Aptamer specifically recognizing CD276, and use thereof

Abstract

An aptamer specifically recognizing CD276, wherein the aptamer has a sequence as shown in SEQ ID NO: 1 or other nucleotide sequences with more than 80% homology thereto. Also disclosed are a use of the aptamer specifically recognizing CD276 in the preparation of a targeted therapeutic reagent, and a use of the aptamer specifically recognizing CD276 in the preparation of a tumor-targeted diagnostic kit. The aptamer has higher binding affinity for CD276 protein and good specificity, is easier to modify and synthesize artificially, has strong stability, and is convenient to use.

Description

Nucleic acid aptamers that specifically recognize CD276 and their applications Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to nucleic acid aptamers that specifically recognize CD276 and their applications. Background Technology

[0002] CD276, also known as B7-H3, belongs to the B7 family of immune costimulatory molecules. As an immune checkpoint, it plays an important role in tumor immunity, regulating the infiltration of immune cells into tumor tissues. [1,2] At the same time, CD276 also plays an important role in tumorigenesis and development, participating in various processes such as tumor progression, metastasis, and drug resistance. [3] CD276 protein is widely expressed at low levels in various organs and tissues of the human body, but highly expressed in a variety of solid tumors, including gastric cancer. Furthermore, its expression level has been shown to be correlated with tumor malignancy and prognosis. [4] Therefore, it may serve as a potential target for tumor treatment.

[0003] Nucleic acid aptamers that specifically recognize the CD276 protein can regulate immune responses by binding to the protein, and can also enable targeted delivery of chemotherapy drugs to achieve better tumor treatment effects.

[0004] Nucleic acid aptamers are DNA or RNA molecules isolated through systematic evolution of ligands by exponential enrichment (SELEX) technology. They can bind with high affinity and specificity to other targets such as proteins, metal ions, small molecules, peptides, and even whole cells, thus showing broad prospects in biochemical analysis, environmental monitoring, basic medicine, and new drug synthesis. [5] Compared to antibodies, nucleic acid aptamers have advantages such as smaller molecular weight, better stability, easier modification, no immunogenicity, shorter production cycle, and can be synthesized artificially, eliminating a series of processes such as animal immunization, feeding, protein extraction, and purification. [6] Therefore, finding a nucleic acid aptamer with higher affinity and higher specificity for the CD276 protein would help achieve high-sensitivity and high-specificity detection of the CD276 protein, and would facilitate the development of aptamer-drug conjugates (ApDCs) targeting the CD276 protein. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, we provide nucleic acid aptamers that specifically recognize CD276 and their applications.

[0006] This invention is achieved through the following scheme:

[0007] An aptamer that specifically recognizes CD276, having the sequence shown in SEQ 1, or other nucleotide sequences that are more than 80% homologous to the sequence shown in SEQ 1.

[0008] In one embodiment, the nucleic acid aptamer is modified, and the modification is a modification of the base or phosphate backbone.

[0009] In one embodiment, the base modification includes drug base modification, F substitution, MOE modification, OMe modification, cEt modification, and inverted T modification. Drug base modification includes substitution or terminal coupling of gemcitabine, fluorouracil, etc.

[0010] In one embodiment, the phosphate backbone is modified to be partially or fully thiolated.

[0011] In one embodiment, the nucleic acid aptamer is terminally coupled, and the coupled material includes a fluorescent group, a drug, a base analog, a sugar modification, a peptide, a radioactive substance, biotin, and a quantum dot.

[0012] In one embodiment, the nucleic acid aptamer is a partially or completely sequence-transformed nucleic acid aptamer; the sequence transformation may be transcribing a DNA sequence into an RNA sequence or translating it into a peptide nucleic acid sequence.

[0013] This invention also relates to a method for preparing a nucleic acid aptamer that specifically recognizes CD276, the method comprising the following steps:

[0014] An aptamer library targeting CD276 was obtained through multiple rounds of protein screening.

[0015] Select the most abundant aptamer samples from the aptamer library;

[0016] We selected well-evolved nucleic acid aptamers for binding verification and obtained nucleic acid aptamers with high affinity.

[0017] The present invention also relates to the application of a nucleic acid aptamer that specifically recognizes CD276 in the preparation of reagents for targeted tumor therapy or diagnostic tumor kits, wherein the reagents or kits contain the nucleic acid aptamer that specifically recognizes CD276, or its derivatives, for the recognition, degradation or drug delivery of CD276 in tissues, cells or blood.

[0018] The beneficial effects of this invention are as follows:

[0019] The nucleic acid aptamer of the present invention has a higher binding affinity for CD276 protein, good specificity, is easy to modify and synthesize artificially, has strong stability, and is convenient to use, and can be better applied to the field of preparing tumor-targeted drugs. Attached Figure Description

[0020] Figure 1 shows the experimental results of verifying the binding effect of the sequence with CD276 protein using SPR.

[0021] Figure 2 shows the SPR affinity experimental data of Aptemer containing the SEQ 1 sequence.

[0022] Figure 3 shows the affinity verification results of AGS and HGC27 cell lines with Aptemer containing the SEQ 1 sequence.

[0023] Figure 4 shows the results of the CCK-8 assay to verify the toxicity of ApDC to CD276-positive AGS cell lines.

[0024] Figure 5 shows the results of in vivo mouse experiments verifying the targeting of Aptemer containing the SEQ 1 sequence.

[0025] Figure 6 shows the experimental results of verifying the efficacy of ApDC targeted therapy in mice.

[0026] Figure 7 is a schematic diagram of the connection between nucleic acid aptamers and chemotherapy drugs.

[0027] Figure 8 is a flowchart of the workflow of the present invention.

[0028] Figure 9 shows the experimental results of the stability test of the modified nucleic acid aptamer of the present invention.

[0029] Figure 10 shows the experimental results of verifying the targeting of ApDC to other tumor cells besides gastric adenocarcinoma cells using the CCK-8 assay. Detailed Implementation

[0030] The preferred embodiments of the present invention are further described below.

[0031] A nucleic acid aptamer that specifically recognizes CD276, characterized in that: the nucleic acid aptamer has a sequence as shown in SEQ 1, or contains other nucleotide sequences that are more than 80% homologous to the sequence shown in SEQ 1.

[0032] In one embodiment, the nucleic acid aptamer may be modified with bases or with a phosphate backbone.

[0033] Base modifications include drug base modifications, F substitution, MOE modification (or RNA analog modification such as OMe), cEt modification (restricted ethyl modification), and inverted T modification, such as 2'-O-Me, 2'-O-MOE, 2'-F, 2'-(s)-restricted ethyl (s-cEt), etc. Drug base modifications include substitution or terminal coupling of other drugs such as gemcitabine and fluorouracil. Phosphate backbone modifications can be partial or complete thiolation.

[0034] Nucleic acid aptamers can also be terminally coupled to form conjugates, which include fluorescent groups, drugs, base analogs, sugar modifications, peptides, radioactive substances, biotin, and quantum dots.

[0035] In this invention, the aforementioned aptamer may be coupled with MMAE (Monomethyl auristatin E), gemcitabine, or 5-fluorouracil. Figure 7 illustrates a mode of linking a nucleic acid aptamer with a chemotherapeutic drug. The nucleic acid aptamer described herein is a partially or completely sequence-transformed aptamer; sequence transformation may involve transcribing a DNA sequence into an RNA sequence or translating it into a peptide nucleic acid sequence.

[0036] Modification of the base sequence is mainly to enhance its resistance to nucleases; to date, many studies have confirmed that the stability of the modified sequence is improved. [7] .

[0037] Drug base modification allows for the precise conjugation of nucleic acid aptamers to drugs directly through DNA solid-phase synthesis technology, and enables modification at specific base positions. [8-10] .

[0038] In the method for preparing nucleic acid aptamers that specifically recognize CD276, the method includes the following steps:

[0039] An aptamer library targeting CD276 was obtained through multiple rounds of protein screening.

[0040] Select the most abundant aptamer samples from the aptamer library;

[0041] We selected well-evolved nucleic acid aptamers for binding verification and obtained nucleic acid aptamers with high affinity.

[0042] On the other hand, this invention relates to the application of a nucleic acid aptamer that specifically recognizes CD276 in the preparation of targeted therapeutic reagents or kits. It can also be used to achieve tumor diagnosis or treatment effects based on this CD276-targeting nucleic acid aptamer, for example, in the preparation of a tumor diagnostic kit that can specifically recognize CD276. The aforementioned reagents or kits contain this nucleic acid aptamer that specifically recognizes CD276 for targeted degradation, targeted drug delivery, and detection analysis of CD276. They may also contain the nucleic acid aptamer and its derivatives for the recognition, degradation, or drug delivery of CD276 in tissues, cells, or blood.

[0043] This invention obtains an Aptamer library targeting the CD276 protein through a seven-round protein screening process. High-throughput sequencing and analysis yielded the 96 most abundant Aptamers. Sequences with good evolutionary characteristics were selected for binding verification, resulting in sequences with high affinity. These Aptamers exhibit excellent targeting ability for the CD276 protein and can be used for targeted degradation, targeted drug delivery, and detection analysis of CD276. The workflow of this application is shown in Figure 8.

[0044] This invention can provide new tools for targeting CD276 protein, new methods for degrading CD276 protein, and new ideas for CD276 protein-based targeted therapy and tumor diagnosis.

[0045] It is important to note that current research on aptamer has demonstrated that the same or highly homologous aptamer can exhibit high targeting specificity for various target protein-expressing cell lines. [11-13] Those skilled in the art will be motivated and can imagine applying the nucleic acid aptamers described in this invention to the targeted therapy or diagnosis of other tumors besides those described in the following embodiments, such as in the preparation of other tumor targeted therapy reagents or diagnostic kits.

[0046] The present application will be further described below with reference to specific embodiments.

[0047] Example 1 Aptamer screening and affinity test for CD276 containing the SEQ 1 sequence.

[0048] The specific process and method described in this invention can be seen in Figure 8.

[0049] As shown in Figure 1, 96 sequences with high enrichment were selected through sequencing analysis. The affinity of these sequences (01-96) for binding to CD276 protein was verified by surface plasmon resonance (SPR). In Figure 1, "03" represents the Aptamer containing the SEQ 1 sequence described in this invention.

[0050] As shown in Figure 2, surface plasmon resonance (SPR) experiments show that the dissociation curves of Aptamer (apt-3) containing the SEQ 1 sequence exhibit slow dissociation rates at different wavelengths, indicating that it has excellent affinity for CD276 protein.

[0051] Flow cytometry experiments confirmed the targeting of the selected SEQ1 nucleotide sequence to CD276-positive AGS and HGC27 cell lines.

[0052] Adherent cell lines (AGS, HGC27) were digested with 0.02% EDTA solution. Each 200 mL cell suspension was incubated with 10 μM Cy5-labeled aptamer on ice for 30 min. The cells were then resuspended in washing water and centrifuged twice. Fluorescence intensity was detected by flow cytometry.

[0053] The experimental results are shown in Figure 3 (CD276-3 shown in Figure 3 is the Aptamer containing the SEQ1 sequence described in this invention). The above flow cytometry experiment confirmed that the SEQ1 sequence obtained by screening also showed excellent affinity compared with the control group.

[0054] Example 2: Aptamer containing the SEQ 1 sequence that specifically recognizes CD276 is used for targeted therapy of CD276-positive tumors.

[0055] As shown in Figure 4, the cytotoxicity of ApDC to CD276-positive AGS cell lines was verified at the cellular level using the CCK-8 assay.

[0056] After digestion, the AGS cell line was centrifuged, resuspended, and counted. It was then appropriately diluted and seeded evenly in 96-well plates at a density of 5000 cells per well. The next day, once the cells had adhered, different concentration gradients of drugs were added, and the plates were incubated at 37°C for 2 days. The cells were then replaced with CCK-8 assay solution, and the plates were incubated for 1 hour before detection. As shown in Figure 4, the experimental results indicate that ApDC exhibits superior toxicity to CD276-positive AGS cell lines compared to the drug monomer.

[0057] As shown in Figure 5, the targeting of Aptamer containing the SEQ 1 sequence was verified through in vivo experiments in mice.

[0058] Female nude mice aged 4-6 weeks were selected, and subcutaneous tumor formation was performed in the right axilla. Each mouse was injected with approximately 1×10⁻⁶ ppm. 7 SNU601 cells were prepared by mixing cell suspension with matrix gel at a 1:1 ratio. When the tumor volume reached 200 mm3, 100 μL of 10 μM Cy5-labeled aptamer was injected via the tail vein. Fluorescence intensity and distribution were detected using a small animal in vivo imaging system at 0 h, 0.5 h, 1 h, 2 h, 3 h, and 4 h post-injection.

[0059] As shown in Figure 6, the targeted therapeutic effect of ApDC was verified through in vivo experiments in mice.

[0060] Female nude mice aged 4-6 weeks were selected, and subcutaneous tumor formation was performed in the right axilla. Each mouse was injected with approximately 1×10⁻⁶ ppm. 7SNU601 cells were prepared by mixing cell suspension and matrix gel at a 1:1 ratio. Drug administration began when the maximum tumor diameter reached 5 mm. Each mouse was injected intravenously with 100 μL of the 75 μM drug twice a week for a total of six administrations. After the drug administration was completed, the mice were fed for another two weeks, and tumor size and body weight were measured and recorded. As shown in Figure 6, the CD276 aptamer described in this invention application exhibits more significant targeted inhibitory activity against tumors.

[0061] Example 3: Specific recognition of the binding of an Aptamer containing the SEQ 1 sequence to a drug for CD276.

[0062] As shown in Figure 7, nucleic acid aptamers and chemotherapy drugs can be connected using several modes illustrated in Figure 7.

[0063] Example 4: Stability test of modified Aptamer containing the SEQ 1 sequence that specifically recognizes CD276.

[0064] DNA aptamer was modified with C12 and incubated at 37°C in 10% FBS. Its stability was then assessed by gel electrophoresis.

[0065] The experimental results are shown in Figure 9. From left to right in Figure 9, the time gradients are represented, which are the gel running results of the aptamer after 0, 2, 4, 6, 8, and 12 hours, respectively. It can be seen that the Aptamer modified by C12 can still maintain good stability over a long period of time.

[0066] Example 5: Verification of the targeted therapeutic effect of Aptamer containing the SEQ 1 sequence in other CD276-positive tumors.

[0067] The experimental procedure in Example 2 was repeated to verify the targeted therapeutic effect of CD276-Aptamer on inhibiting the growth of other tumors.

[0068] The experimental results are shown in Figure 10. The pancreatic cancer cell line Mia Paca-2, the non-small cell lung cancer cell line H1299, and the colon cancer cell line SW620 were treated with two ApDC drugs and the control group CCK8 experiment results confirmed that ApDC can better inhibit the growth of various cancer cell lines in vitro compared with other drug monomers, and the targeted therapy effect is better than that of drug monomers.

[0069] Although the technical solutions of the present invention have been described and enumerated in detail, it should be understood that modifications to the above embodiments or the adoption of equivalent alternatives are obvious to those skilled in the art. Such modifications or improvements made without departing from the spirit of the present invention are all within the scope of protection claimed by the present invention.

[0070] References

[0071] [1].Ulase, D., et al., Gastric Carcinomas with Stromal B7-H3 Expression Have Lower Intratumoural CD8+ T Cell Density. Int J Mol Sci, 2021. 22(4).

[0072] [2].Picarda, E., K.C. Ohaegbulam and X. Zang, Molecular Pathways: Targeting B7-H3 (CD276) for Human Cancer Immunotherapy. Clin Cancer Res, 2016. 22(14): p. 3425-3431.

[0073] [3].Li, Y., et al., B7-H3 promotes gastric cancer cell migration and invasion. Oncotarget, 2017. 8(42): p. 71725-71735.

[0074] [4].Zang, X., et al., Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas. Mod Pathol, 2010. 23(8): p. 1104-12.

[0075] [5].Zhao, Y., D. Xu and W. Tan, Aptamer-functionalized nano / micro-materials for clinical diagnosis: isolation, release and bioanalysis of circulating tumor cells. Integr Biol (Camb), 2017. 9(3): p. 188-205.

[0076] [6].Jin, C., et al., Cancer biomarker discovery using DNA aptamers. Analyst, 2016. 141(2): p. 461-6.

[0077] [7].KUWAHARA M, SUGIMOTO N. Molecular evolution of functional nucleic acids with chemical modifications [J]. Molecules, 2010, 15(8): 5423-5444.

[0078] [8]. Li Yazhou. Construction and application of chemically modified nucleic acid aptamers based on solid-phase synthesis [D]; Hunan University, 2024.

[0079] [9]. [YOON S, HUANG KW, REEBYE V, et al. Aptamer-Drug Conjugates of Active Metabolites of Nucleoside Analogs and Cytotoxic Agents Inhibit Pancreatic Tumor Cell Growth [J]. Mol Ther Nucleic Acids, 2017, 6: 80-88.

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Claims

1. A nucleic acid aptamer that specifically recognizes CD276, characterized in that: The nucleic acid aptamer has a sequence as shown in SEQ 1, or other nucleotide sequences that are more than 80% homologous to the sequence shown in SEQ 1.

2. The aptamer that specifically recognizes CD276 according to claim 1, characterized in that: The nucleic acid aptamer is modified with bases or with a phosphate backbone.

3. The aptamer that specifically recognizes CD276 according to claim 2, characterized in that: The base modifications include drug base modifications, F substitution, MOE modification, OMe modification, cEt modification, and inverted T modification; the drug base modifications include substitution or terminal coupling of gemcitabine and fluorouracil.

4. The aptamer that specifically recognizes CD276 according to claim 2, characterized in that: The phosphate backbone is modified to be partially or fully thiolated.

5. The aptamer that specifically recognizes CD276 according to claim 3, characterized in that: The conjugates obtained by terminal coupling of the nucleic acid aptamers contain fluorescent groups, drugs, base analogs, sugar modifications, peptides, radioactive substances, biotin, and quantum dots.

6. The aptamer that specifically recognizes CD276 according to claim 1, characterized in that: The nucleic acid aptamer contains nucleotide sequences that are transcribed or translated from DNA to form RNA sequences or peptide nucleic acid sequences.

7. The use of a nucleic acid aptamer that specifically recognizes CD276 as described in any one of claims 1-6 in the preparation of reagents for targeted tumor therapy or diagnostic tumor kits.

8. The use of the nucleic acid aptamer that specifically recognizes CD276 according to claim 7 in the preparation of reagents for targeted tumor therapy or diagnostic tumor kits, characterized in that: The reagents and kits further contain derivatives of the nucleic acid aptamers for the recognition, degradation, or drug delivery of CD276 in tissues, cells, or blood.

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