A nucleic acid aptamer targeting claudin 4 and a preparation method and application thereof

Abstract

The application discloses a nucleic acid aptamer targeting CLDN4 and a preparation method and application thereof. Specifically, the application provides a nucleic acid aptamer binding to CLDN4, wherein a core sequence of the nucleic acid aptamer binding to the CLDN4 protein comprises a sequence as shown in SEQ ID NO: 9, 10, 11, 12, 13, 14, 15 or 16. The nucleic acid aptamer has the advantages of small molecular weight, high binding affinity, good specificity, easy modification and artificial synthesis, good stability and convenient use.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nucleic acid aptamer targeting CLDN4, its preparation method, and its application. Background Technology

[0002] Claudin 4 (CLDN4) is a member of the Claudins protein family and an important component of tight junctions. It is highly expressed in tumor cells of various solid tumors and has an adverse effect on patient prognosis.

[0003] Nucleic acid aptamers have molecular recognition functions similar to antibodies, but they have more application advantages compared to antibodies. For example, they can be screened in vitro without relying on organisms, have a wide range of target molecules (metal ions, toxins, viruses, bacteria, cells, tissues, etc.), small molecular weight, low immunogenicity, easy solid-phase synthesis and labeling, good chemical stability, and can be stored at room temperature.

[0004] Therefore, developing a nucleic acid aptamer that can specifically recognize the CLDN4 protein for the diagnosis and targeted therapy of CLDN4-positive tumors is of great significance in this field. Summary of the Invention

[0005] This invention provides a nucleic acid aptamer targeting CLDN4, its preparation method, and its application.

[0006] In a first aspect of the invention, a nucleic acid aptamer that binds to CLDN4 is provided, wherein the core sequence of the nucleic acid aptamer binding to CLDN4 comprises a sequence selected from the group consisting of:

[0007] (1) The sequence shown in SEQ ID NO: 9;

[0008] (2) The sequence shown in SEQ ID NO: 10;

[0009] (3) The sequence shown in SEQ ID NO: 11;

[0010] (4) The sequence shown in SEQ ID NO: 12;

[0011] (5) The sequence shown in SEQ ID NO: 13;

[0012] (6) The sequence shown in SEQ ID NO: 14;

[0013] (7) The sequence shown in SEQ ID NO: 15;

[0014] (8) The sequence shown in SEQ ID NO: 16.

[0015] In another preferred embodiment, the core sequence of the nucleic acid aptamer binding to the CLDN4 includes the sequence shown in SEQ ID NO: 9.

[0016] In another preferred embodiment, the core sequence has flanking sequences at the 5' end and / or 3' end.

[0017] In another preferred embodiment, the length of the flanking sequence is 1-80 bases, preferably extended by 5-50 bases, and most preferably extended by 10-30 bases.

[0018] In another preferred embodiment, the affinity index of the nucleic acid aptamer to CLDN4 is... K D The value is 1-100 nM, preferably 1-50 nM, and optimally 1-10 nM.

[0019] In another preferred embodiment, the nucleic acid aptamer comprises a sequence selected from the group consisting of:

[0020] (a) The sequence shown in SEQ ID NO: 6;

[0021] (b) The sequence shown in SEQ ID NO: 1;

[0022] (c) The sequence shown in SEQ ID NO: 2;

[0023] (d) The sequence shown in SEQ ID NO: 3;

[0024] (e) The sequence shown in SEQ ID NO: 4;

[0025] (f) The sequence shown in SEQ ID NO: 5;

[0026] (g) The sequence shown in SEQ ID NO: 7;

[0027] (h) The sequence shown in SEQ ID NO: 8.

[0028] In another preferred embodiment, the nucleic acid aptamer further comprises a DNA sequence having ≥85%, preferably ≥90%, and most preferably ≥95% sequence identity with SEQ ID NO: 6, 1, 2, 3, 4, 5, 7, and 8.

[0029] In another preferred embodiment, the nucleic acid aptamer is a modified nucleic acid aptamer, and the modification is a base modification or a phosphate backbone modification.

[0030] In another preferred embodiment, the base modification is selected from the group consisting of F substitution, MOE modification, OME modification, or a combination thereof.

[0031] In another preferred embodiment, the skeleton is modified to be partially or fully thiolated.

[0032] In another preferred embodiment, one or more positions in the nucleic acid aptamer sequence are modified by a selection from the group consisting of phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium, isotopization, or combinations thereof.

[0033] In another preferred embodiment, some or all of the nucleotides in the nucleic acid aptamer are converted to levorotatory nucleotides.

[0034] In another preferred embodiment, the number of one or more is 1-10, more preferably 1-5, and most preferably 1-3.

[0035] In another preferred embodiment, the modified nucleic acid aptamer has an affinity for binding CLDN4 equal to or higher than that of the unmodified parent nucleic acid aptamer sequence, and / or has higher stability.

[0036] In another preferred embodiment, the modification enhances the stability of the nucleic acid aptamer sequence.

[0037] In a second aspect of the invention, a coupling is provided, the coupling comprising:

[0038] (a) the nucleic acid aptamer as described in the first aspect of the invention; and

[0039] (b) A coupling portion coupled to the nucleic acid aptamer portion, the coupling portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0040] In another preferred embodiment, the nucleic acid aptamer portion and the coupling portion are coupled by chemical bonds or adapters.

[0041] In another preferred embodiment, the detectable marker is a chemical marker, a biological marker, or a combination thereof.

[0042] In another preferred embodiment, the chemical label is an isotope, an immunotoxin, and / or a chemical drug.

[0043] In another preferred embodiment, the biomarker is biotin, avidin, or an enzyme label.

[0044] In another preferred embodiment, the drug is a small molecule drug, a biological factor, an oligonucleotide, or a combination thereof.

[0045] In another preferred embodiment, the oligonucleotide is an antisense oligonucleotide, small interfering RNA, microRNA, nucleic acid aptamer, or a combination thereof.

[0046] In another preferred embodiment, the drug is a cytotoxic drug (toxin).

[0047] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs (such as MMAF or MMAE), DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0048] In another preferred embodiment, the coupling portion is a detectable marker.

[0049] In another preferred embodiment, the detectable marker includes biotin, chemiluminescent groups, chemifluorescent groups, fluorescent proteins, enzymes, colloidal gold, radioisotopes, latex particles, antibodies, ligands, antigens, receptors, or combinations thereof.

[0050] In a third aspect of the invention, a biochip is provided, the biochip comprising the nucleic acid aptamer described in the first aspect of the invention, or the conjugate described in the second aspect of the invention.

[0051] In a fourth aspect of the invention, the use of the nucleic acid aptamer described in the first aspect of the invention, the conjugate described in the second aspect of the invention, or the biochip described in the third aspect of the invention is provided for the following purposes:

[0052] (Z1) A kit for preparing the detection of CLDN4;

[0053] (Z2) is used to prepare reagents for imaging CLDN4;

[0054] (Z3) Preparation of pharmaceutical compositions targeting CLDN4; and / or

[0055] (Z4) was used to purify CLDN4.

[0056] In another preferred embodiment, CLDN4 is detected in biological samples.

[0057] In another preferred embodiment, the biological sample is selected from the group consisting of serum samples, plasma samples, tissue samples, cell samples, secretion samples, or combinations thereof.

[0058] In a fifth aspect of the invention, a detection kit is provided, the detection kit comprising: the nucleic acid aptamer described in the first aspect of the invention, or the conjugate described in the second aspect of the invention.

[0059] In a sixth aspect of the invention, the use of the detection kit described in the fifth aspect of the invention is provided for preparing a kit for diagnosing CLDN4-positive tumors.

[0060] In another preferred embodiment, the CLDN4-positive tumor is a tumor that highly expresses CLDN4.

[0061] In another preferred embodiment, the tumors that highly express CLDN4 are selected from the group consisting of pancreatic cancer, colorectal cancer, gastric cancer, breast cancer, thyroid cancer, ovarian cancer, bladder cancer, or combinations thereof.

[0062] In a seventh aspect of the invention, a composition is provided, the composition comprising:

[0063] (a) The nucleic acid aptamer described in the first aspect of the present invention or the conjugate described in the third aspect of the present invention; and

[0064] (b) Optional, pharmaceutically acceptable excipients.

[0065] In another preferred embodiment, the composition is a pharmaceutical composition.

[0066] In another preferred embodiment, the dosage form of the composition is a liquid formulation.

[0067] In another preferred embodiment, the dosage form of the composition is an injectable formulation.

[0068] In an eighth aspect of the invention, a method for detecting CLDN4 for non-disease diagnosis and / or treatment is provided, comprising the following steps:

[0069] (S1) Provide the sample to be tested;

[0070] (S2) The sample to be tested is mixed with the nucleic acid aptamer described in the first aspect of the present invention or the conjugate described in the second aspect of the present invention to form a mixture;

[0071] (S3) Detect the presence or absence of the “CLDN4-nucleic acid aptamer complex” in the mixture, wherein if the complex is present, it indicates that CLDN4 is present in the sample; if the complex is not present, it indicates that CLDN4 is not present in the sample.

[0072] In another preferred embodiment, the detection includes qualitative detection and quantitative detection.

[0073] In another preferred embodiment, the method is an in vitro method.

[0074] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0075] In a ninth aspect of the present invention, a method for preparing the nucleic acid aptamer described in the first aspect of the present invention is provided, comprising the steps of:

[0076] (i) An aptamer library targeting CLDN4 was obtained through multiple rounds of protein screening;

[0077] (ii) Select the most abundant aptamers from the aptamer library;

[0078] (iii) Selecting well-evolved aptamers for binding verification yielded aptamers with high affinity; and

[0079] (iv) Optionally, the selected aptamers with higher affinity are truncated and verified.

[0080] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0081] Figure 1 This shows a sequence library of proteins targeting CLDN4 obtained after five rounds of protein screening.

[0082] Figure 2 The results show the affinity differences between 90 highly enriched sequences selected through SPR verification and CLDN4.

[0083] Figure 3 The flow cytometry results showed the affinity of eight favorable nucleic acid aptamer sequences and their corresponding truncated sequences for CLDN4 human cells.

[0084] Figure 4 A schematic diagram of constructing ApDC is shown.

[0085] Figure 5 The study demonstrated the cytotoxicity of ApDC against pancreatic cancer cell lines at the cellular level using a CCK-8 assay.

[0086] Figure 6 The study demonstrated the cytotoxicity of ApDC against colorectal cancer cell lines at the cellular level using a CCK-8 assay.

[0087] Figure 7 The in vivo fluorescence imaging experiment in mice demonstrated that aptamer and ApDC have good tumor targeting properties in mice.

[0088] Figure 8 The results of statistical analysis of in vivo fluorescence imaging experiments in mice using LivingImage 4.4 software are shown.

[0089] Figure 9The results show an in vitro fluorescence imaging experiment in mice, which verifies that aptamer and ApDC are well enriched in tumor tissue.

[0090] Figure 10 The results of statistical analysis of mouse ex vivo fluorescence imaging experiments using LivingImage 4.4 software are shown.

[0091] Figure 11 The results showed that in vivo experiments in mice demonstrated that ApDC has a good tumor-targeting therapeutic effect.

[0092] Figure 12 The determination of subcutaneous tumor quality in mice is shown.

[0093] Figure 13 The results show the body weight measurements taken in mice during the in vivo experiment.

[0094] Figure 14 The results of homology analysis of the eight nucleic acid aptamers screened in this invention are shown.

[0095] Figure 15 The mass spectrometry results after ApDC was constructed are shown.

[0096] Figure 16 The diagram shows the secondary structure of the eight nucleic acid aptamers screened in this invention. Detailed Implementation

[0097] Through extensive and in-depth research and screening, the inventors have developed, for the first time, a nucleic acid aptamer targeting CLDN4. A total of eight nucleic acid aptamers with excellent affinity were obtained through screening (CLDN4-29, CLDN4-02, CLDN4-05, CLDN4-14, CLDN4-23, CLDN4-26, CLDN4-36, and CLDN4-55). Experimental results show that the nucleic acid aptamers of this invention possess excellent CLDN4 targeting and tumor targeting capabilities, and when used in combination with drugs or toxins, they exhibit very good tumor-targeted therapeutic effects. Based on this, the present invention was completed.

[0098] the term

[0099] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0100] The term "about" can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101.

[0101] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “made of”.

[0102] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0103] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0104] As used herein, the "core sequence" refers to the critical sequence that binds to the CLDN4 protein and is the determining factor in the affinity between the aptamer and the CLDN4 protein. In some embodiments, replacing the core sequence with any DNA sequence will result in the loss or significant reduction of affinity between the aptamer and the CLDN4 protein. The significant reduction is statistically significant.

[0105] As used herein, the term "flanking sequence" refers to a DNA sequence present at the 5' and / or 3' end of the core sequence of the aptamer. In some embodiments, the flanking sequence participates in the binding of the aptamer to the CLDN4 protein. In some embodiments, the flanking sequence does not participate in the binding of the aptamer to the CLDN4 protein. In some embodiments, the presence or absence of the flanking sequence does not significantly differ the affinity between the aptamer and the CLDN4 protein. In some embodiments, the flanking sequence may be replaced with any DNA sequence.

[0106] As used herein, “nucleic acid aptamer of the present invention”, “aptamer of the present invention”, “aptamer of the present invention” and “aptamer of the present invention” have the same meaning and all refer to nucleic acid aptamers targeting CLDN4, and can be used interchangeably.

[0107] As used in this article, "tight junction protein 4", "Claudin 4", "CLDN4" and "CLDN4 protein" have the same meaning and can be used interchangeably.

[0108] As used in this article, "a nucleic acid sequence with more than 90% but less than 100% homology" refers to a nucleic acid sequence in which one or more nucleotides are added, deleted or substituted to have more than 90% but less than 100% homology and exhibit similar CLDN4 binding ability.

[0109] As used herein, nucleic acid aptamers CLDN4-29, CLDN4-02, CLDN4-05, CLDN4-14, CLDN4-23, CLDN4-26, CLDN4-36, and CLDN4-55 can also be directly represented by their corresponding designations: 29, 2, 5, 14, 23, 26, 36, and 55.

[0110] As used in this article, “the truncated sequence of CLDN4-29”, “C29s”, or “C29 shorten” have the same meaning and can be used interchangeably. They refer to the core sequence that binds to the CLDN4 protein, as shown in SEQ ID NO: 9.

[0111] CLDN4

[0112] Claudin 4 (CLDN4), a member of the Claudins protein family, is a crucial component of tight junctions. It is highly expressed in tumor cells of various solid tumors and negatively impacts patient prognosis. The Claudins protein family plays a role in regulating epithelial cell permeability and maintaining cell polarity. Changes in Claudins protein levels affect intercellular permeability, influencing disease development, progression, and subsequent treatment.

[0113] Studies have shown that CLDN4 may represent an early stage in the progression of various cancers. For example, in colorectal cancer, CLDN4 activation of the YAP signaling pathway is a characteristic of the precancerous lesion stage. In epithelial malignancies, changes in cell and tissue polarity, and the tight junctions of malignant epithelial cells involving CLDN4, help maintain the tumor microenvironment, retain cytokines secreted by tumor cells, and form micro-niches suitable for cancer growth, thereby promoting the malignant phenotype of cancer.

[0114] Nucleic acid aptamers

[0115] Nucleic acid aptamers are single-stranded oligonucleotides obtained from artificially synthesized DNA / RNA libraries through SELEX technology, capable of binding to target molecules with high specificity and high affinity. They fold into secondary and tertiary structures and, through intermolecular forces such as electrostatic attraction, hydrophobic interaction, and van der Waals forces, specifically recognize target molecules by matching their spatial structure with the conformation of the target molecule.

[0116] Nucleic acid aptamers have molecular recognition functions similar to antibodies, but they have more application advantages compared to antibodies. For example, they can be screened in vitro without relying on organisms, have a wide range of target molecules (metal ions, toxins, viruses, bacteria, cells, tissues, etc.), small molecular weight, low immunogenicity, easy solid-phase synthesis and labeling, good chemical stability, and can be stored at room temperature.

[0117] Nucleic acid aptamers serve as nucleic acid or protein recognition modules in molecular biology experiments; they are used for biomarker discovery, molecular diagnosis, targeted molecular medicine, and molecular pathology in diseases; and they show great promise for clinical applications in multi-omics research fields such as genomics, transcriptomics, and proteomics.

[0118] The nucleic acid aptamers used in this invention refer to nucleic acid molecules that specifically bind to target substances. The binding strength between the nucleic acid aptamer and the target substance needs to be constant. Examples of nucleic acid aptamers include DNA aptamers and RNA aptamers, but DNA aptamers are preferred from a stability point of view. The length of the nucleic acid base sequence is not particularly limited, but is preferably 20 to 100 bases. Secondary structures are known to include hairpin, convex, and G-quartet types, but there are no particular restrictions on the secondary structure.

[0119] Each nucleotide contained in a nucleic acid aptamer may include one or more identical or different chemical modifications. For example, at the 2' position of the ribose, the hydroxyl group may be any nucleotide in which any atom or group is substituted. Such arbitrary atoms or groups include, for example, hydrogen atoms, fluorine atoms, or -O-alkyl groups (e.g., -O-CH3), -O-acyl groups (e.g., -O-CHO), and amino groups (e.g., -NH2). Furthermore, the nucleic acid aptamer is provided in the form of single-stranded RNA or DNA. In this invention, when the nucleic acid is DNA, the U in the nucleic acid sequence is represented by T, and such sequences are also included within the scope of this invention. This invention will be readily apparent to those skilled in the art.

[0120] Composition

[0121] The present invention also provides compositions comprising the nucleic acid aptamers of the present invention. These compositions can be used for the diagnosis or treatment of tumors with high CLDN4 expression. It is a known fact in the art that cancer mechanisms can be inhibited and cancer can be treated by binding aptamers that specifically bind to cancer cell lines. The mechanisms by which specifically binding aptamers bind to tumor cells or tissues and inhibit tumors are readily apparent to those skilled in the art. Therefore, compositions comprising these aptamers can be provided as compositions for the treatment of tumors.

[0122] The pharmaceutical dosage forms of the compositions of the present invention can be used in the form of their pharmaceutically acceptable salts, or they can be used alone or in combination with other pharmaceutically active compounds or as a set.

[0123] The pharmaceutical compositions of the present invention can be formulated into powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injections using conventional methods. Carriers, excipients, and diluents that may be included in the compositions include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0124] During formulation, diluents or excipients are used, such as commonly used fillers, expanders, binders, wetting agents, disintegrants, and surfactants. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used as non-aqueous solvents and suspending agents. As a base for suppositories, Witepsol, polyethylene glycol, Tween 61, cocoa butter, lauryl oleate, glyceryl keratin, etc., can be used.

[0125] The preferred dosage of the components of this invention depends on the patient's condition and weight, the severity of the disease, the type of drug, the route of administration, and the duration of administration, but can be appropriately selected by those skilled in the art. The compositions of this invention can be administered to mammals such as rats, mice, livestock, and humans via various routes, and all routes of administration are contemplated, such as rectal or intravenous, intramuscular, subcutaneous, intrauterine, or intradural. It can also be administered via intracerebral injection.

[0126] The nucleic acid aptamers of the present invention

[0127] The nucleic acid aptamers of this invention refer to those obtained by the inventors based on SELEX technology, which involve designing and synthesizing a random single-stranded DNA library and corresponding primers, and screening for small molecular weight, chemical stability, ease of storage and labeling, and the ability to bind to CLDN4 protein with high affinity. Their dissociation constant (Ki) was determined by surface plasmon resonance (SPR) experiments. D This allows for accurate determination of its binding affinity, demonstrating that the nucleic acid aptamer of the present invention has high affinity and good specificity for the CLDN4 protein.

[0128] The nucleic acid aptamers of the present invention comprise a core sequence as shown in SEQ ID NO: 9, 10, 11, 12, 13, 14, 15 or 16.

[0129] In another preferred embodiment, the core sequence has flanking sequences at the 5' end and / or 3' end.

[0130] In another preferred embodiment, the length of the flanking sequence is 1-80 bases, preferably extended by 5-50 bases, and most preferably extended by 10-30 bases.

[0131] In another preferred embodiment, the CLDN4 protein is the human CLDN4 protein.

[0132] In another preferred embodiment, the affinity index between the nucleic acid aptamer and the CLDN4 protein is... K D The value is 1-100 nM, preferably 1-50 nM, and optimally 1-10 nM.

[0133] In another preferred embodiment, the nucleic acid aptamer comprises a sequence as shown in SEQ ID NO: 6, 1, 2, 3, 4, 5, 7 or 8.

[0134] In another preferred embodiment, the nucleic acid aptamer further comprises an RNA sequence having ≥85%, preferably ≥90%, and most preferably ≥95% sequence identity with SEQ ID NO: 6, 1, 2, 3, 4, 5, 7, or 8.

[0135] In another preferred embodiment, the ≥95% sequence identity includes 95%, 96%, 97%, 98%, and 99% sequence identity.

[0136] In another preferred embodiment, the nucleic acid aptamer comprises a sequence obtained by extending SEQ ID NO: 6, 1, 2, 3, 4, 5, 7 or 8.

[0137] In another preferred embodiment, the extension refers to an extension of 1-20 bases, more preferably 1-10 bases, and most preferably 1-5 bases.

[0138] In another preferred embodiment, the nucleic acid aptamer sequence is as shown in SEQ ID NO: 6, 1, 2, 3, 4, 5, 7 or 8.

[0139] In another preferred embodiment, one or more positions in the nucleic acid aptamer sequence are modified by a selection from the group consisting of phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium, isotopization, or combinations thereof.

[0140] In another preferred embodiment, the number of one or more is 1-10, more preferably 1-5, and most preferably 1-3.

[0141] In another preferred embodiment, the modification enhances the stability of the nucleic acid aptamer sequence.

[0142] In another preferred embodiment, the nucleic acid aptamer blocks the activity of the CLDN4 protein.

[0143] In another preferred embodiment, the nucleic acid aptamer inhibits the activity of the CLDN4 protein.

[0144] In another preferred embodiment, the nucleic acid aptamer, its conjugate, or its derivative may be used for one or more applications selected from the group consisting of:

[0145] (1) Quantitative or qualitative detection of CLDN4 protein;

[0146] (2) Purify CLDN4 protein;

[0147] (3) Imaging of the CLDN4 protein;

[0148] (4) As an inhibitor of CLDN4 protein;

[0149] (5) Preparation of drugs targeting the CLDN4 protein;

[0150] (6) Prepare reagents or drugs for the diagnosis and treatment of abnormal CLDN4 expression.

[0151] Preparation method of nucleic acid aptamers of the present invention

[0152] This invention also provides a method for preparing an aptamer targeting CLDN4, the method comprising the following steps:

[0153] (i) An aptamer library targeting CLDN4 was obtained through multiple rounds of protein screening;

[0154] (ii) Select the most abundant aptamers from the aptamer library;

[0155] (iii) Select the better evolved aptamers for binding verification to obtain aptamers with higher affinity;

[0156] (iv) The selected aptamers with high affinity were truncated and verified.

[0157] The aptamer described herein can be used for the diagnosis and targeted therapy of pancreatic and colorectal tumors. The prepared aptamer targets the CLDN4 protein and ensures high binding affinity, good specificity, ease of artificial synthesis and modification, good stability, and convenient use.

[0158] Reagent test kit

[0159] This invention also provides a kit for detecting CLDN4 or tumors with high CLDN4 expression. The kit contains nucleic acid aptamers that specifically bind to tumor cells or tissues.

[0160] Kits for diagnosing tumors may include buffer solutions and containers for detection and analysis, such as bottles, tubs, sachets, envelopes, test tubes, ampoules, etc., if desired. These may be of the same form and may be partially or entirely made of plastic, glass, paper, foil, wax, etc. Containers may be equipped with a fully or partially removable closure, which may initially be part of the container or attached to it mechanically, adhesively, or otherwise. Containers may also be equipped with a stopper to allow access to the contents via a needle. Kits may include outer packaging, which may include instructions for use of the components.

[0161] application

[0162] The nucleic acid aptamers of this invention have undergone rigorous screening and possess stable chemical properties and good environmental adaptability. They can be used for qualitative and quantitative analysis of CLDN4 protein through fluorescent labeling, electrochemical signals, or other chemical detection methods, and are widely applicable to fields such as medical diagnostics, drug development, and blood monitoring.

[0163] The nucleic acid aptamers of this invention can be used as alternatives to traditional antibodies in immunoassays. Their high specificity binding to the CLDN4 protein gives them extremely high detection sensitivity and stability in immunoassays. By introducing nucleic acid aptamers into immunoassay technology, the detection process can achieve faster and more accurate target molecule capture. Furthermore, the chemical modification properties of the nucleic acid aptamers make them compatible with existing immunoassay platforms, thereby expanding the application range of traditional immunoassays.

[0164] When used for diagnostic purposes, this involves methods for detecting tumors (such as pancreatic cancer) that highly express CLDN4 using the nucleic acid aptamer. In this case, the method may be characterized by a step of contacting the nucleic acid aptamer with a sample selected from tissues, cells, blood, serum, plasma, saliva, sputum, and urine. The sample is isolated from mammals, preferably human, but not limited to the above; any sample that can be obtained minimally invasively, or that may contain CLDN4, such as secretory fluids or in vitro cell culture components.

[0165] When a nucleic acid aptamer comes into contact with a sample, the CLDN4 protein present in the sample specifically binds to the aptamer. Therefore, tumors can be detected by labeling the aptamer with fluorescence and confirming the presence or absence of a signal.

[0166] In another preferred aspect, the nucleic acid aptamer that specifically binds to cells or tissues is immobilized on a solid support, such as conventional supports like beads, particles, test strips, fibers, filters, membranes, and glass slides, as well as silane or silicate supports. Provided as a detection sensor, it can be used to diagnose CLDN4-positive or highly expressed tumors.

[0167] In another preferred aspect, the present invention relates to a sensor for diagnosing tumors with high CLDN4 expression, wherein an aptamer specifically binding to cells or tissues is immobilized.

[0168] The solid support includes at least one substantially rigid surface on which nucleic acid aptamers can be immobilized. In this case, the nucleic acid aptamers can be immobilized using any conventional chemical coupling method. For example, biotin binds to the ends of nucleic acid aptamers to form complexes; streptavidin is immobilized on the surface of a matrix such as a chip; the interaction between biotin and streptavidin immobilized on the matrix surface allows the nucleic acid aptamers to be immobilized on the matrix surface.

[0169] The main advantages of this invention include:

[0170] (a) The present invention targets the aptamer of the CLDN4 protein, which has high binding affinity, good specificity, is easy to modify and synthesize artificially, has good stability, and is convenient to use.

[0171] (b) The present invention targets the aptamer of the CLDN4 protein, which can be used for efficient diagnosis and targeted therapy of pancreatic tumors.

[0172] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0173] Example 1: aptamer screening targeting CLDN4

[0174] In this embodiment, an enriched library was obtained through five rounds of CLDN4 protein Aptamer screening, such as... Figure 1 As shown. Figure 1 The data represents the retention rate after 5 rounds of screening, where E(Elution)- represents the nucleic acid aptamers retained after elution in the negative screening group, and E(Elution)+ represents the nucleic acid aptamers retained after elution in the positive screening group.

[0175] like Figure 1 As shown, each round of screening retained a large number of nucleic acid aptamers. With the increase of screening pressure and the number of screening rounds, the retention rate of the Elution negative group gradually decreased, while the retention rate of the Elution positive group gradually increased. In the 4th and 5th rounds, it can be seen that the retention rate of the positive group was much higher than that of the negative group.

[0176] Through sequencing analysis, 90 sequences with high enrichment were selected, and their binding ability to CLDN4 protein was determined by SPR (Single-clone sequencing). Figure 2 ).

[0177] Figure 2 The binding curves of CLDN4 protein to 90 highly enriched aptamer sequences were detected using the SPR method. The results showed that most sequences had good affinity for CLDN4 protein, with eight sequences standing out. Homology analysis of these eight sequences is shown below. Figure 14 As shown, the secondary deconstruction diagram is as follows: Figure 16 As shown.

[0178] The concentration gradient KD values ​​of eight promising sequences were determined using a single-clone aptamer (SPR) assay. Table 1 shows the KD values ​​of the eight selected aptamer sequences bound to the CLDN4 protein as determined by SPR.

[0179] Table 1

[0180]

[0181] Example 2

[0182] Flow cytometry experiments were conducted to compare the binding differences of eight selected sequences and their corresponding truncated sequences between the CLDN4 small interfering RNA knockdown group (si2, si3) and the negative control group (siNC) of the same tumor cell lines (BXPC-3, CAPAN2, CW2, HCT116, SW620) and the overexpression group (OE) and the negative control group (Vector) of the normal pancreatic ductal epithelial cell line (HPNE). This confirmed the targeting of the selected sequences and their corresponding truncated sequences after removing the primer regions for CLDN4-positive human pancreatic cancer and colorectal cancer cell lines.

[0183] The specific steps are as follows: Digest and collect cells (including: human pancreatic cancer cell lines with high CLDN4 protein expression (BXPC-3, CAPAN2), normal human pancreatic ductal epithelium (HPNE), human colorectal cancer cell lines (CW2, HCT116, SW620) and corresponding CLDN4 knockdown or overexpression cell lines), and incubate them with Cy5-labeled Aptamer at 4 degrees for 1 hour. Detect cell surface fluorescence by flow cytometry and perform quantitative analysis of the results using FlowJo software.

[0184] The results are as follows Figure 3 As shown, Figure 3The image shows a heatmap of the binding affinity of selected Aptamer cells to human pancreatic cancer cell lines (BXPC-3, CAPAN2) with high CLDN4 protein expression, normal human pancreatic ductal epithelium (HPNE), human colorectal cancer cell lines (CW2, HCT116, SW620), and corresponding CLDN4 knockdown or overexpression cell lines, as determined by flow cytometry.

[0185] The results showed that the eight selected nucleic acid aptamer sequences all had a more significant average intensity than the random library sequences, indicating better cell binding ability.

[0186] Example 3: Tumor Diagnosis and Targeted Therapy Based on CLDN4

[0187] In this embodiment, a truncated sequence of CLDN4-29 (named C29s or C29shorten), which is the core sequence for CLDN4 protein binding, will be used to conduct the following experiments.

[0188] Cell experiments

[0189] By linking C29s to siRNA or drugs, Aptamer-drug conjugates (ApDC) were constructed, and the results were as follows: Figure 4 As shown. The mass spectrometry results after ApDC construction are as follows. Figure 15 As shown. Figure 4 The diagram illustrates methods for loading drugs onto nucleic acid aptamers, including drug-terminal linkage, base substitution, complementary pairing, nucleic acid sequence linkage, and drug embedding.

[0190] The cytotoxicity of ApDC to pancreatic cancer cell line (Capan2) and colorectal cancer cell line (CW2) at the cellular level was verified using the CCK-8 assay.

[0191] Capan2 and CW2 cell lines were seeded into 96-well plates at a density of 4000 cells per well. After 24 hours, the medium was replaced with the corresponding concentrations of drugs (aptamer, monomeric drug, and aptamer-conjugated drug, respectively). After incubation for 72 hours, cell viability was detected using CCK8 reagent and IC50 curves were plotted.

[0192] The results are as follows: Figure 5 and Figure 6 As shown in the results, the nucleic acid aptamer-drug conjugate exhibits a high inhibitory effect on the proliferation of cell lines expressing CLDN4, and this inhibitory effect is higher than that of the corresponding monomeric drug.

[0193] Animal experiments

[0194] The targeting of aptamer and ApDC to tumors was confirmed using in vivo fluorescence imaging experiments in mice. The specific steps are as follows:

[0195] The human colorectal cancer cell line HCT116, which highly expresses CLDN4, was subcutaneously implanted into nude mice. After tumor formation, Cy5-labeled aptamers were injected via the tail vein. The fluorescence intensity of the subcutaneous tumor tissue was detected using a small animal imaging system at 1, 2, 3, 4, and 6 hours post-injection.

[0196] The results are as follows Figure 7 As shown, compared with the random library sequence, C29s and the corresponding ApDC C29s-MMAE showed increased fluorescence intensity in tumor tissue, indicating that they were significantly enriched in the subcutaneous tumor region, suggesting that they have good targeting of colorectal cancer subcutaneous tumors in mice.

[0197] like Figure 8 As shown, statistical analysis of the results using LivingImage 4.4 software revealed that, compared to the library sequences, the screened nucleic acid aptamers and their conjugate drugs were significantly enriched in mouse subcutaneous tumors, and the difference remained significant even 6 hours after injection. In vitro fluorescence imaging experiments in mice further demonstrated the enrichment of aptamer and ApDC in tumor tissue. Figures 9-10 ).

[0198] Six hours after injection, the mice were dissected, and the fluorescence intensity of the subcutaneous tumor tissue was detected using a small animal imaging system. The results are as follows: Figure 9 As shown, compared with the random library sequence, C29s and the corresponding ApDC C29s-MMAE showed higher fluorescence intensity in tumor tissue, indicating that the nucleic acid aptamers were significantly enriched in tumor tissue.

[0199] Figure 10 Table 2 shows the results of statistical analysis performed using LivingImage 4.4 software. The results indicate that nucleic acid aptamers and their corresponding ApDCs are mainly metabolized by the kidneys, and there is no significant enrichment in other non-tumor regions.

[0200] Table 2

[0201]

[0202] In vivo experiments in mice revealed that ApDC exhibits excellent tumor-targeting therapeutic effects in tumor models. The specific steps are as follows: HCT116 cell lines were subcutaneously inoculated into nude mice until the tumor volume was estimated at 100 mm². 3 Tumors were administered via tail vein injection every 3 days for a total of 4 doses, and tumor size was monitored. PBS served as the control group, the C29s group received the aptamer alone, the VC-MMAE group received the single drug, and the C29s-MMAE group received the aptamer-conjugated drug.

[0203] result Figure 11As shown in the figure. The results indicate that, compared to the single nucleic acid aptamer or monomeric drug groups, the nucleic acid aptamer-conjugated drug group has a more significant inhibitory effect on tumors. In vivo experiments in mice verified that ApDC has good tumor-targeting therapeutic effects.

[0204] Mice were euthanized and dissected to weigh subcutaneous tumors. Results were as follows: Figure 12 As shown, the nucleic acid aptamer-drug conjugate combination exhibits a more significant inhibitory effect on tumors.

[0205] The body weight of the mice was measured during the treatment period. The results are as follows: Figure 13 As shown, no significant weight loss was observed, and no significant weight difference was observed between the groups, suggesting that the nucleic acid aptamer conjugate has a certain degree of safety.

[0206] The sequence information of the present invention is shown in Table A below.

[0207] Table A

[0208]

[0209] Note: The underlined portions represent the single-stranded DNA (ssDNA) of the constant 5' and 3' primers corresponding to Aptamer; SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, and 16 are the core sequences of CLDN4-29, 02, 05, 14, 23, 26, 36, and 55 that bind to the target protein CLDN4, respectively. SEQ ID NO: 18 is the core sequence of the control (SEQ ID NO: 17).

[0210] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A nucleic acid aptamer that binds to CLDN4, characterized in that, It is a core sequence that is incorporated into the CLDN4, and the core sequence is shown in SEQ ID NO:

9.

2. A nucleic acid aptamer that binds to CLDN4, characterized in that, The sequence of the nucleic acid aptamer is shown in SEQ ID NO:

6.

3. A coupling agent, characterized in that, The coupling contains: (a) The nucleic acid aptamer as described in claim 1 or 2; and (b) A coupling portion coupled to the nucleic acid aptamer portion, the coupling portion being selected from the group consisting of: detectable markers, drugs, toxins, or combinations thereof.

4. The coupling as described in claim 3, characterized in that, The detectable markers are selected from the group consisting of: biotin, chemiluminescent groups, chemifluorescent groups, fluorescent proteins, enzymes, colloidal gold, radioisotopes, latex particles, antibodies, ligands, antigens, receptors, or combinations thereof.

5. A biochip, characterized in that, The biochip comprises the nucleic acid aptamer as described in claim 1 or 2, or the conjugate as described in claim 3.

6. The use of the nucleic acid aptamer according to claim 1 or 2, the conjugate according to claim 3, or the biochip according to claim 5, characterized in that, For the following purposes: (Z1) A kit for preparing the detection of CLDN4; (Z2) is used to prepare reagents for imaging CLDN4; (Z3) Preparation of pharmaceutical compositions targeting CLDN4; and / or (Z4) was used to purify CLDN4.

7. A test kit, characterized in that, The detection kit includes: the nucleic acid aptamer as described in claim 1 or 2, or the conjugate as described in claim 3.

8. The use of the test kit according to claim 7, characterized in that, A kit for preparing a diagnostic tool for CLDN4-positive tumors.

9. A composition, characterized in that, The composition comprises: (a) The nucleic acid aptamer according to claim 1 or 2; and (b) Optional, pharmaceutically acceptable excipients.

10. An in vitro method for detecting CLDN4 for non-disease diagnosis and / or treatment, characterized in that, Includes the following steps: (S1) Provide the sample to be tested; (S2) The sample to be tested is mixed with the nucleic acid aptamer of claim 1 or 2, or the conjugate of claim 3, to form a mixture; (S3) Detect the presence or absence of "CLDN4-nucleic acid aptamer complex" in the mixture, wherein if the complex is present, it indicates that CLDN4 is present in the sample; if the complex is not present, it indicates that CLDN4 is not present in the sample.

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