, presented as SEQ ID NO 1-SEQ ID NO 5.

Example 1. To prove the potential use of aptamers for the detection of human lung cancer by analysis of peripheral blood samples of patients, the method of flow cytometry was used.

The following reagents were used to identify the decay products of the tumor and circulating cancer cells in blood samples.

1. DNA aptamers specific for human lung cancer tissues, labeled with a fluorescent FAM tag.

2. Lysing solution OptiLyse.

3. Phosphate buffer with Ca ²⁺ and Mg ²⁺ .

4. Masking yeast RNA.

The following peripheral blood samples of people taken from a vein with the addition of an anticoagulant were used to implement the example.

1. A sample of the peripheral blood of a patient with lung cancer.

2. A sample of the peripheral blood of a healthy person.

3. A sample of the peripheral blood of a patient with kidney cancer.

Blood samples were incubated with masking yeast RNA for 15 minutes at room temperature. Then synthetic aptamers selected from SEQ ID NO 1-SEQ ID NO 5 labeled with a fluorescent label were added to the samples, after which the sample was incubated for another 20 minutes in a dark place. Red blood cells in the sample, which were difficult to analyze, were destroyed using a lysis solution. The sample was brought to 300 μl with phosphate buffer with Ca ²⁺ and Mg ²⁺ , after which the fluorescence of the aptamers bound to the target was determined using an FC-500 flow cytometer (Beckman Coulter Inc., USA).

The results of the analysis of human peripheral blood samples using aptamers having the nucmeotide sequences shown as SEQ ID NO 1-SEQ ID NO 5 labeled with a fluorescent label showed that molecular targets binding to the aptamers are present in the blood of a patient with lung cancer (Fig. 2, type A), which indicates the presence in the peripheral blood of the patient of the tumor tumor cells of the lung and the decay products of the lung tumor. Thus, the analysis of a peripheral blood sample of a patient with lung cancer using aptamers confirmed the diagnosis established by clinical, laboratory and histological methods.

In the blood of a healthy person (Fig. 2, view B) no molecular targets binding to the aptamers were detected, which indicates the absence of lung tumor cells and decay products of the lung tumor in the blood.

In the blood of a patient with kidney cancer (Fig. 2, view C), no molecular targets that bind to aptamers were also detected, which shows the specificity of aptamers to cancer cells and decay products of a tumor of pulmonary origin.

The results obtained prove the potential use of aptamers of the claimed sequences of SEQ ID NO 1-SEQ ID NO 34 for the detection of lung cancer.

Claims (3)

1. Aptamer specific for lung tumor tissue obtained as a result of selection of aptamers, including the alternation of rounds of positive selection of aptamers to ground tumor tissues of the lung of a person, which were taken from cancer patients after surgery, and negative selection to healthy tissues of the lung and whole blood of healthy people, with the identification of the aptamer pool with the highest affinity, its cloning, sequencing, testing for the specificity of binding to lung tumor cells, it has an established nucleotide acid sequence selected from SEQ ID NO 1-SEQ ID NO 34. 2. The aptamer specific for tumor tissue of a human lung according to claim 1, characterized in that it is selected from a pool of aptamers with the highest affinity for given targets detected by flow cytometry. 3. The aptamer specific for tumor tissue of a human lung according to claim 1, characterized in that the aptamer having the highest affinity is characterized by an established nucleotide sequence selected from SEQ ID NO 1-SEQ ID NO 5.

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