CN118460549B - Aptamer capable of specifically recognizing CD276 and application thereof - Google Patents
Aptamer capable of specifically recognizing CD276 and application thereof Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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Abstract
The invention discloses a nucleic acid aptamer for specifically recognizing CD276, which has a sequence shown in SEQ1 or a nucleotide sequence with homology of more than 80% with other nucleic acid aptamers; the invention also discloses application of the aptamer specifically recognizing the CD276 in preparing a targeted therapeutic reagent and in preparing a tumor targeted diagnostic kit. The aptamer has higher binding affinity for CD276 protein, has good specificity, is easier to modify and artificially synthesize, has strong stability and is convenient to use.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a nucleic acid aptamer specifically recognizing CD276 and application thereof.
Background
CD276, also known as B7-H3, belongs to the family of immune co-stimulatory molecules B7 and plays an important role in tumor immunity as an immune checkpoint, regulating immune cell infiltration [1,2] of tumor tissue. At the same time, CD276 also plays an important role in tumorigenesis, progression, and participation in various processes [3] such as tumor progression, metastasis, and drug resistance. CD276 protein is widely expressed in various organs and tissues of human body, but is highly expressed in various solid tumors including gastric cancer, and the expression level of CD276 protein has been proved to be related to malignancy degree and prognosis [4] of the tumor, so CD276 protein can be used as a potential target point of tumor treatment.
The aptamer specifically recognizing the CD276 protein can regulate immune response by combining the protein, and can realize directional administration of chemotherapeutic drugs to play a better role in tumor treatment.
The Aptamer (Aptamer) refers to a DNA or RNA molecule obtained by screening and separating by an exponential enrichment ligand system evolution (SELEX) technology, and can be combined with other targets such as proteins, metal ions, small molecules, polypeptides and even whole cells with high affinity and specificity, so that the Aptamer has wide prospect [5] in the aspects of biochemical analysis, environmental monitoring, basic medicine, new medicine synthesis and the like. Compared with an antibody, the aptamer has the advantages of small molecular weight, better stability, easy modification, no immunogenicity, short preparation period, capability of being synthesized artificially and the like, and is free of a series of processes [6] such as animal immunization, feeding, protein extraction and purification and the like. Thus, if a nucleic acid Aptamer with higher affinity to the CD276 protein and high specificity can be found, it would be helpful to achieve high sensitivity and high specificity detection of the CD276 protein, and to develop an Aptamer-drug conjugate (ApDC) targeted to the CD276 protein.
Disclosure of Invention
To overcome the deficiencies in the prior art, a nucleic acid aptamer specifically recognizing CD276 and uses thereof are provided.
The invention is realized by the following scheme:
A nucleic acid aptamer specifically recognizing CD276, which has a sequence shown in SEQ1, and may be other nucleotide sequences with homology of 80% or more with the sequence shown in SEQ 1.
In one embodiment, the aptamer is modified, the modification being a modification of a base or phosphate backbone.
In one embodiment, the base modification comprises a drug base modification, an F substitution, a MOE modification, an OMe modification, a cEt modification, an inverted T modification, a drug base modification comprising a substitution or a terminal coupling of gemcitabine, fluorouracil.
In one embodiment, the phosphate backbone is modified to be partially thio or fully thio.
In one embodiment, the aptamer is terminally conjugated, and the conjugates include fluorophores, drugs, base analogues, sugar modifications, peptides, radioactive materials, biotin, and quantum dots.
In one embodiment, wherein the nucleic acid aptamer is a partially or fully sequence-converted nucleic acid aptamer; the sequence conversion may be transcription of a DNA sequence into an RNA sequence or translation into a peptide nucleic acid sequence.
The invention also relates to a preparation method of the aptamer specifically recognizing the CD276, which comprises the following steps:
Obtaining a CD 276-targeted aptamer library by means of multiple rounds of protein screening;
Selecting a plurality of most abundant aptamers from the aptamer library;
and selecting the aptamer with better evolution for binding verification to obtain the aptamer with higher affinity.
The invention also relates to application of the aptamer specifically recognizing the CD276 in preparing a reagent for targeted treatment of tumors or a diagnostic tumor kit, wherein the reagent or the kit comprises the aptamer specifically recognizing the CD276 and also can be a derivative thereof, and is used for recognizing, degrading or delivering medicines of the CD276 in tissues, cells and blood.
The beneficial effects of the invention are as follows:
The aptamer provided by the invention has higher binding affinity for CD276 protein, has good specificity, is easy to modify and artificially synthesize, has strong stability and convenient use, and can be better applied to the field of preparing tumor targeted drugs.
Drawings
FIG. 1 is a graph showing the effect of SPR to verify binding of sequences to CD276 protein;
FIG. 2 is a graph of SPR affinity assay data for Aptemer comprising the sequence of SEQ 1;
FIG. 3 is a graph showing the results of the verification of the affinity of AGS and HGC27 cell lines with Aptemer comprising the sequence of SEQ 1;
FIG. 4 is a graph showing the results of an AGS cell line toxicity test to CD276 positive by the test CCK-8 to verify ApDC;
FIG. 5 is a graph of targeting experiments validated by in vivo experiments in mice to contain Aptemer of the SEQ1 sequence;
FIG. 6 is a graph of experimental results of in vivo experiments in mice to verify the effect of ApDC targeted therapy;
FIG. 7 is a schematic representation of the pattern of aptamer ligation with a chemotherapeutic agent;
FIG. 8 is a block diagram of the workflow of the present invention;
FIG. 9 is a graph showing the results of a modified aptamer stability test according to the present invention;
FIG. 10 is a graph of experimental results of the CCK-8 experiment to verify the targeting of ApDC to other tumor cells outside gastric adenocarcinoma cells.
Detailed Description
Preferred embodiments of the present invention are further described below.
A nucleic acid aptamer that specifically recognizes CD276, characterized by: the nucleic acid aptamer has a sequence shown as SEQ1 or comprises other nucleotide sequences with the sequence homology of more than 80% with the sequence shown as SEQ 1.
In one embodiment, the aptamer may be base modified or modified with a phosphate backbone.
Base modifications include drug base modifications, F substitutions, MOE modifications (or RNA analog modifications such as OMe), cEt modifications (constrained ethyl modifications), inverted T modifications, e.g., 2'-O-Me,2' -O-MOE,2'-F,2' -(s) -constrained ethyl (s-cEt), etc., drug base modifications include substitution or terminal coupling of other drugs such as gemcitabine, fluorouracil, etc., and the phosphate backbone modifications may be partial thio or holothiothio.
The aptamer may also be coupled via a terminus to form a conjugate, the conjugate comprising a fluorophore, a drug, a base analog, a sugar modification, a peptide, a radioactive substance, biotin, and a quantum dot.
In the present invention, the aforementioned aptamer may be conjugated to MMAE (Monomethyl auristatin E), gemcitabine, 5 fluorouracil. The pattern of aptamer ligation to chemotherapeutic agents is shown in FIG. 7. Wherein the aptamer is a partially or fully sequence-converted aptamer; the sequence conversion may be transcription of a DNA sequence into an RNA sequence or translation into a peptide nucleic acid sequence.
The modification of the base sequence is mainly to enhance its resistance to nucleases, and many studies so far confirm that the stability of the modified sequence improves [7].
Drug base modification nucleic acid aptamers can be precisely coupled to drugs directly by DNA solid phase synthesis techniques and allow modification [8-10] of specific base positions.
In a method for preparing a nucleic acid aptamer specifically recognizing CD276, the method for preparing the nucleic acid aptamer comprises the steps of:
Obtaining a CD 276-targeted aptamer library by means of multiple rounds of protein screening;
Selecting a plurality of most abundant aptamers from the aptamer library;
and selecting the aptamer with better evolution for binding verification to obtain the aptamer with higher affinity.
In another aspect, the invention relates to the use of a aptamer specifically recognizing CD276 in the preparation of a targeted therapeutic agent or kit, or to the achievement of a tumor diagnosis or therapeutic effect based on the aptamer targeting CD276, for example, in the preparation of a kit for diagnosing a tumor with a aptamer specifically recognizing CD 276. The reagent or the kit comprises the aptamer specifically recognizing the CD276, which is used for targeted degradation, targeted administration and detection analysis of the CD276, and can also comprise the aptamer and derivatives thereof, which are used for recognizing, degrading or delivering medicines of the CD276 in tissues, cells and blood.
The application obtains the Aptamer library targeting the CD276 protein by a 7-round protein screening mode. Through high-throughput sequencing and analysis, 96 Aptamers with highest abundance are obtained. And selecting a sequence with better evolution for binding verification to obtain a sequence with higher affinity. The Aptamer has good capability of targeting CD276 protein, and can be used for targeted degradation, targeted drug delivery, detection analysis and the like of CD 276. The workflow of the present application is shown in fig. 8.
The invention can provide a novel tool for targeting CD276 protein, a novel method for degrading CD276 protein and a novel thought for targeting treatment and diagnosis of tumor based on CD276 protein.
It should be noted that the results of the related studies of the aptamer to date all prove that the aptamer with the same or high homology can have higher targeting [11-13] for a plurality of target protein high expression cell lines. The person skilled in the art is motivated and can envision the application of the aptamer described in the present invention to targeted therapy or diagnosis of other tumors than those described in the examples below, such as the preparation of other tumor targeted therapeutic agents or diagnostic kits.
The application will be further illustrated with reference to specific examples.
Example 1 Aptamer screening and affinity test comprising the sequence of SEQ1 specifically recognizing CD276
The specific flow and method of the present invention can be seen in fig. 8.
As shown in FIG. 1, 96 sequences with higher enrichment degree are selected through sequencing analysis, and the binding affinity of the sequences (01-96) to CD276 protein is verified through surface plasmon resonance SPR, wherein "03" in FIG. 1 represents an Aptamer containing SEQ1 sequence described in the invention.
As shown in fig. 2, it can be seen from the surface plasmon resonance SPR experiment that the dissociation curve of the Aptamer (apt-3) comprising the sequence of SEQ1 shows a slow dissociation rate at different wavelengths, and thus shows excellent affinity with the CD276 protein.
The targeting of the selected SEQ1 nucleotide sequence to CD276 positive AGS and HGC27 cell lines was confirmed by flow experiments.
The adherent cell lines (AGS, HGC 27) were digested with 0.02% edta solution, incubated with 10 μm Cy 5-labeled aptamer on ice for 30min per 200mL of cell suspension, resuspended in wash water and centrifuged 2 times, and the fluorescence intensity was detected with a flow cytometer.
The experimental results are shown in fig. 3 (the CD276-3 shown in fig. 3 is the Aptamer containing the sequence of SEQ1 described in the present invention), and the above flow experiment proves that the sequence of SEQ1 obtained by screening also shows excellent affinity compared with the control group.
EXAMPLE 2 Aptamer comprising the sequence of SEQ1 specifically recognizing CD276 for targeted treatment of CD 276-positive tumors
As shown in FIG. 4, cell level was verified by CCK-8 experiments for the toxicity of ApDC to CD276 positive AGS cell lines.
The AGS cell line is digested, centrifuged, resuspended and counted, after proper dilution, the AGS cell line is evenly spread on a 96-well plate at a density of 5000 cells per well, and after the next day, medicines with different concentration gradients are added, the AGS cell line is incubated for 2 days at 37 ℃, CCK-8 detection liquid is changed, and the AGS cell line is incubated for 1 hour for detection. As can be seen from the experimental results in fig. 4, apDC was more toxic to the CD276 positive AGS cell line than the drug monomer.
As shown in fig. 5, targeting of the Aptamer comprising the sequence of SEQ1 was verified by in vivo experiments in mice.
Female nude mice of 4-6 weeks of age were selected and subjected to subcutaneous oncology at the right armpit. About 1×10 7 SNU601 cells were injected per nude mouse, and the cell suspension was mixed with matrigel 1:1. When the tumor volume reaches 200mm3, 100 mu L and 10 mu M of the aptamer marked by Cy5 are injected through a tail vein, and the fluorescence intensity and distribution are detected by a small animal living body imager at 0h, 0.5h, 1h, 2h, 3h and 4h after injection.
As shown in fig. 6, the targeted therapeutic effect of ApDC was verified by in vivo experiments in mice.
Female nude mice of 4-6 weeks of age were selected and subjected to subcutaneous oncology at the right armpit. About 1×10 7 SNU601 cells were injected per nude mouse, and the cell suspension was mixed with matrigel 1:1. When the maximum diameter of the tumor reaches 5mm, the administration is started, and each mouse is injected with 100 mu L of the drug and 75 mu M of the drug through tail vein, and the administration is carried out 2 times per week for 6 times. After the end of the administration, the animals were kept for 2 weeks, and the tumor size and body weight of the mice were measured and recorded. As shown in the results of fig. 6, the CD276 aptamer described in the present application has more significant targeted inhibition on tumors.
Example 3 binding of an Aptamer comprising the sequence SEQ1 specifically recognizing CD276 to a drug
As shown in FIG. 7, the aptamer and chemotherapeutic agent may be linked in several modes as illustrated in FIG. 7.
Example 4 stability test of modified Aptamer comprising the sequence of SEQ1 specifically recognizing CD276
DNA APTAMER was subjected to C12 modification, incubated at 37℃in 10% FBS, and its stability was examined by gel electrophoresis.
The experimental results are shown in fig. 9, and the time gradients from left to right in fig. 9 are respectively 0, 2,4, 6, 8 and 12h of the Aptamer, so that the C12 modified Aptamer can still maintain good stability for a long time.
Example 5 validation of the Targeted therapeutic effect of Aptamer comprising the SEQ1 sequence in other CD 276-positive tumors
The experimental procedure in example 2 was repeated to verify the targeted therapeutic effect of CD276-Aptamer on inhibition of other tumor growth.
The experimental results are shown in fig. 10, and the pancreatic cancer cell line Mia Paca-2, the non-small cell lung cancer cell line H1299 and the colon cancer cell line SW620 are subjected to 2 ApDC drug treatment and control group CCK8 experimental results, so that the ApDC can better inhibit the growth of various cancer tumor cell lines in vitro compared with other drug monomers, and the targeted therapeutic effect is better than that of the drug monomers.
While the invention has been described and illustrated in considerable detail, it should be understood that modifications and equivalents to the above-described embodiments will become apparent to those skilled in the art, and that such modifications and improvements may be made without departing from the spirit of the invention.
Reference to the literature
[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).
[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.
[3].Li, Y., et al., B7-H3 promotes gastric cancer cell migration and invasion. Oncotarget, 2017. 8(42): p. 71725-71735.
[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.
[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.
[6].Jin, C., et al., Cancer biomarker discovery using DNA aptamers. Analyst, 2016. 141(2): p. 461-6.
[7].KUWAHARA M, SUGIMOTO N. Molecular evolution of functional nucleic acids with chemical modifications [J]. Molecules, 2010, 15(8): 5423-5444.
[8] Li Yazhou construction of chemically modified nucleic acid aptamers based on solid phase synthesis and research on their use [ D ]; university of Hunan, 2024.
[9]. [YOON S, HUANG K W, 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.
[10]. XIANG W, PENG Y, ZENG H, et al. Targeting treatment of bladder cancer using PTK7 aptamer-gemcitabine conjugate [J]. Biomater Res, 2022, 26(1): 74.
[11]. BOHRMANN L, BURGHARDT T, RODRIGUEZ-RODRIGUEZ C, et al. Quantitative Evaluation of a Multimodal Aptamer-Targeted Long-Circulating Polymer for Tumor Targeting [J]. ACS Omega, 2023, 8(12): 11003-11020.
[12]. VAN DEN AVONT A, SHARMA-WALIA N. Anti-nucleolin aptamer AS1411: an advancing therapeutic [J]. Front Mol Biosci, 2023, 10: 1217769.
[13]. MAHLKNECHT G, MARON R, MANCINI M, et al. Aptamer to ErbB-2/HER2 enhances degradation of the target and inhibits tumorigenic growth [J]. Proc Natl Acad Sci U S A, 2013, 110(20): 8170-8175.
Claims (6)
1. A nucleic acid aptamer that specifically recognizes CD276, characterized by: the nucleic acid aptamer is a sequence shown as SEQ 1.
2. The aptamer specifically recognizing CD276 according to claim 1, wherein: the aptamer is modified by a base or a phosphate skeleton.
3. The aptamer specifically recognizing CD276 according to claim 2, wherein: the base modification comprises drug base modification, F substitution, MOE modification, OMe modification, cEt modification and inverted T modification; the drug base modification includes substitution or terminal coupling of gemcitabine and fluorouracil.
4. The aptamer specifically recognizing CD276 according to claim 2, wherein: the phosphate backbone is modified to be partially thio or fully thio.
5. The aptamer specifically recognizing CD276 according to claim 3, wherein: the conjugate obtained by the end coupling of the aptamer contains a fluorescent group, a drug, a base analogue, a sugar modification, a peptide, a radioactive substance, biotin and quantum dots.
6. Use of a nucleic acid aptamer specifically recognizing CD276 according to any of claims 1-5 for the preparation of a reagent for targeted treatment of tumors or a diagnostic kit for tumors.
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