Nectin-4 targeted aptamer nuclide diagnosis and treatment integrated probe

Abstract

The invention discloses a Nectin-4 targeted nucleic acid aptamer nuclide diagnosis and treatment integrated probe, and relates to the field of biotechnology and diagnosis and treatment integration and treatment, and the diagnosis and treatment integrated probe comprises a Nectin-4 targeted nucleic acid aptamer, a nuclide chelating ligand and a nuclide for marking; wherein the Nectin-4 targeting nucleic acid aptamer has a base sequence as shown in any one of SEQ ID NO.1-3, or has a base sequence of which the homology with the base sequence as shown in any one of SEQ ID NO.1-3 is more than 80%. The diagnosis and treatment integrated probe prepared by the invention can be applied to PET / CT diagnosis and internal irradiation radioactive therapy, on one hand, non-invasive visual molecular imaging of Nectin-4 receptor protein overexpression tumors is realized, and on the other hand, radioactive therapy of malignant bladder cancer is further realized; the probe is good in specific targeting effect and high in tumor uptake and has a diagnosis and treatment integrated function.

Description

Technical Field

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

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

[0003] Nucleic acid aptamers (Apt) are known as the antibodies of chemists. Compared with antibodies, they have the advantages of short screening time, easy chemical modification, low cost, high affinity, and good biocompatibility, showing great application potential in the field of tumor targeted diagnosis and treatment. In tumor detection, aptamers can target tumor-specific proteins, providing a new approach for non-invasive diagnosis and targeted therapy.

[0004] In radioactive diagnosis and treatment, PET examination can provide detailed functional and metabolic molecular information of lesions, and CT scan can provide accurate spatial positioning, and the two complement each other's advantages. Since the human body cannot emit γ photons, the signal-to-noise ratio of PET / CT is higher than that of other imaging methods. At the same time, compared with traditional examination methods, it has higher sensitivity and accuracy and is a powerful means in early tumor screening.

[0005] Therefore, those skilled in the art are committed to developing a probe with a nucleic acid aptamer as the target head, which has good specific targeting effect, high tumor uptake, through reasonable design of molecular probes, and finally realizes the integration of radionuclide diagnosis and treatment of tumors. Summary of the Invention

[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to obtain an integrated diagnosis and treatment probe with a nucleic acid aptamer as the target head, which has good specific targeting effect, low radiation dose, high tumor uptake, short imaging cycle and is more easily clinically translated and applied.

[0007] To achieve the above object, the present invention provides a Nectin-4 targeted nucleic acid aptamer radionuclide integrated diagnosis and treatment probe. The present invention selects the Nectin-4 nucleic acid aptamer, uses PET / CT imaging without background interference and accurate positioning for radionuclide radiotherapy and obtains good curative effects, and finally provides a radionuclide probe with the ability of integrated diagnosis and treatment.

[0008] Diagnostic and therapeutic integrated probe In one aspect, the present invention provides a Nectin-4 targeting aptamer-based theranostic probe, comprising a Nectin-4 targeting aptamer, a radionuclide chelating ligand, and a labeled radionuclide; Wherein, the Nectin-4 targeting aptamer has a base sequence shown in any one of SEQ ID NO.1-3, or has a base sequence with a homology of more than 80% to the base sequence shown in any one of SEQ ID NO.1-3.

[0009] In some embodiments, the Nectin-4 targeting aptamer of the present invention has a DNA or RNA base sequence shown in SEQ ID No.1, 2 or 3, and the specific aptamer names corresponding thereto in the present invention are N4A-6, N4A-7T, and N4A-8T.

[0010] In some other embodiments, the present invention further provides a variant of the Nectin-4 targeting aptamer, and the variant 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 aptamer from which it is derived (such as the biological activity of specifically binding to Nectin-4).

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

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

[0013] As used herein, a Nectin-4 targeting nucleic acid aptamer can specifically bind to Nectin-4.

[0014] The term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (K D ). In the present invention, the term "K D " refers to the dissociation equilibrium constant of a specific nucleic acid aptamer-antigen interaction, which is used to describe the binding affinity between a nucleic acid aptamer and an antigen. The smaller the equilibrium dissociation constant, the tighter the nucleic acid aptamer-antigen binding and the higher the affinity between the nucleic acid aptamer and the antigen.

[0015] The specific binding property between two molecules can be determined using methods well known in the art. One method involves measuring the rates of antigen binding site / antigen complex formation and dissociation. The "association rate constant" (k a or k on ) and the "dissociation rate constant" (k dis or k off ) can both be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of k dis / k on is equal to the dissociation constant K D (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). Any effective method can be used to measure K D , k on and k dis values. In certain embodiments, surface plasmon resonance (SPR) in a Biacore can be used to measure the dissociation constant. In addition, bioluminescence interferometry or Kinexa can also be used to measure the dissociation constant.

[0016] In some embodiments, the radionuclide chelating ligand includes at least one of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

[0017] In some embodiments, the radionuclide for the labeling is any one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, I-124, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, Y-86, Mn-52, Sc-44; or any one of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra-223, Ru-106, Na-24, Sr-89, Tb149, Th-227, Xe-133, Yb-169, Yb-177.

[0018] In a specific embodiment, the radionuclide for the labeling is Ga-68 or Lu-177.

[0019] In a specific embodiment, the present invention provides a novel Nectin-4 specific nucleic acid aptamer-radionuclide molecular probe 68 Ga]NOTA-Apt, specifically including 68 Ga]NOTA-N4A-6 probe, 68 Ga]NOTA-N4A-7T probe, 68 Ga]NOTA-N4A-8T probe, which realizes non-invasive visual detection of cells with overexpressed Nectin-4 receptor protein, and further realizes non-invasive molecular imaging of malignant bladder cancer.

[0020] In a specific embodiment, the present invention provides a novel Nectin-4 specific nucleic acid aptamer-radionuclide molecular probe 177 Lu]DOTA-Apt, specifically including 177 Lu]DOTA-N4A-6 probe, 177 Lu]DOTA-N4A-7T probe, 177 Lu]DOTA-N4A-8T probe, which realizes internal irradiation of tumors with overexpressed Nectin-4 receptor protein, and further realizes radiotherapy of malignant bladder cancer.

[0021] Preparation method In one aspect, the present invention provides a method for preparing a Nectin-4-targeted nucleic acid aptamer-based theranostic probe, which comprises chelating a radionuclide with a specific short single-stranded nucleic acid after coupling a radionuclide chelating ligand with the short single-stranded nucleic acid. The method specifically includes the following steps: S1. Perform a coupling reaction between a radionuclide chelating ligand with NHS and a Nectin-4-targeted nucleic acid aptamer modified with an amino group at the end to obtain a radionuclide chelating ligand-nucleic acid aptamer conjugate; S2. Dissolve the radionuclide to be labeled in a hydrochloric acid solution, and then add a pH regulator to adjust the pH to obtain a radionuclide-containing solution; S3. Mix the radionuclide chelating ligand-nucleic acid aptamer conjugate prepared in step S1 with the radionuclide-containing solution prepared in step S2, and purify the reaction product to obtain the theranostic probe.

[0022] In some embodiments, in step S2, the pH regulator is selected as a sodium acetate buffer solution, and the pH is adjusted to 4-4.5. Specifically, the pH can be adjusted to any one of 4, 4.1, 4.2, 4.3, 4.4, 4.5 or a range formed by any two of these values. The pH regulator can also be of other types as long as the purpose of pH adjustment can be achieved, and the present invention does not make specific limitations.

[0023] In some embodiments, in step S2, the concentration of the hydrochloric acid solution used is 0.1 mol / L.

[0024] In some embodiments, in step S3, the specific conditions of the reaction are: shaking reaction at 93-97 °C for 13-18 minutes. More specifically, the specific conditions of the reaction are: shaking reaction at 95 °C for 15 minutes.

[0025] In some embodiments, in step S3, when the radionuclide is 68 Ga, the radioactive dose of the 68 Ga hydrochloric acid solution in the prepared radionuclide-containing solution is 2 mCi to 10 mCi. Specifically, the radioactive dose can be any one of 2 mCi, 3 mCi, 4 mCi, 5 mCi, 6 mCi, 7 mCi, 8 mCi, 9 mCi, 10 mCi or a range formed by any two of these values; when the radionuclide is 177 Lu, the radioactive dose of the 177 Lu hydrochloric acid solution in the prepared radionuclide-containing solution is 3 mCi to 5 mCi. Specifically, the radioactive dose can be any one of 3 mCi, 4 mCi, 5 mCi or a range formed by any two of these values.

[0026] In some embodiments, in step S3, a GE Life MiniTrip G-25 desalting and purification column is used for purification.

[0027] Composition In another aspect, the present invention provides a composition comprising the Nectin-4-targeting nucleic acid aptamer radionuclide theranostic probe as described herein. The composition can be used for visualizing the expression of Nectin-4, diagnosing Nectin-4-related tumors, predicting the progression and prognosis of Nectin-4-related tumors, predicting the therapeutic effect of Nectin-4-related tumors, and / or treating Nectin-4-related tumors.

[0028] In some embodiments, the composition can be a pharmaceutical composition.

[0029] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient.

[0030] In some embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

[0031] In some embodiments, the additional pharmaceutically active agent is an anti-inflammatory drug or an immunosuppressant.

[0032] In some embodiments, in the pharmaceutical composition, the Nectin-4-targeting nucleic acid aptamer radionuclide theranostic probe as described herein and the additional pharmaceutically active agent can be provided as separate components or as mixed components. Thus, the Nectin-4-targeting nucleic acid aptamer radionuclide theranostic probe as described herein and the additional pharmaceutically active agent can be administered simultaneously, separately, or sequentially.

[0033] In some embodiments, the pharmaceutically acceptable carrier and / or excipient may comprise a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w / v) NaCl), glucose solution (such as 5% glucose), solution containing a surfactant (such as 0.01% polysorbate 20), pH buffer solution (such as phosphate buffer solution), Ringer's solution, and any combination thereof.

[0034] The pharmaceutical composition of the present invention may include a "therapeutically effective amount" of the Nectin-4-targeted nucleic acid aptamer radionuclide theranostic probe as described herein. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially arrest 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 to be treated, the overall status of the patient's own immune system, the general condition of the patient such as age, weight and gender, the mode of administration of the drug, and other treatments administered concomitantly, etc.

[0035] Kit The present invention also provides a kit comprising the Nectin-4-targeted nucleic acid aptamer radionuclide theranostic probe as described herein.

[0036] 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 therapeutic effect of Nectin-4-related tumors and / or treat Nectin-4-related tumors.

[0037] The kit may further comprise a container, instructions for use, and other reagents and buffers required for practical applications, such as a lysis medium for dissolving samples, various buffers, detection labels, detection substrates, etc.

[0038] Diagnostic and therapeutic applications In the Nectin-4-targeted nucleic acid aptamer radionuclide theranostic probe of the present invention, the Nectin-4-targeted nucleic acid aptamer used has extremely high affinity for Nectin-4, and thus can be used to visualize the expression of Nectin-4, diagnose Nectin-4-related tumors, radiotherapy Nectin-4-related tumors, predict the progression and prognosis of Nectin-4-related tumors, predict the therapeutic effect of Nectin-4-related tumors and / or treat Nectin-4-related tumors.

[0039] As used herein, Nectin-4-related tumors may include various tumors or cancers well known in the art. For example, Nectin-4-related tumors are tumors with abnormally high expression of Nectin-4, which may include bladder tumors with abnormally high expression of Nectin-4. The present invention uses tumor-bearing mice with a malignant bladder cancer tumor model to verify the molecular radioimaging and therapeutic efficacy of the targeted nucleic acid aptamer radionuclide theranostic probe.

[0040] Advantages of the present invention: The sequence of the nucleic acid aptamer used in the present invention can be solid-phase synthesized using a DNA synthesizer; the sequence can be customized; the difference between batches is small. Compared with the preparation of traditional antibody probes, the reaction conditions are simple. At the same time, the radionuclide-labeled PET imaging probe 68 Ga]NOTA-Apt can be used for non-invasive visual molecular imaging of malignant bladder cancer, 177 Lu]DOTA-Apt probe can be used for the radiotherapy of malignant bladder cancer. The probe preparation method disclosed in the invention can be generally used for the creation of molecular probes targeting other molecular targets and different radionuclides, and has broad application prospects and very high clinical transformation value in the future diagnosis and precision treatment of tumor tissues. It has the advantages of simple preparation process, low cost, high specificity, high stability, high imaging signal-to-noise ratio, and easy clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a schematic diagram of the coupling of the nucleic acid aptamer of Example 1 of the present invention with radionuclide chelating ligands (NOTA and DOTA); Figure 2 is the high performance liquid chromatography (HPLC) chromatogram of the nucleic acid aptamer of Example 1 of the present invention after coupling with the NOTA ligand; Figure 3 is the high performance liquid chromatography (HPLC) chromatogram of the nucleic acid aptamer of Example 1 of the present invention after coupling with the DOTA ligand; Figure 4 is the high resolution mass spectrometry (HRMS) chromatogram of the nucleic acid aptamer of Example 1 of the present invention after coupling with the NOTA ligand; Figure 5 is the high resolution mass spectrometry (HRMS) chromatogram of the nucleic acid aptamer of Example 1 of the present invention after coupling with the DOTA ligand; Figure 6 is of Example 3 of the present invention 68 PET / CT imaging of the bladder in nude mice bearing orthotopic tumors two hours after intravesical perfusion of three Apt probes ( 68 Ga]NOTA-Apt) labeled with Figure 7 is the body weight change curve of nude mice bearing orthotopic tumors of the in situ cell line HT1376 in Example 4 of the present invention, and the volume ratio of the bladder (tumor-bearing and healthy) to the healthy bladder and the ratio of the weight of the bladder (tumor-bearing and healthy) to the body weight of the corresponding mice at the end time of radionuclide radiotherapy of the bladder; Figure 8 is the body weight change curve of C57 mice bearing orthotopic tumors of the in situ cell line MB49 in Example 4 of the present invention, and the volume ratio of the bladder (tumor-bearing and healthy) to the healthy bladder and the ratio of the weight of the bladder (tumor-bearing and healthy) to the body weight of the corresponding mice at the end time of radionuclide radiotherapy of the bladder; Figure 9It is the digital image of the excised tumor of tumor-bearing mice in different groups of Example 4 of the present invention 14 days after bladder perfusion radiotherapy. Detailed implementation mode

[0042] The following introduces multiple preferred embodiments of the present invention with reference to the accompanying drawings of the specification, making its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0043] The nucleic acid aptamer involved in the embodiments of the present invention is a synthetic 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 with the target receptor.

[0044] In the embodiments of the present invention, a Nectin-4-targeted nucleic acid aptamer radionuclide diagnosis and treatment integrated probe 68 Ga]NOTA-Apt and 177 Lu]DOTA-Apt are specifically disclosed, where Apt is a nucleic acid aptamer, specifically at least one of N4A-6, N4A-7T, and N4A-8T (the base sequences of each nucleic acid aptamer are shown in Table 1 below), DOTA is a ligand coupling agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and NOTA is a ligand coupling agent 1,4,7-triazacyclononane-1,4,7-triacetic acid. 68 Ga (T1 / 2 = 67.71 min) is a radionuclide used for positron emission tomography (PET). 177 Lu (T1 / 2 = 6.65 days) is a metal radionuclide used for radiotherapy.

[0045] Table 1

[0046] In the nucleic acid aptamer radionuclide diagnosis and treatment integrated probe, the Nectin-4-targeted nucleic acid aptamer N4A-6 used is obtained by screening through the systematic evolution of ligands of Nectin-4 protein. Specifically, from a large number (10 12 ~10 15In a random nucleic acid sequence (i.e., when synthesizing DNA, after selecting a specific number of bases, the base types are not specified, and a library with the same length but random sequences is obtained through random combination during the synthesis process), by screening sequences that specifically bind to Nectin-4 protein and do not bind to human serum albumin, and after multiple rounds of screening, a specific Nectin-4 targeting nucleic acid sequence is obtained. The Nectin-4 targeting nucleic acid aptamers N4A-7T and N4A-8T are obtained by mimicking two other specific Nectin-4 targeting nucleic acid sequences screened by the same method as described above, obtaining their conformational information, and then gradually truncating the full-length sequences while ensuring that the specific conformation of each sequence remains unchanged and the change in Gibbs free energy △G is not significant, resulting in truncated nucleic acid sequences.

[0047] The binding affinity (Kd) between the nucleic acid aptamer N4A-6 and Nectin-4 protein was tested, and the measured Kd value was: Kd = 1.51e-07 M.

[0048] In a specific embodiment, the preparation process of the Nectin-4 targeting nucleic acid aptamer radionuclide diagnosis and treatment integrated probe is described in detail.

[0049] In another specific embodiment, the process of constructing a Nectin-4 expressing positive bladder cancer tumor-bearing mouse model is described in detail. The tumor-bearing mice with the constructed malignant bladder cancer tumor model are used to verify the molecular radioimaging and therapeutic efficacy of the Nectin-4 targeting nucleic acid aptamer radionuclide diagnosis and treatment integrated probe molecule.

[0050] In another specific embodiment, the molecular imaging process of the Nectin-4 targeting nucleic acid aptamer radionuclide diagnosis and treatment integrated ( 68 Ga]NOTA-Apt) PET / CT molecular probe for bladder cancer is described in detail.

[0051] In another specific embodiment, the radioactive internal irradiation treatment process of the aptamer-based specific radionuclide diagnosis and treatment integrated ( 177 Lu]DOTA-Apt) PET / CT molecular probe for bladder cancer is described in detail.

[0052] In the following examples, the HT376 cells used were derived from ATCC, the MB49 cells were derived from Millipore, and the mice were purchased from Unilever; all the reagents used were commercially available or prepared by existing methods.

[0053] In the following examples, all the nucleic acid sequences were synthesized by solid-phase synthesis.

[0054] Example 1 Preparation of a Nectin-4-targeted aptamer-based radionuclide theranostic probe, specifically including the following steps: (1) Preparation of 5'-amino-modified aptamer: Using a conventional method, 6-amino-1-hexanol was condensed with a 5'-phosphorylated nucleic acid strand to obtain a 5'-amino-modified aptamer; (2) As shown in the schematic diagram of the coupling of the nucleic acid short single strand as Figure 1 with NOTA or DOTA, NOTA or DOTA with NHS was dissolved in DMSO, and the dissolved NOTA-NHS or DOTA-NHS was added to the 5'-amino-modified aptamer (dissolved in carbonate). The reaction was carried out at room temperature for 120 min, and then purified by high-performance liquid chromatography (HPLC), and the molecular weight of the product was confirmed by high-resolution mass spectrometry. As shown in the high-performance liquid chromatography diagrams of the aptamers (N4A-6, N4A-7T, N4A-8T in sequence) after NOTA ligand coupling as Figure 2 , the main peak of the retention time is the main product NOTA-aptamer. As shown in the high-performance liquid chromatography diagrams of the aptamers (N4A-6, N4A-7T, N4A-8T in sequence) after DOTA ligand coupling as Figure 3 , the main peak of the retention time is the main product DOTA-aptamer. As shown in the high-resolution mass spectrometry diagrams of the aptamers (N4A-6, N4A-7T, N4A-8T in sequence) after NOTA ligand coupling as Figure 4 , the main peak shown is the main product NOTA-aptamer. As shown in the high-resolution mass spectrometry diagrams of the aptamers (N4A-6, N4A-7T, N4A-8T in sequence) after DOTA ligand coupling as Figure 5 , the main peak shown is the main product DOTA-aptamer; (3) Take 1 ml of 68 Ga or 177 Lu in 0.1 M hydrochloric acid solution with a radioactive dose of 6 mCi, and add 0.125 ml of 1 M sodium acetate solution to adjust the pH value to about 4 - 4.5. After standing for 5 min, a reaction solution was formed; (4) Mix the reaction solution obtained in step (3) with the NOTA-aptamer or DOTA-aptamer solution obtained in step (2), heat to 95 degrees Celsius and shake for 15 minutes, and then use PBS as the mobile phase to purify and separate the free 68 Ga or 177Lu ions, and finally radio-thin layer chromatography (Radio-TLC, Eckert & Ziegler Radiopharma Inc) was used to measure the concentration of each probe ([ 68 Ga]NOTA-Apt or [ 177 The radiochemical purity (RCP) of the probes was greater than 99%.

[0055] Example 2 The orthotopic bladder cancer-bearing mouse model with positive expression of Nectin-4 was constructed, which specifically included the following steps: (1) Using HT1376 and MB49 cell lines, which were confirmed to be nectin-4 positive by Western blot, for pre-culture; (2) The cultured HT1376 and MB49 tumor cells were digested with 0.25% trypsin digestion solution, resuspended in cell culture medium with DPBS, and then mixed with matrix gel (Corning 354234) at a volume ratio of 1:1. The mixed cell suspension was placed on ice and the tumor was implanted as soon as possible; (3) Take 20 μl of the cell suspension prepared in step 2 (containing 400,000 cells) and inject it into the bladder of 5- to 6-week-old Balb / c nude mice or C57 black mice (Unilever) to establish an orthotopic xenograft tumor model (Nude-HT1376, C57-MB49).

[0056] Example 3 A Nectin-4 targeted nucleic acid aptamer nuclide diagnostic and therapeutic integrated probe ([ 68 Ga]NOTA-Apt) PET / CT molecular imaging of bladder cancer. The specific steps include: (1) Each tumor-bearing mouse was instilled with approximately 3.7 MBq of 68 Ga]NOTA-Apt molecular probe (3–6 per group); (2) Two hours after perfusion, the tumor-bearing nude mice were anesthetized with isoflurane (3%) mixed with oxygen, and the deeply anesthetized nude mice were placed in a prone position on the IRIS small animal PET / CT scanning bed. PET and CT images were acquired sequentially, and image reconstruction was completed using the IRIS system's own software. Figure 6 As shown, 68 Ga-labeled aptamer ([ 68PET / CT diagnostic and therapeutic integration images of tumor-bearing mice two hours after intravesical perfusion of [Ga]NOTA-Apt (Apt is N4A-6, N4A-7T, and N4A-8T respectively). One mouse per group is shown, and each group shows a separate PET / CT fusion image (left), PET image (middle), and CT image (right). It can be seen that 68 All [Ga]NOTA-Apt molecular probes can accurately perform molecular imaging of in-situ bladder cancer tumors, namely HT1376 and MB49 tumors. Figure 6 In the middle, Library is the comparison of the injected control probe ( 68 [Ga]NOTA-Library). Its PET / CT diagnostic and therapeutic integration image shows that the enrichment amount of the control probe in the bladder area is very low. The control sequence used for the control probe is a random sequence (a library with the same length but random sequences is obtained by randomly combining specific bases without specifying the base type after selecting 76 specific bases during DNA synthesis). The preparation method of the control probe refers to Example 1.

[0057] Example 4 A radionuclide diagnostic and therapeutic integration ( 177 [Lu]DOTA-Apt) molecular probe for Nectin-4-targeted nucleic acid aptamer in a tumor-bearing mouse model. Specifically, it includes the following steps: (1) The treatment group intravesically perfused about 3.7 MBq of 177 [Lu]DOTA-Apt molecular probe into each tumor-bearing nude mouse (5 mice per group). The control group (PBS) perfused an equal volume of normal saline. Control group 1 (Library) was set to replace the nucleic acid aptamer Nectin-4 with the control sequence of the nucleic acid aptamer Nectin-4 and perfused about 3.7 MBq of the control probe; Control group 2 (177Lu) perfused an equal amount of 177 [Lu]; The healthy group (Health) was set to healthy Balb / c nude mice or C57 black mice and perfused an equal volume of PBS; Among them, the base sequence of the control sequence (Library) used in control group 1 is a random sequence (a library with the same length but random sequences is obtained by randomly combining specific bases without specifying the base type after selecting 76 specific bases during DNA synthesis). The preparation method of the control probe ( 177 [Lu]DOTA-Library) refers to Example 1; (2) Record the body weight of the mice every two days after intravesical perfusion radiotherapy. Continuously observe and terminate the experiment when the body weight of the mice drops by more than 20%. In this experiment, all mice were euthanized on the 14th day of radiotherapy. Figure 7 and Figure 8Among them, the left figures are all the change curves of the body weights of the mice in each experimental group; the middle figures are all the volume ratios of the bladder volumes of the orthotopic tumor-bearing mice taken on the 14th day of radiotherapy to the bladder volumes of healthy mice; the right figures are all the weight ratios of the bladder weights of the orthotopic tumor-bearing mice taken on the 14th day of radiotherapy to the bladder weights of healthy mice. It was experimentally found that in the HT1376 and MB49 xenograft orthotopic tumor models, the volume and weight ratios (B / W) of the saline control group were much greater than those of healthy mice, and the volume and weight ratios (B / W) of Control Group 1 and Control Group 2 were also much greater than those of healthy mice, without inhibiting the growth of tumors; in the HT1376 xenograft orthotopic tumor model, there was no significant difference in the B / W ratio of the bladder tumors between the treatment group using the N4A-7T nucleic acid aptamer and the control group (PBS), but there was a significant difference in the volume ratio of the bladder tumors between the treatment group using the N4A-7T nucleic acid aptamer and the control group (PBS) in the HT1376 xenograft orthotopic tumor model, and both its volume ratio and B / W ratio of the bladder tumors to the control group (PBS) showed significant differences in the MB49 xenograft orthotopic tumor model; in both tumor models, there were significant differences in the volume ratio and B / W ratio of the bladder tumors between the treatment groups using the N4A-6 and N4A-8T nucleic acid aptamers and the control group (PBS), showing an effective ability to inhibit tumor growth; compared with Control Group 1 and Control Group 2 respectively, the volume ratio and B / W ratio of the bladder tumors of the mice in each treatment group decreased, indicating that the nucleic acid aptamer probe prepared by the present invention has an effective therapeutic effect in bladder cancer, such as Figure 7 , Figure 8 shown. After the treatment was completed, the mice in each group were euthanized to end the experiment. Finally, the tumor volumes of the mice in each treatment group (N4A-6, N4A-7T, N4A-8T) were all smaller than those of the control group, Control Group 1, and Control Group 2, as Figure 9 shown. In addition, the body weights of all the mice in this experiment fluctuated within a small range, and no serious toxic side effects occurred. This experiment verified the ability of the Nectin-4-targeted nucleic acid aptamer radionuclide diagnosis and treatment integrated probe to inhibit tumor growth, and no obvious physiological toxicity was found.

[0058] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations according to the concept of the present invention without creative labor. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention based on the concept of the present invention through logical analysis, reasoning or limited experiments should be within the protection scope determined by the claims.

Claims

1. A Nectin-4-targeting nucleic acid aptamer-based theranostic probe, characterized in that, The diagnosis and treatment integrated probe includes a Nectin-4 targeting nucleic acid aptamer, a radionuclide chelating ligand, and a labeling radionuclide; Among them, the Nectin-4 targeting nucleic acid aptamer has a base sequence shown in any one of SEQ ID NO.1 - 3.

2. The Nectin-4-targeting nucleic acid aptamer-based radionuclide diagnosis and treatment integrated probe according to claim 1, wherein The radionuclide chelating ligand includes at least one of 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and 1,4,7-triazacyclononane-1,4,7-triacetic acid.

3. The Nectin-4-targeting nucleic acid aptamer radionuclide diagnosis and treatment integrated probe according to claim 1, wherein The labeling radionuclide is any one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, I-124, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, Y-86, Mn-52, Sc-44; or is any one of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra-223, Ru-106, Na-24, Sr-89, Tb149, Th-227, Xe-133, Yb-169, Yb-177.

4. The Nectin-4 targeted nucleic acid aptamer radionuclide diagnosis and treatment integrated probe according to claim 3, wherein The labeling radionuclide is Ga-68 or Lu-177.

5. A preparation method of the Nectin-4-targeting nucleic acid aptamer radionuclide diagnosis and treatment integrated probe according to any one of claims 1 to 4, characterized in that, It includes the following steps: S1. Couple the radionuclide chelating ligand with NHS to the Nectin-4 targeting nucleic acid aptamer modified with an amino group at the end to obtain a radionuclide chelating ligand-nucleic acid aptamer conjugate; S2. Dissolve the labeling radionuclide in a hydrochloric acid solution, and then add a pH regulator to adjust the pH to obtain a radionuclide-containing solution; S3. Mix the radionuclide chelating ligand-nucleic acid aptamer conjugate prepared in step S1 with the radionuclide-containing solution prepared in step S2, and purify after reaction to obtain the diagnosis and treatment integrated probe.

6. The preparation method of the targeted nucleic acid aptamer radionuclide diagnosis and treatment integrated probe according to claim 5, characterized in that, In step S2, the pH regulator is selected from sodium acetate buffer solution, and the pH value is adjusted to 4 - 4.

5.

7. The preparation method of the Nectin-4-targeted nucleic acid aptamer radionuclide diagnosis and treatment integrated probe according to claim 5, characterized in that, In step S3, the specific conditions of the reaction are: shake and react at 93 - 97 °C for 13 - 18 minutes.

8. Use of the Nectin-4 targeting nucleic acid aptamer radionuclide diagnosis and treatment integrated probe according to any one of claims 1 - 4 in the preparation of a kit or composition for visualizing the expression of Nectin-4, Nectin-4 positive tumor targeted radiotherapy, diagnosing Nectin-4-related tumors, predicting the progression and prognosis of Nectin-4-related tumors, predicting the treatment effect of Nectin-4-related tumors, and / or treating Nectin-4-related tumors; The Nectin-4-related tumor is a bladder tumor with abnormally high expression of Nectin-4 protein.

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