Aptamer conjugate for targeting triple negative breast cancer as well as preparation method and application of aptamer conjugate

Abstract

The invention provides an aptamer conjugate targeting triple negative breast cancer as well as a preparation method and application of the aptamer conjugate. A non-natural nucleic acid aptamer capable of entering triple negative breast cancer cells in a targeted internalization manner is identified by using a cell internalization screening technology, and the aptamer is further coupled with a cytotoxic agent through a pH-responsive degradable linker CL2A to construct an aptamer conjugate based on non-natural nucleic acid. The aptamer conjugate can specifically target and internalize to enter triple negative breast cancer cells, and accurately release cytotoxic agents. The aptamer conjugate disclosed by the invention can better target a tumor lesion in a triple-negative breast cancer tumor-bearing mouse, efficiently inhibits the growth of the tumor of the triple-negative breast cancer tumor-bearing mouse, has no obvious biological toxicity, and provides a new means for targeted therapy of triple-negative breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an aptamer conjugate targeting triple-negative breast cancer, and a preparation method and application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is a highly malignant breast cancer subtype clinically defined as lacking the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). The conventional treatment options for TNBC are mainly limited to chemotherapy due to the lack of specific molecular targets, which makes endocrine therapy and HER2 targeted therapy difficult to work. Antibody-drug conjugates (ADCs), as innovative therapeutic agents, have emerged in the treatment of TNBC and have shown good efficacy in clinical applications. Sacituzumabgovitecan (SG) is an ADC that combines anti-trophoblast cell surface antigen-2 (Trop-2) antibodies with the cytotoxic drug SN38. It is currently the only ADC approved by the US Food and Drug Administration (FDA) for the treatment of metastatic TNBC. With the specificity of monoclonal antibodies, ADCs can accurately deliver cytotoxic drugs to cancer cells, while maximizing the killing of cancer cells and minimizing the damage to normal cells. This targeted therapy has significant advantages in improving treatment effects and reducing side effects.

[0003] However, the large molecular weight of antibodies limits the internalization efficiency of ADCs, thereby causing off-target effects and immunotoxicity, which may have an adverse effect on their clinical performance. In addition, the production process of antibodies is complicated and time-consuming, covering many steps such as animal immunization, cell isolation, fusion with myeloma cells, hybridoma screening, and antibody purification. In contrast, aptamers are oligonucleotides that bind to target molecules with high specificity and affinity by folding to form a unique three-dimensional (3D) structure. Usually composed of 30 to 80 nucleotides, their size is only about one-tenth that of antibodies. These molecules can be generated in just two to three months through an in vitro screening (systematic evolution of ligands exponential enrichment, SELEX) process, and the subsequent manufacturing process is even faster. Aptamers that bind to cell surface targets can easily enter the cytoplasm, providing an attractive alternative tool for targeted delivery. Aptamers have low immunogenicity and therefore have significant advantages over traditional antibodies. In addition, chemical modifications such as 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), and polyethylene glycol (PEG) can enhance the resistance of aptamers to nuclease degradation, prolong the circulation half-life of aptamers, and improve their binding affinity with target molecules. In particular, 2'-F modification significantly improves the stability and resistance to nucleases of aptamers, while enhancing their binding affinity and specificity with target molecules. For this reason, 2'-F modified aptamers have been widely used in drug delivery, diagnosis, and biosensing.

[0004] Aptamer-drug conjugates (ApDCs) have attracted much attention due to their diverse targeting capabilities and low production costs. Similar to ADCs, ApDCs use aptamers instead of monoclonal antibodies as the targeting moiety. Structurally, ApDCs are composed of aptamers, cytotoxic drugs, and linkers, in which aptamers act as recognition ligands, target diseased tissues, and promote the delivery of targeted drugs. The unique properties of aptamers give ApDCs potential advantages in targeted therapy, including high specificity, high drug delivery efficiency, and low production costs. Therefore, it is necessary to further explore the application of aptamer-drug conjugates in triple-negative breast cancer. Summary of the invention

[0005] The purpose of the present invention is to provide an aptamer conjugate based on non-natural nucleic acids and a preparation method thereof, and to provide the use of the aptamer conjugate in the treatment of triple-negative breast cancer, thereby solving the problem that triple-negative breast cancer in the prior art lacks targeted treatment methods, and the large molecular weight of the corresponding ADC drug antibody component limits the internalization efficiency of ADCs, thereby causing off-target effects and immunotoxicity.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides an aptamer conjugate targeting triple-negative breast cancer, the aptamer conjugate comprising a non-natural nucleic acid aptamer that targets internalization into triple-negative breast cancer, a degradable linker connected to the non-natural nucleic acid aptamer, and a cytotoxic agent connected to the degradable linker, wherein the non-natural nucleic acid aptamer comprises a fluorine-modified nucleotide.

[0008] Preferably, the fluorine-modified nucleotides are 2'-F-modified cytosine nucleotides and / or uracil nucleotides.

[0009] Preferably, the degradable linker is a pH-responsive degradable linker CL2A linker.

[0010] Preferably, the cytotoxic agent is selected from SN38, irinotecan, Capecitabine, topoisomerase I.

[0011] In a second aspect, the present invention provides a method for preparing an aptamer conjugate targeting triple-negative breast cancer as described above, the method comprising: in vitro screening of non-natural nucleic acid aptamers targeting triple-negative breast cancer cells; high-throughput sequencing of the non-natural nucleic acid aptamers obtained by screening, and selecting sequences with a high copy number for targeted internalization ability testing; and connecting a degradable linker CL2A and a cytotoxic agent to the ends of the non-natural nucleic acid aptamer to obtain a non-natural nucleic acid aptamer conjugate targeting triple-negative breast cancer cells.

[0012] Preferably, the in vitro screening of non-natural nucleic acid aptamers targeting triple-negative breast cancer cells comprises:

[0013] 1) Using a biotin-labeled single-stranded DNA library containing 40 random bases as a template, a non-natural nucleic acid library modified with fluorine elements was synthesized by in vitro "transcription", separated by streptavidin resin, and then collected by ultrafiltration;

[0014] 2) Constructing normal mammary epithelial cell and triple-negative breast cancer cell models in vitro, incubating them with non-natural nucleic acid libraries, and extracting non-natural nucleic acid components in the cell models;

[0015] 3) Using the recovered non-natural nucleic acid component as a template, a non-natural nucleic acid polymerase is used to "reverse transcribe" the non-natural nucleic acid into a natural DNA sequence for the next round of screening.

[0016] Preferably, the targeted internalization ability test of the non-natural nucleic acid aptamer includes:

[0017] 1) Under the catalysis of non-natural nucleic acid polymerase, "transcription" synthesizes non-natural nucleic acid sequences:

[0018] 2) separation of non-natural nucleic acid chains by streptavidin resin;

[0019] 3) The non-natural nucleic acid chain was incubated with normal breast epithelial cells and triple-negative breast cancer cell models for 1 hour, and the targeted internalization ability was analyzed by flow cytometry.

[0020] Preferably, connecting the degradable linker CL2A and the cytotoxic agent to the end of the non-natural nucleic acid aptamer comprises: connecting the degradable linker CL2A and the cytotoxic agent to the end of the non-natural nucleic acid aptamer by amidation reaction to form an aptamer conjugate molecule based on non-natural nucleic acid.

[0021] In a third aspect, the present invention provides use of the aptamer conjugate targeting triple-negative breast cancer as described above for preparing a drug for treating triple-negative breast cancer.

[0022] In a fourth aspect, the present invention provides a drug for treating triple-negative breast cancer, wherein the drug comprises the aptamer conjugate targeting triple-negative breast cancer as described above.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: the non-natural nucleic acid-based aptamer conjugate of the present invention utilizes non-natural nucleic acid as a molecular tool, combined with the pH-responsive degradable linker CL2A to couple AptT1 with the cytotoxic agent SN38, thereby achieving specific killing of triple-negative breast cancer. Compared with the existing antibody conjugates, it has a similar anti-tumor effect, and is significantly better than SN38 administered alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a technical principle diagram of the solution of the present invention;

[0025] Figure 2 This is a diagram illustrating the principle of connecting the non-natural nucleic acid aptamer AptT1 with the degradable linker CL2A and the cytotoxic agent SN38;

[0026] Figure 3 This is a schematic diagram of screening non-natural nucleic acid aptamer AptT1 for internalization into triple-negative breast cancer: Figure 3 A is a schematic diagram of the screening process; Figure 3 B is a schematic diagram of the two-dimensional structure of AptT1; Figure 3 C is the test result of flow cytometry testing the ability of AptT1 to target and internalize into triple-negative breast cancer and normal breast epithelial cells; Figure 3 D is a schematic diagram of flow cytometry testing the ability of AptT1 and its control sequence to target and internalize into triple-negative breast cancer; Figure 3 E is a photograph of the internalization ability of AptT1 in normal mammary epithelial cells and triple-negative breast cancer cell models tested by confocal microscopy;

[0027] Figure 4This is a schematic diagram of the secondary structure prediction of the four families with the highest enrichment after screening and their representative sequences;

[0028] Figure 5 This is a diagram of the mechanism of internalization of non-natural nucleic acid aptamers into triple-negative breast cancer, in which: Figure 5 A: The effect of four different endocytosis inhibitors on the internalization ability of AptT1; Figure 5 B and Figure 5 C is a schematic diagram of the Pull-Down experiment and molecular docking simulation of the binding of AptT1 and cell membrane protein ANXA2; Figure 5 D is a Western blot experiment demonstrating the binding of AptT1 to the cell membrane protein ANXA2; Figure 5 E and Figure 5 F is the result of flow cytometry to test the change of AptT1 internalization ability in triple-negative breast cancer after knocking down ANXA2 protein with ANXA2-specific siRNA; Figure 5 G and Figure 5 H is the dissociation constant of AptT1 and ANXA2 determined by dot blot method;

[0029] Figure 6 This is a diagram of the construction of aptamer conjugates based on non-natural nucleic acids. Figure 6 A shows the gel electrophoresis migration method to test the construction efficiency of non-natural nucleic acid aptamer conjugates; Figure 6 B is the molecular weight of the non-natural nucleic acid aptamer conjugate product tested by mass spectrometry; Figure 6 C. Figure 6 D and Figure 6 E is the flow cytometry test of the internalization ability of the non-natural nucleic acid aptamer conjugates in the triple-negative breast cancer cell model; Figure 6 F is a photograph of the internalization ability of the non-natural nucleic acid aptamer conjugates in normal breast epithelial cells and triple-negative breast cancer cell models tested by confocal microscopy;

[0030] Figure 7 It is a test of the anti-tumor ability of non-natural nucleic acid aptamer conjugates in triple-negative breast cancer-bearing mice. Figure 7 A is a schematic diagram of interval dosing in tumor-bearing mice; Figure 7 B is the growth curve of tumor volume of mice in different drug administration groups; Figure 7 C is a photo comparing the sizes of tumors in mice in different drug-dosing groups after intraperitoneal injection. Figure 7 D is the tumor weight of mice in different drug administration groups; Figure 7 E is the growth curve of the body weight of mice in different drug administration groups; Figure 7 F is the hematoxylin & eosin (H&E) staining of the tumors of mice in different drug-administered groups. DETAILED DESCRIPTION

[0031] We first used the cell internalization SELEX technology to generate a chemically modified aptamer AptT1 for triple-negative breast cancer, which showed excellent cell specificity in vitro. Subsequently, we constructed ApDC (T1-CL2A-SN38) by coupling AptT1, CL2A linker and cytotoxic drug SN38. This ApDC showed good safety and efficacy both in vitro and in vivo. Overall, we provide a promising model for the precision treatment of triple-negative breast cancer.

[0032] In an exemplary embodiment, the present invention provides a non-natural nucleic acid-based aptamer conjugate, which includes an aptamer AptT1 that targets internalization into triple-negative breast cancer cells, and a degradable linker CL2A and a cytotoxic agent SN38 are connected to the ends thereof.

[0033] The above-mentioned aptamer conjugate based on non-natural nucleic acid, wherein the non-natural nucleic acid comprises 2'-F modified cytosine nucleotides and / or uracil nucleotides.

[0034] In the above-mentioned non-natural nucleic acid-based aptamer conjugate, the cytotoxic agent includes SN38, irinotecan, Capto or topoisomerase I.

[0035] In another exemplary embodiment, the present invention provides a method for preparing a non-natural nucleic acid aptamer conjugate, which includes the following steps: in vitro screening of non-natural nucleic acid aptamers targeting triple-negative breast cancer cells; high-throughput sequencing of the non-natural nucleic acid aptamers, and selecting sequences with high copy numbers for targeted internalization ability testing; connecting the degradable linker CL2A and the cytotoxic agent SN38 to the end of the non-natural nucleic acid aptamer AptT1, thereby preparing a non-natural nucleic acid aptamer conjugate that targets and kills triple-negative breast cancer cells.

[0036] In the above-mentioned method for preparing the aptamer conjugate based on non-natural nucleic acid, the in vitro screening of non-natural nucleic acid aptamers comprises the following steps:

[0037] 1) Using a biotin-labeled single-stranded DNA library containing 40 random bases as a template, a non-natural nucleic acid library modified with fluorine elements was synthesized by in vitro "transcription", separated by streptavidin resin, and then collected by ultrafiltration;

[0038] 2) constructing normal mammary epithelial cells and triple-negative breast cancer cell models in vitro, incubating them with the non-natural nucleic acid library, the incubation time between the non-natural nucleic acid library and the cell model is 60 minutes from the first to the fourth round, and 30 minutes from the fifth to the ninth round, and extracting the non-natural nucleic acid components in the cell model by the Trizol method;

[0039] 3) Using the recovered non-natural nucleic acid component as a template, a non-natural nucleic acid polymerase is used to "reverse transcribe" the non-natural nucleic acid into a natural DNA sequence for the next round of screening.

[0040] In the above-mentioned method for preparing the aptamer conjugate based on non-natural nucleic acid, the targeted internalization ability test comprises the following steps:

[0041] 1) Under the catalysis of non-natural nucleic acid polymerase, "transcription" synthesizes non-natural nucleic acid sequences:

[0042] 2) separation of non-natural nucleic acid chains by streptavidin resin;

[0043] 3) The non-natural nucleic acid chain was incubated with normal breast epithelial cells and triple-negative breast cancer cell models for 1 hour, and the targeted internalization ability was analyzed by flow cytometry.

[0044] In the above-mentioned preparation method based on non-natural nucleic acid aptamer conjugate, the specific method of connecting the degradable linker CL2A and the cytotoxic agent SN38 to the end of the non-natural nucleic acid aptamer is: using an amidation reaction to connect the degradable linker CL2A and the cytotoxic agent SN38 to the end of the non-natural nucleic acid aptamer AptT1 to form an aptamer conjugate molecule based on a non-natural nucleic acid.

[0045] The present invention further discloses the application of the aptamer conjugates based on the non-natural nucleic acid. The aptamer conjugates based on the non-natural nucleic acid can be used for triple-negative breast cancer tumor imaging and inhibiting the growth of triple-negative breast cancer.

[0046] The following will be combined with the specific embodiments of the present invention and the accompanying drawings to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front end", "rear end", "two ends", "one end", "the other end" and the like indicating the orientation or positional relationship are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0048] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "provided with", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0049] The embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0050] Example 1

[0051] like Figure 1 , Figure 2 and Figure 3 As shown, the present invention provides an aptamer conjugate based on non-natural nucleic acids, including a non-natural nucleic acid sequence that specifically targets and internalizes into triple-negative breast cancer cells, and at the same time, a degradable linker CL2A and a cytotoxic agent SN38 are connected to the end of the non-natural nucleic acid aptamer. The non-natural nucleic acid includes 2'-F modified cytosine nucleotides and uracil nucleotides. Cytotoxic agents include SN38, irinotecan, Capto, and topoisomerase I. Nucleic acid aptamers are a class of biological macromolecules with targeting functions. The introduction of fluorine-modified non-natural nucleic acids enhances the anti-degradation ability of nucleic acid aptamers and the ability to target and internalize into triple-negative breast cancer cells, and has better targeting for triple-negative breast cancer. The aptamer conjugate based on non-natural nucleic acids realizes the precise delivery and release of cytotoxic agents, and has a good therapeutic effect on triple-negative breast cancer.

[0052] The method for preparing the aptamer conjugate based on non-natural nucleic acid of the present invention comprises the following steps:

[0053] Step 1: In vitro screening of non-natural nucleic acid aptamers, including the following steps:

[0054] 1) Prepare a non-natural nucleic acid library, refer to Table 1 and Figure 1 , Figure 3 As shown, a single-stranded DNA library containing 40 random bases labeled with biotin was used as a template to exogenously add a Cy5-labeled DNA primer Primer2R, and a non-natural nucleic acid library was synthesized by in vitro "transcription" under the catalysis of SFM4-3 polymerase, and the single strands of the non-natural nucleic acid library were separated by streptavidin resin separation. Normal breast epithelial cells and triple-negative breast cancer cell models were constructed in vitro and incubated with non-natural nucleic acid libraries under simulated physiological conditions (136mMNaCl, 2.6mM KCl, 4mM Na,HPO, 1.8mM KH,P04, 2mM MgC1, pH 7.4);

[0055] 2) Recovering non-natural nucleic acids, refer to Figure 1 and Figure 3 , removing the supernatant, recovering the cells that have internalized the non-native nucleic acid, and recovering the non-native nucleic acid sequence internalized by the cells according to the instructions of the kit;

[0056] 3) Using the recovered components as templates, use M-MLV to "reverse transcribe" the non-natural nucleic acid sequence into a corresponding DNA sequence: perform PCR amplification, separate the single strands, recover the DNA template, and then perform a "transcription" reaction to prepare a non-natural nucleic acid library, thereby generating a non-natural nucleic acid library that can be used for the next round of screening;

[0057] The steps of "transcription-strand separation-incubation-target binding component separation-reverse transcription-amplification-strand separation" are carried out in a cycle.

[0058] The above experimental steps were repeated for 9 rounds.

[0059] Step 2: The non-natural nucleic acid aptamers after screening are subjected to high-throughput sequencing, and sequences with high copy numbers are selected for targeted internalization ability testing, including the following steps:

[0060] 1) Using the biotin-labeled DNA sequence as a template, the non-natural nucleic acid sequence was "transcribed" and synthesized under the catalysis of SFM4-3: the enriched DNA library was subjected to high-throughput sequencing, and the sequences with higher copy numbers were selected for targeted internalization ability testing and sequence statistical analysis. The secondary structures of the three sequences with the highest copy numbers are as follows: Figure 3 As shown;

[0061] 2) Separation of Cy5-AptT1 by streptavidin column;

[0062] 3) The non-natural nucleic acid chain was incubated with normal breast epithelial cells and triple-negative breast cancer cell models for 1 hour, and the targeting internalization ability was analyzed by flow cytometry and confocal microscopy. The results are as follows Figure 3 As shown in CE.

[0063] Step 3: Connecting the pH-responsive degradable linker CL2A and the cytotoxic agent SN38 to the end of the non-natural aptamer AptT1 targeting triple-negative breast cancer to obtain a non-natural nucleic acid aptamer conjugate targeting triple-negative breast cancer, comprising the following steps:

[0064] 1) The pH-responsive degradable linker CL2A and the cytotoxic agent SN38 were connected to the end of the non-natural aptamer AptT1 targeting triple-negative breast cancer by amidation reaction to form T1-CL2A-SN38 molecules, such as Figure 2 As shown;

[0065] 2) Treat triple-negative breast cancer cells with T1-CL2A-SN38 molecules.

[0066] Reference Figure 5-Figure 7 , the test results of this embodiment are as follows:

[0067] (1) Testing of AptT1 targeting and internalization mechanism.

[0068] 1) Flow cytometry

[0069] Cells were seeded in 12-well plates and incubated overnight at 37°C. Libraries or sequences were modified with Cy5 fluorophores. After washing twice with 500 μL PBS, cells were incubated with each round of libraries or increasing concentrations of aptamers at 37°C for 1 hour. After 3 washes with PBS, cells were suspended with 1 mL PBS and analyzed by flow cytometry.

[0070] 2) Confocal microscopy analysis

[0071] 2×10 5 The relevant cell models were seeded in confocal dishes at a density of 10 cells / dish and incubated overnight. Cy5-labeled nucleic acid sequences were added to the dishes and incubated with the cells for 1 hour. After three PBS washes, the cells were incubated with 4% paraformaldehyde for 30 minutes to fix them, and then the cell nuclei were stained with Hoechst33258 for 5 minutes. Finally, fluorescent images of the cells were obtained using an Olympus laser scanning confocal microscope.

[0072] 3) Structure prediction and molecular docking

[0073] The secondary structure of the aptamer was predicted by RNAfold, while the tertiary structure prediction was completed by RNAcomposer. The tertiary structure of ANXA2 was obtained from the PDB website. Molecular docking simulation was performed using the HDock website.

[0074] 4) Pull-Down Experiment

[0075] After washing triple-negative breast cancer cells twice in PBS, they were lysed with lysis buffer for 10 minutes. The cell lysate was collected and incubated on ice for 30 minutes. Subsequently, the supernatant was collected by centrifugation. The biotin-labeled aptamer AptT1 (DNA version and scrambled version as negative controls) were incubated with streptavidin-coated magnetic beads at room temperature for 30 minutes. Then, the aptamer-magnetic bead conjugate was mixed with the cell lysate supernatant to allow the aptamer to interact with the potential target protein at room temperature for 30 minutes. After washing three times with washing buffer (10mmol / L NaCl, 20mmol / L pH7.5 Tris and 0.1% Tween-20) to remove proteins that could not bind, the magnetic beads with proteins were boiled in PBS. The resulting supernatant was analyzed by LC-MS / MS.

[0076] 5) Dot blot analysis

[0077] ANXA2 protein was incubated with Cy5-labeled AptT1 in 50 μL reaction buffer (1×PBS containing 1 mmol / L MgCl2 and 1 mmol / L CaCl2) at room temperature for 60 min. Filter binding was performed using a dot blot apparatus, nitrocellulose membrane, and nylon membrane. The samples were vacuum adsorbed and washed with 50 μL PBS buffer. After air drying, the membrane strips were imaged and quantified using the Odyssey CLx imaging system. Dissociation constants were calculated using GraphPad Prism 10.

[0078] (2) Construction of non-natural nucleic acid aptamer conjugates

[0079] AptT1 was mixed with a 100-fold excess of Tris (2-carboxyethyl) hydrogen phosphate hydrochloride (TCEP) solution and incubated on ice for 90 minutes. The mixture was placed in an ultrafiltration tube and centrifuged at 12,000 rpm for 5 minutes, and this process was repeated three times to remove TCEP. Subsequently, a three-fold excess of CL2A-SN38, one-fifth volume of 3 mol / L sodium acetate (NaOAc, pH 6.5), and one-fifth volume of 10× PBS were added in sequence, and the total volume was doubled with N,N-dimethylformamide (DMF). After incubation with shaking at room temperature for 30 minutes, the sample was placed in an ultrafiltration tube and centrifuged at 12,000 rpm for 5 minutes, and this operation was repeated three times. The product was confirmed by 12% denaturing polyacrylamide gel electrophoresis (PAGE) and mass spectrometry.

[0080] (3) Anti-tumor ability test of non-natural nucleic acid aptamer conjugates

[0081] 1) Construction of triple-negative breast cancer xenograft model

[0082] Female BALB / c nude mice aged 3-4 weeks were placed under a 12-h light / dark cycle with free access to food and water. The concentration of MDA-MB-231 cells was adjusted to 1 × 10 7 100 μL of cell suspension was injected into the mammary fat pad of each nude mouse. Tumor growth was observed every 3 days.

[0083] 2) In vivo anti-tumor efficacy test

[0084] On day 7 after triple-negative breast cancer tumor implantation, all tumor-positive nude mice were randomly assigned to different groups for subsequent treatment. These groups were treated with 0.9% saline (negative control), SN38 (1 mg / kg), T1-CL2A-SN38 (6 mg / kg), S1-CL2A-SN38 (equimolar to T1-CL2A-SN38) or ADC drug SG (equimolar to T1-CL2A-SN38). SN38, T1-CL2A-SN38, S1-CL2A-SN38 and ADC drug SG were all dissolved in sterile 0.9% sodium chloride solution. Treatment was performed every three days for a total of seven times. Before each treatment, the tumor growth and body weight changes of nude mice were systematically monitored. After the treatment, the tumors were removed, weighed and stained to determine the therapeutic effect.

[0085] 3) Hematoxylin and eosin (H&E) staining

[0086] Tumors and major organs were removed from nude mice. They were first fixed with tissue fixatives and then dehydrated in ethanol solutions of varying concentrations (75-100%). After washing with xylene, the organs or tumors were paraffin-embedded and cut into 4 μm sections. The tissues were flattened in 40°C water, removed and baked at 60°C. After the water dried and the wax melted, they were removed and stained according to the standard H&E staining protocol.

[0087] The non-natural nucleic acid-based aptamer conjugate of the present invention uses non-natural nucleic acids as molecular tools, and combines the pH-responsive degradable linker CL2A to couple AptT1 with the cytotoxic agent SN38 to achieve specific killing of triple-negative breast cancer. Compared with existing antibody conjugates, it has similar anti-tumor effects and is significantly better than SN38 alone. The introduction of fluorine-modified non-natural nucleic acids enhances the anti-degradation ability of the aptamer conjugate and its ability to target and internalize triple-negative breast cancer cells, making it more suitable for tumor-bearing mice and human studies, and provides a new means for the targeted treatment of triple-negative breast cancer.

[0088] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

[0089] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented and modified over a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. This application is intended to include any modifications, uses or improvements of the present invention.

[0090] The description of the sequences used in the specification is shown in Table 1 below.

[0091] Table 1, Description of sequences used in the specification

[0092]

[0093]

[0094] The present invention belongs to the field of non-natural nucleic acid screening and targeted treatment of triple-negative breast cancer, and in particular, relates to a method for preparing an aptamer-drug conjugate (ApDC) based on a non-natural nucleic acid modified with fluorine and its application in targeted treatment of triple-negative breast cancer. The present invention first uses the cell internalization screening technology (Cellinternalization SELEX) to identify the non-natural nucleic acid aptamer AptT1 that can be targeted and internalized into triple-negative breast cancer cells, and further couples AptT1 with the cytotoxic agent SN38 through the pH-responsive degradable linker CL2A to construct an aptamer conjugate (T1-CL2A-SN38) based on non-natural nucleic acid. The aptamer conjugate can specifically target and internalize into triple-negative breast cancer cells, and then accurately release the cytotoxic agent SN38 through the pH-responsive linker CL2A. Further data show that T1-CL2A-SN38 can better target tumor lesions in triple-negative breast cancer tumor-bearing mice, and efficiently inhibit the growth of tumors in triple-negative breast cancer tumor-bearing mice, without obvious biological toxicity. The non-natural nucleic acid-based aptamer conjugates in the present invention have similar anti-tumor effects compared to existing antibody conjugates, and are significantly better than SN38 alone. The introduction of fluorine-modified non-natural nucleic acids enhances the aptamer conjugates' ability to resist degradation and target and internalize into triple-negative breast cancer cells, making them more suitable for tumor-bearing mice and human studies, providing a new means for targeted treatment of triple-negative breast cancer.

[0095] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0096] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified and replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. An aptamer conjugate targeting triple-negative breast cancer, characterized in that: The aptamer conjugate comprises a non-natural nucleic acid aptamer that targets internalization into triple-negative breast cancer, a degradable linker connected to the non-natural nucleic acid aptamer, and a cytotoxic agent connected to the degradable linker, wherein the non-natural nucleic acid aptamer comprises a fluorine-modified nucleotide.

2. The aptamer conjugate targeting triple-negative breast cancer according to claim 1, characterized in that The fluorine-modified nucleotides are 2'-F-modified cytosine nucleotides and / or uracil nucleotides.

3. The aptamer conjugate targeting triple-negative breast cancer according to claim 1, characterized in that: The degradable linker is a pH-responsive degradable linker CL2A linker.

4. The aptamer conjugate targeting triple-negative breast cancer according to claim 1, characterized in that The cytotoxic agent is selected from SN38, irinotecan, Capto, and topoisomerase I.

5. The method for preparing an aptamer conjugate targeting triple-negative breast cancer according to any one of claims 1 to 4, characterized in that: The method comprises: in vitro screening of non-natural nucleic acid aptamers targeting triple-negative breast cancer cells; high-throughput sequencing of the screened non-natural nucleic acid aptamers, and selecting sequences with high copy numbers for targeted internalization ability testing; and connecting a degradable linker CL2A and a cytotoxic agent to the ends of the non-natural nucleic acid aptamer, thereby preparing a non-natural nucleic acid aptamer conjugate targeting triple-negative breast cancer cells.

6. The method according to claim 5, characterized in that In vitro screening of non-natural nucleic acid aptamers targeting triple-negative breast cancer cells includes: 1) Using a biotin-labeled single-stranded DNA library containing 40 random bases as a template, a non-natural nucleic acid library modified with fluorine elements was synthesized by "transcription" in vitro, separated by streptavidin resin, and then collected by ultrafiltration; 2) Constructing normal mammary epithelial cell and triple-negative breast cancer cell models in vitro, incubating them with non-natural nucleic acid libraries, and extracting non-natural nucleic acid components in the cell models; 3) Using the recovered non-natural nucleic acid component as a template, a non-natural nucleic acid polymerase is used to "reverse transcribe" the non-natural nucleic acid into a natural DNA sequence for the next round of screening.

7. The method according to claim 5, characterized in that The targeted internalization ability test of non-natural nucleic acid aptamers includes: 1) Under the catalysis of non-natural nucleic acid polymerase, "transcribe" and synthesize non-natural nucleic acid sequences: 2) separation of non-natural nucleic acid chains by streptavidin resin; 3) The non-natural nucleic acid chain was incubated with normal breast epithelial cells and triple-negative breast cancer cell models for 1 hour, and the targeted internalization ability was analyzed by flow cytometry.

8. The method according to claim 5, characterized in that Connecting the degradable linker CL2A and the cytotoxic agent to the end of the non-natural nucleic acid aptamer includes: connecting the degradable linker CL2A and the cytotoxic agent to the end of the non-natural nucleic acid aptamer by amidation reaction to form an aptamer conjugate molecule based on non-natural nucleic acid. 9 . Use of the aptamer conjugate targeting triple-negative breast cancer according to any one of claims 1 to 4 for preparing a drug for treating triple-negative breast cancer.

10. A drug for treating triple-negative breast cancer, characterized in that: The drug comprises the aptamer conjugate targeting triple-negative breast cancer according to any one of claims 1 to 4.

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