A Nectin-4 targeted nucleic acid aptamer integrated diagnosis and treatment probe

Abstract

The present invention discloses a Nectin-4 targeted nucleic acid aptamer diagnosis and treatment integrated probe, which relates to the fields of biotechnology and diagnosis and treatment integration and treatment, including a Nectin-4 targeted nucleic acid aptamer and Vc-MMAE; the Nectin-4 targeted nucleic acid aptamer and Vc-MMAE are connected by coupling; wherein the Nectin-4 targeted nucleic acid aptamer has a base sequence as shown in any one of SEQ ID NOs. 1 to 3, or has a base sequence with a homology of more than 80% to the base sequence described in any one of SEQ ID NOs. 1 to 3. The diagnosis and treatment integrated probe prepared by the present invention realizes targeted diagnosis and treatment of tumors overexpressing the Nectin-4 receptor protein, and the probe has good specific targeting effect and low toxic and side effects.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and integrated diagnosis and treatment, and in particular to a Nectin-4 targeted nucleic acid aptamer integrated diagnosis and treatment probe. Background Art

[0002] At present, there are still limitations in the early clinical diagnosis of bladder cancer. For example, traditional cystoscopy is invasive and may cause complications, and urine cytology testing has low sensitivity and is prone to false negatives or false positives, which in turn affect clinical diagnosis and treatment decisions. There is an urgent need to develop new targeted diagnosis and treatment technologies.

[0003] Recent studies have revealed that nectin-4 is overexpressed in a variety of malignant tumors, including bladder, breast, ovarian, colorectal, prostate, and lung cancers. Due to its high expression in various tumors, nectin-4 has become a key target for antibody-drug conjugates (ADCs). MMAE, a highly toxic microtubule inhibitor, cannot be developed as a drug alone. Currently, it is primarily used in conjunction with antibodies to form antibody-drug complexes (ADCs) for targeted tumor therapy. However, antibodies present numerous challenges, including high immunogenicity, high cost, large batch-to-batch variability, difficulty in modification, and the need for low temperatures for storage and transportation. Aptamers (Apts), often called "chemists' antibodies," offer advantages over antibodies, including shorter screening times, ease of chemical modification, low cost, high affinity, and excellent biocompatibility. They demonstrate significant potential in targeted tumor diagnosis and therapy. In tumor detection, aptamers can target tumor-specific proteins, offering new avenues for non-invasive diagnosis and targeted therapy.

[0004] Therefore, technicians in this field are committed to developing integrated diagnosis and treatment probes with nucleic acid aptamers as target heads through the rational design of molecular probes, which have good specific targeting effects and low toxic side effects, and ultimately achieve integrated diagnosis and treatment of tumors. Summary of the Invention

[0005] In view of the deficiencies of the existing technology, the present invention provides a Nectin-4 targeted nucleic acid aptamer integrated diagnosis and treatment probe.

[0006] Integrated diagnosis and treatment probe

[0007] In one aspect, the present invention provides a Nectin-4 targeting nucleic acid aptamer integrated diagnosis and treatment probe, comprising a Nectin-4 targeting nucleic acid aptamer and Vc-MMAE; the Nectin-4 targeting nucleic acid aptamer and Vc-MMAE are connected by coupling;

[0008] The nectin-4 targeting nucleic acid aptamer has a base sequence as shown in any one of SEQ ID NOs. 1 to 3, or has a base sequence with a homology of more than 80% with the base sequence as shown in any one of SEQ ID NOs. 1 to 3.

[0009] In some embodiments, the nectin-4 targeting nucleic acid aptamer of the present invention has a DNA or RNA base sequence as shown in SEQ ID No. 1, 2 or 3, and the specific corresponding nucleic acid aptamers in the present invention are named NT1, NT2, and NT3.

[0010] In other embodiments, the present invention also provides a variant of a nectin-4 targeting nucleic acid aptamer, which is a DNA or RNA sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology to the base sequence shown in SEQ ID No. 1, 2 or 3, and substantially retains the biological function of the nucleic acid aptamer from which it is derived (e.g., biological activity of specifically binding to nectin-4).

[0011] More specifically, the variant differs from the nectin-4 targeting nucleic acid aptamer described herein only in the deletion, substitution, and / or insertion of one or more nucleic acid bases (e.g., deletion, substitution, and / or insertion of up to 20, up to 15, up to 10, up to 5, or up to 1 nucleic acid base). The nucleic acid base deletion refers to the deletion of one or more bases in the nucleic acid sequence. The substitution of nucleic acid bases refers to replacing one or more bases in a nucleic acid sequence with another one or more bases, specifically referring to changing adenine (A), guanine (G), cytosine (C), thymine (T), hydroxypurine or uracil (U) to another base, for example, replacing adenine with cytosine, thymine, uracil, hydroxypurine or guanine; replacing cytosine with adenine, thymine, uracil, hydroxypurine or guanine; replacing guanine with cytosine, thymine, uracil, hydroxypurine or adenine; replacing thymine with adenine, cytosine, uracil, hydroxypurine or guanine; replacing uracil with cytosine, thymine, adenine, hydroxypurine or guanine; replacing hydroxypurine with adenine, thymine, uracil or guanine. The insertion of nucleic acid bases refers to inserting one or more bases at one or more positions in a nucleic acid sequence.

[0012] As used herein, sequence "homology" refers to the percentage of corresponding bases that are identical by position, which can indicate the level of similarity (also known as sequence identity, similarity, or identity) between two or more nucleic acids.

[0013] As used herein, the nectin-4 targeting aptamer can specifically bind to nectin-4.

[0014] The term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D The smaller the equilibrium dissociation constant, the tighter the aptamer-antigen binding and the higher the affinity between the aptamer and the antigen.

[0015] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate at which the antigen binding site / antigen complex forms and dissociates. The "association rate constant" (k a or k on ) and the “dissociation rate constant” (k dis or k off ) Both can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). dis / k on The ratio is equal to the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D 、k on and k dis In certain embodiments, the dissociation constant can be measured using surface plasmon resonance (SPR) in Biacore. Alternatively, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0016] In the present invention, the Vc-MMAE, also known as mc-vc-PAB-MMAE, is an MMAE derivative with a valine-citrulline (Vc) linker and a drug-linker conjugate (Drug-linker Conjugate) with anti-tumor activity, and is used in the synthesis of antibody-drug conjugates (ADCs).

[0017] In some embodiments, the Vc-MMAE is coupled to the terminal thiol group of the nucleic acid aptamer by reaction.

[0018] In a specific embodiment, the present invention provides a Nectin-4 targeted nucleic acid aptamer diagnostic and therapeutic integrated probe Apt-MMAE, which enables non-invasive visual detection of cells overexpressing Nectin-4 receptor protein, and further achieves the treatment of malignant bladder cancer.

[0019] Preparation method

[0020] In one aspect, the present invention provides a method for preparing a Nectin-4 targeted nucleic acid aptamer diagnostic and therapeutic integrated probe, which specifically comprises the following steps:

[0021] S1. Preparing a stability-modified nectin-4 targeting aptamer;

[0022] S2. The stability-modified Nectin-4 targeting nucleic acid aptamer is coupled with Vc-MMAE to obtain the integrated diagnosis and treatment probe.

[0023] Composition

[0024] In another aspect, the present invention provides a composition comprising the Nectin-4-targeting aptamer diagnostic and therapeutic probe described herein. The composition can be used to visualize Nectin-4 expression, diagnose Nectin-4-associated tumors, predict the progression and prognosis of Nectin-4-associated tumors, predict the therapeutic efficacy of Nectin-4-associated tumors, and / or treat Nectin-4-associated tumors.

[0025] In some embodiments, the composition may be a pharmaceutical composition.

[0026] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient.

[0027] In some embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

[0028] In some embodiments, the additional pharmaceutically active agent is an anti-inflammatory drug or an immunosuppressant.

[0029] In some embodiments, in the pharmaceutical composition, the Nectin-4 targeting nucleic acid aptamer integrated diagnostic and therapeutic probe as described herein and the additional pharmaceutically active agent can be provided as separate components or as mixed components. Therefore, the Nectin-4 targeting nucleic acid aptamer integrated diagnostic and therapeutic probe as described herein and the additional pharmaceutically active agent can be administered simultaneously, separately, or sequentially.

[0030] In some embodiments, the pharmaceutically acceptable carrier and / or excipient may comprise a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0031] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" of the Nectin-4-targeting nucleic acid aptamer diagnostic and therapeutic integrated probe described herein. A "therapeutically effective amount" is an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on factors such as the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the method of drug administration, and any other concurrently administered treatments.

[0032] Reagent test kit

[0033] The present invention also provides a kit comprising the Nectin-4 targeted nucleic acid aptamer diagnosis and treatment integrated probe as described herein.

[0034] The kit can be used to visualize the expression of Nectin-4, diagnose Nectin-4 related tumors, predict the progression and prognosis of Nectin-4 related tumors, predict the treatment effect of Nectin-4 related tumors and / or treat Nectin-4 related tumors.

[0035] The kit may also include a container, instructions for use, and other reagents and buffers required for practical applications, such as a lysis medium for dissolving the sample, various buffers, detection labels, detection substrates, etc.

[0036] Diagnostic and therapeutic applications

[0037] In the Nectin-4 targeted nucleic acid aptamer integrated diagnosis and treatment probe of the present invention, the Nectin-4 targeted nucleic acid aptamer used has an extremely high affinity for Nectin-4, and can therefore be used to visualize Nectin-4 expression, diagnose Nectin-4 related tumors, predict the progression and prognosis of Nectin-4 related tumors, predict the treatment effect of Nectin-4 related tumors and / or treat Nectin-4 related tumors.

[0038] As used herein, "nectin-4-associated tumors" may include various tumors or cancers known in the art. For example, nectin-4-associated tumors are tumors with abnormally high nectin-4 expression, including bladder tumors. The present invention utilizes a mouse model of advanced bladder cancer to validate the radioactive imaging and therapeutic efficacy of a targeted nucleic acid aptamer-based integrated diagnostic and therapeutic probe.

[0039] Beneficial effects of the present invention:

[0040] The aptamer sequences used in the present invention can be synthesized using a DNA synthesizer through solid-phase synthesis; the sequences can be customized, and batch-to-batch variability is minimal. Aptamers inherently possess targeting and tumor specificity, enabling tumor-specific drug enrichment and tumor killing. Furthermore, the integrated diagnostic and therapeutic probe Apt-MMAE, formed by coupling the Vc-MMAE drug to the aptamer, can be used for noninvasive molecular imaging and treatment of malignant bladder cancer, achieving targeted tumor therapy and reducing drug toxicity and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 1 is a schematic diagram of the results of examining the targeting binding ability of nucleic acid aptamers by flow cytometry in Example 1 of the present invention;

[0042] Figure 2 This is a schematic diagram of the results of examining the toxicity of Apt-MMAE to T24 cells through CCK8 experiments in Example 1 of the present invention;

[0043] Figure 3 This is a schematic diagram of the results of examining the toxicity of Apt-MMAE to BFTC905 cells through CCK8 experiments in Example 1 of the present invention;

[0044] Figure 4 These are fluorescent imaging images of animals in Example 3 of the present invention, obtained after in situ drug administration at different times, in the experimental group (NT1-MMAE, NT3-MMAE), blank group (Saline), control group (Vc-MMEA), and control group (lib-MMAE);

[0045] Figure 5 3 is a schematic diagram of the results of the in vivo mouse experiment examining the targeted therapeutic effects of the experimental group (NT1-MMAE, NT3-MMAE), the blank group (Saline), the control group (Vc-MMEA) and the control group (lib-MMAE) in Example 3 of the present invention; wherein, Figure 5 A is the fluorescence intensity change curve of the in situ tumor in fluorescence imaging; Figure 5 B shows the results of tumor weight and tumor photos. DETAILED DESCRIPTION

[0046] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0047] The nucleic acid aptamer involved in the embodiments of the present invention is a segment of artificially synthesized single-stranded DNA or RNA, which is usually obtained by in vitro screening using the systematic evolution of ligands by exponential enrichment (SELEX) method, and has high affinity and specificity for the target ligand.

[0048] In an embodiment of the present invention, a nectin-4-targeted nucleic acid aptamer diagnostic and therapeutic integrated probe, Apt-MMAE-, is disclosed. The probe comprises a nectin-4-targeted nucleic acid aptamer (Apt) and a drug. The Apt (Aptmer) is any one of NT1, NT2, and NT3, and the drug is Vc-MMAE. The chemical formula of the nucleic acid aptamer diagnostic and therapeutic integrated probe is:

[0049] ;

[0050] Among them, Aptamer represents Nectin-4 targeting nucleic acid aptamer.

[0051] In the nucleic acid aptamer diagnosis and treatment integrated probe, the Nectin-4 targeting nucleic acid aptamer NT1 was obtained by screening the ligand evolution technology of exponential enrichment of Nectin-4 protein, specifically from a large number of (10 12 ~10 15 Specific nectin-4-targeting nucleic acid sequences were obtained after multiple rounds of screening by selecting sequences that specifically bind to the nectin-4 protein and do not bind to human serum albumin from a library of random nucleic acid sequences (i.e., a library of uniform length but random sequences is randomly combined during DNA synthesis after selecting a specific number of bases without specifying base types). The nectin-4-targeting nucleic acid aptamers NT2 and NT3 were simulated by obtaining conformational information from two other specific nectin-4-targeting nucleic acid sequences obtained through screening using the same method described above. Truncated nucleic acid sequences were then obtained by gradually truncating the full-length sequences while ensuring that the specific conformation of each sequence remained unchanged and that the Gibbs free energy ΔG did not change significantly.

[0052] In one specific embodiment, the preparation process of the Nectin-4 targeted nucleic acid aptamer integrated diagnostic and therapeutic probe is described in detail, and its retained toxicity is verified.

[0053] In another embodiment, the process of constructing a nectin-4-positive bladder cancer mouse model is described in detail. This malignant bladder cancer model is used to validate the molecular imaging and therapeutic efficacy of the nectin-4-targeted aptamer diagnostic and therapeutic probe for bladder cancer.

[0054] In the following examples, T24 cells, BFTC905 cells, and Scaber-luc cells were purchased from the Cell Bank of the Chinese Academy of Sciences. All reagents used were commercially available or prepared using existing methods.

[0055] In the following examples, the nucleic acid sequences used were synthesized by solid phase synthesis.

[0056] Example 1

[0057] A method for preparing a Nectin-4-targeted nucleic acid aptamer diagnostic and therapeutic integrated probe (Apt-MMAE) specifically comprises the following steps:

[0058] (1) Preparation of terminally modified thiol aptamers: Prepared by conventional commercial modification methods, the thiol groups are modified at the sequence ends (5') of the aptamers NT1, NT2, and NT3, respectively, to obtain terminally modified thiol aptamers;

[0059] The sequence of the nucleic acid aptamer NT1 (SEQ ID NO.1) is:

[0060] 5'-CGTACGGTCGACGCTAGCCGAGGTGCTCGTTGGAGTGGATTGCAAAGGACTGCTGTTCCACGTGGAGCTCGGATCC-3';

[0061] The sequence of the nucleic acid aptamer NT2 (SEQ ID NO.2) is:

[0062] 5'-AGCAGTCAAAGGGCCTGGCGTCCCGATTGGGGAAGTTGATTGCCAC-3';

[0063] The sequence of the nucleic acid aptamer NT3 (SEQ ID NO.3) is:

[0064] 5'-ACGCTAGCAGTCAAAGGGCCTGGCGTCCCGATTGGGGAAGTTGATTGCCACGTGGA-3'.

[0065] (2) Conjugation of Vc-MMAE with terminal thiol-modified nucleic acid aptamers: This step was commissioned to Shanghai Bioengineering Co., Ltd. for preparation.

[0066] (3) Verification of target binding ability: The prepared nucleic acid aptamer diagnosis and treatment integrated probe was used to examine its target binding ability by flow cytometry. The specific steps were as follows: 250nM nucleic acid aptamer diagnosis and treatment integrated probes (NT1-MMAE, NT2-MMAE, NT3-MMAE), simple nucleic acid aptamers (NT1, NT2, NT3) and control probes (Control) were linked to the fluorescent group Cy5 and then incubated with bladder cancer cells T24 in a 200ul buffer system for 30 minutes. After washing, the fluorescence on the cell surface was detected.

[0067] The control probe is a conjugate of a fluorescently labeled random sequence and Vc-MMAE, wherein the random sequence is selected after a specific number of bases of 76 is selected during DNA synthesis, without specifying the base type, and a library with consistent length but random sequence is obtained by random combination during the synthesis process.

[0068] The results are as follows Figure 1 shown. Figure 1 The results showed that compared with simple nucleic acid aptamers, the prepared nucleic acid aptamer integrated diagnosis and treatment probes retained the specific targeting of nucleic acid aptamers.

[0069] (4) Test of T24 cell retention toxicity: Different concentrations of nucleic acid aptamer diagnostic and therapeutic integrated probes (NT1-MMAE, NT2-MMAE, NT3-MMAE) and Vc-MMAE were incubated with bladder cancer cells T24 in a 200ul buffer system. After 24 hours, CCK-8 was added, the absorbance value was detected, and the cell survival rate was calculated. The results are as follows: Figure 2 As shown. Figure 2 The results show that compared with pure Vc-MMAE (IC50 value is 1247 nM), the three nucleic acid aptamer integrated diagnosis and treatment probes prepared in the embodiments of the present invention all retain their toxicity. Due to the improvement in the ability to bind to target cells, the drug efficacy is improved (compared with pure Vc-MMAE, the IC50 values ​​of the three nucleic acid aptamer integrated diagnosis and treatment probes decreased to 103.4~327.1 nM).

[0070] (5) Test of the toxicity retention of BFTC905 cells: Different concentrations of nucleic acid aptamer diagnostic and therapeutic integrated probe (NT1-MMAE) and Vc-MMAE were incubated with BFTC905 cells in 200ul buffer system. After 24 hours, CCK-8 was added, the absorbance value was detected, and the cell survival rate was calculated. The results are as follows: Figure 3 As shown. Figure 3The results showed that compared with simple Vc-MMAE (IC50 value was 93.32 nM), the efficacy of the integrated nucleic acid aptamer diagnostic and therapeutic probe NT1-MMAE was improved (the IC50 value of NT1-MMAE decreased to 1.418 nM).

[0071] Example 2

[0072] Establishment of a bladder cancer mouse model with positive Nectin-4 expression. The specific steps include:

[0073] (1) Using the Scaber-luc cell line, which was confirmed to be Nectin-4 positive by Western blot, the cells were pre-cultured;

[0074] (2) Digest the cultured Scaber-luc tumor cells with 0.25% trypsin digestion solution and resuspend them in cell culture medium with DPBS. Then mix them with Matrigel (Corning 354234) at a volume ratio of 1:1. Place the mixed cell suspension on ice and perform tumor implantation as soon as possible.

[0075] (3) Take 20 μl of the cell suspension prepared in step (2) (containing 4×10 5 The orthotopic xenograft tumor model was established by injecting the cells into the bladder of 4-5 week-old Balb / c nude mice (purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.).

[0076] Example 3

[0077] A Nectin-4-targeted aptamer diagnostic and therapeutic integrated probe (Apt-MMAE) is used for bladder tumor instillation treatment. The specific steps include:

[0078] (1) Each bladder tumor-bearing mouse was perfused with 50 ul of 0.2 uM Apt-MMAE through the bladder (8 mice per group, once a week, for 4 weeks) as the experimental group. The blank group (Saline) was perfused with an equal volume of normal saline, the control group 1 (Vc-MMAE) was perfused with an equal volume of 10 μM Vc-MMAE, and the control group 2 (Lib-MMAE) was perfused with an equal volume of 0.2 μM control probe. The control probe was formed by replacing the nucleic acid aptamer Nectin-4 of the present invention with the random sequence lib of the nucleic acid aptamer Nectin-4 coupled with Vc-MMAE; the random sequence Lib of Nectin-4 is formed by selecting a specific number of 76 bases during DNA synthesis without specifying the base type, and randomly combining them during the synthesis process to obtain a library with consistent length but random sequence.

[0079] (2) In this example, the tumors of the tumor-bearing mice were constructed using Scaber-luc cells. Therefore, the tumors can be made to emit spontaneous fluorescence by intraperitoneal injection of luciferase substrate. The size of the tumors can be observed by observing the fluorescence of the tumor site of the mice using a small animal imaging device. The fluorescence of the tumor site of the mice can be observed every 7 days after administration. The observation was continued for 4 weeks, and the experiment was terminated and all mice were euthanized. Figure 4 As shown, the experimental results show that the nucleic acid aptamer drug conjugates prepared in Example 1 (i.e., NT1-MMAE, NT3-MMAE) have the ability to inhibit in situ tumor growth compared with the control random sequence drug conjugate (Lib-MMAE).

[0080] After the treatment, the mice in each group were euthanized to terminate the experiment, and the bladder tumors were weighed and photographed after dissection. Figure 5 As shown in A, the tumor treatment effect was evaluated by measuring tumor autofluorescence. The nucleic acid aptamer drug conjugate has similar efficacy to Vc-MMAE and can effectively inhibit tumors. Figure 5 As shown in Figure B, tumor weights in the experimental groups were significantly lower than those in the saline blank group and the randomized drug conjugate control group 2. Compared to the drug-only control group 1, the experimental groups demonstrated comparable therapeutic efficacy. The NT3-MMAE group, in particular, achieved the closest therapeutic effect to the drug-only control group 1, despite using only one-fiftieth (0.02 times) the drug concentration used in the NT3-MMAE group. This suggests that drug aggregation due to targeting enhances therapeutic efficacy compared to the drug-only control group 1. Therefore, the aptamer-based integrated diagnostic and therapeutic probe constructed by this invention demonstrates effective therapeutic effects against bladder cancer at low doses. Furthermore, the body weights of all mice in this experiment fluctuated within a small range, and no serious side effects were observed. This experiment validates the ability of the described nectin-4-targeted aptamer-based integrated diagnostic and therapeutic probe to inhibit tumor growth without significant physiological toxicity.

[0081] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A Nectin-4 targeted nucleic acid aptamer integrated diagnosis and treatment probe, characterized in that: The integrated diagnosis and treatment probe includes a Nectin-4 targeting nucleic acid aptamer and Vc-MMAE; the Nectin-4 targeting nucleic acid aptamer and Vc-MMAE are connected by coupling; The base sequence of the Nectin-4 targeting nucleic acid aptamer is shown in any one of SEQ ID NOs. 1 to 3.

2. The Nectin-4 targeted nucleic acid aptamer diagnosis and treatment integrated probe according to claim 1, characterized in that: The base sequence of the Nectin-4 targeting nucleic acid aptamer is shown in SEQ ID NO.

1.

3. The Nectin-4 targeted nucleic acid aptamer diagnosis and treatment integrated probe according to claim 1, characterized in that: The base sequence of the Nectin-4 targeting nucleic acid aptamer is shown in SEQ ID NO.

2.

4. The Nectin-4 targeted nucleic acid aptamer diagnosis and treatment integrated probe according to claim 1, characterized in that: The base sequence of the Nectin-4 targeting nucleic acid aptamer is shown in SEQ ID NO.

3.

5. The Nectin-4 targeted nucleic acid aptamer integrated diagnosis and treatment probe according to any one of claims 1 to 4, characterized in that: The Nectin-4 targeting nucleic acid aptamer needs to be modified for stability before being coupled with Vc-MMAE.

6. The Nectin-4 targeted nucleic acid aptamer integrated diagnosis and treatment probe according to claim 5, characterized in that: The stability modification is to perform thiol modification and / or C6 modification on the end of the Nectin-4 targeting nucleic acid aptamer.

7. The Nectin-4 targeted nucleic acid aptamer integrated diagnosis and treatment probe according to claim 1, characterized in that: The integrated diagnosis and treatment probe has the chemical structure shown in the following formula I: ； Among them, Aptamer represents Nectin-4 targeting nucleic acid aptamer.

8. A method for preparing the Nectin-4 targeted nucleic acid aptamer diagnosis and treatment integrated probe according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Preparing a stability-modified nectin-4 targeting aptamer; S2. The stability-modified Nectin-4 targeting nucleic acid aptamer is coupled with Vc-MMAE to obtain the integrated diagnosis and treatment probe.

9. Use of the Nectin-4 targeted nucleic acid aptamer diagnostic and therapeutic integrated probe according to any one of claims 1 to 7 in the preparation of a kit for visually detecting Nectin-4 expression, diagnosing Nectin-4-related tumors, and predicting the progression and prognosis of Nectin-4-related tumors; The Nectin-4 related tumor is a bladder tumor with abnormally high expression of Nectin-4 protein.

10. Use of the Nectin-4 targeted nucleic acid aptamer integrated diagnosis and treatment probe according to any one of claims 1 to 7 in preparing a pharmaceutical composition for treating Nectin-4 related tumors, wherein the Nectin-4 related tumors are bladder tumors with abnormally high expression of Nectin-4 protein.

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