A Nectin-4-targeted nucleic acid aptamer radionuclide diagnostic and therapeutic integrated probe

Abstract

The present invention discloses a Nectin‑4 targeted nucleic acid aptamer nuclide diagnosis and treatment integrated probe, which relates to the fields of biotechnology and diagnosis and treatment integration and treatment. The diagnosis and treatment integrated probe includes a Nectin‑4 targeted nucleic acid aptamer, a nuclide chelating ligand and a labeling nuclide; wherein the Nectin‑4 targeted nucleic acid aptamer has a base sequence as shown in any one of SEQ ID NO.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 NO.1 to 3. The diagnosis and treatment integrated probe prepared by the present invention can be applied to PET / CT diagnosis and internal irradiation radiotherapy. On the one hand, it realizes non-invasive visual molecular imaging of tumors overexpressing Nectin‑4 receptor protein, and on the other hand, it further realizes radiotherapy of malignant bladder cancer; and the probe has good specific targeting effect, high tumor uptake and has both diagnosis and treatment integration functions.

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 nuclide 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] Aptamers (Apts), often called the chemists' antibodies, offer advantages over antibodies, including shorter screening times, ease of chemical modification, lower costs, higher affinity, and improved biocompatibility. Aptamers have shown tremendous potential in targeted tumor diagnosis and therapy. In tumor detection, aptamers can target tumor-specific proteins, providing a new avenue for noninvasive diagnosis and targeted therapy.

[0004] Among radiological diagnostic and therapeutic methods, PET scans can provide detailed functional and metabolic information about lesions, while CT scans can provide precise spatial localization, complementing each other's strengths. Because the human body cannot emit gamma photons, PET / CT offers a higher signal-to-noise ratio than other imaging modalities. Furthermore, compared to traditional examination methods, PET / CT offers greater sensitivity and accuracy, making it a powerful tool for early tumor screening.

[0005] Therefore, technicians in this field are committed to developing probes with nucleic acid aptamers as target heads through the rational design of molecular probes, which have good specific targeting effects and high tumor uptake, and ultimately realize the integrated nuclear 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 diagnostic and therapeutic 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 easier to clinically transform and apply.

[0007] To achieve these objectives, the present invention provides a nectin-4-targeted aptamer-based radionuclide diagnostic and therapeutic probe. This approach utilizes the nectin-4 aptamer and utilizes background-free, precisely targeted PET / CT imaging for radionuclide therapy, achieving excellent therapeutic efficacy. Ultimately, this provides a radionuclide probe with integrated diagnostic and therapeutic capabilities.

[0008] Integrated diagnosis and treatment probe

[0009] In one aspect, the present invention provides a Nectin-4 targeted nucleic acid aptamer nuclide diagnosis and treatment integrated probe, comprising a Nectin-4 targeted nucleic acid aptamer, a nuclide chelating ligand, and a labeling nuclide;

[0010] 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.

[0011] 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. In the present invention, the corresponding nucleic acid aptamers are named N4A-6, N4A-7T, and N4A-8T.

[0012] 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).

[0013] 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.

[0014] 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.

[0015] As used herein, the nectin-4 targeting aptamer can specifically bind to nectin-4.

[0016] 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 ) indicates. In the present invention, the term "K D " refers to the dissociation equilibrium constant of a specific aptamer-antigen interaction, which is used to describe the binding affinity between the aptamer and the antigen. The smaller the equilibrium dissociation constant, the tighter the aptamer-antigen binding and the higher the affinity between the aptamer and the antigen.

[0017] 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.

[0018] In some embodiments, the nuclide chelating ligand includes at least one of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

[0019] In some embodiments, the labeling nuclide 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, and Sc-44; or Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Any one of 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.

[0020] In a specific embodiment, the labeling nuclide used is Ga-68 or Lu-177.

[0021] In a specific embodiment, the present invention provides a novel nucleic acid aptamer-nuclide molecular probe specific for Nectin-4 [ 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 enables non-invasive visual detection of cells overexpressing nectin-4 receptor protein, and further realizes non-invasive molecular imaging of malignant bladder cancer.

[0022] In a specific embodiment, the present invention provides a novel nucleic acid aptamer-nuclide molecular probe specific for Nectin-4 [ 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 enables internal irradiation of tumors overexpressing nectin-4 receptor protein, further realizing radiotherapy of malignant bladder cancer.

[0023] Preparation method

[0024] In one aspect, the present invention provides a method for preparing a nectin-4-targeted nucleic acid aptamer radionuclide diagnostic and therapeutic integrated probe, which involves first coupling a radionuclide chelating ligand to a specific short single-stranded nucleic acid and then chelating it with the labeling radionuclide. Specifically, the method comprises the following steps:

[0025] S1. A nuclide chelating ligand with NHS is coupled with a nectin-4 targeting nucleic acid aptamer with an amino group modified at the end to obtain a nuclide chelating ligand-nucleic acid aptamer conjugate;

[0026] S2, dissolving the labeling nuclide in a hydrochloric acid solution, and then adding a pH adjuster to adjust the pH to obtain a solution containing the nuclide;

[0027] S3. Mixing the nuclide chelating ligand-nucleic acid aptamer conjugate prepared in step S1 with the nuclide-containing solution prepared in step S2, and purifying the mixture after reaction to obtain the integrated diagnosis and treatment probe.

[0028] In some embodiments, in step S2, the pH adjuster is a sodium acetate buffer solution, and the pH value is adjusted to 4-4.5. Specifically, the pH value can be adjusted to any one of 4, 4.1, 4.2, 4.3, 4.4, and 4.5, or a range formed by any two of these values. The pH adjuster can also be other types that can achieve the purpose of pH adjustment, and the present invention does not impose any particular limitation.

[0029] In some embodiments, in step S2, the concentration of the hydrochloric acid solution used is 0.1 mol / L.

[0030] In some embodiments, in step S3, the specific reaction conditions are: shaking reaction at 93-97° C. for 13-18 minutes. More specifically, the specific reaction conditions are: shaking reaction at 95° C. for 15 minutes.

[0031] In some embodiments, in step S3, the nuclide is 68 Ga, the prepared solution containing nuclides contains 68 The radioactive dose of Ga hydrochloric acid solution is 2 mCi~10 mCi. Specifically, the radioactive dose can be any one point value among 2 mCi, 3 mCi, 4 mCi, 5 mCi, 6 mCi, 7 mCi, 8 mCi, 9 mCi, and 10 mCi, or a range formed by any two point values. The nuclide is 177 Lu, the prepared solution containing nuclides contains 177 The radioactive dose of the Lu hydrochloric acid solution is 3 mCi to 5 mCi. Specifically, the radioactive dose can be any one point value among 3 mCi, 4 mCi, and 5 mCi, or a range value formed by any two point values.

[0032] In some embodiments, in step S3, purification is performed using a GE Life MiniTrip G-25 desalting purification column.

[0033] Composition

[0034] In another aspect, the present invention provides a composition comprising the Nectin-4-targeting aptamer and therapeutic probe as 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.

[0035] In some embodiments, the composition may be a pharmaceutical composition.

[0036] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient.

[0037] In some embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

[0038] In some embodiments, the additional pharmaceutically active agent is an anti-inflammatory drug or an immunosuppressant.

[0039] In some embodiments, in the pharmaceutical composition, the Nectin-4 targeting nucleic acid aptamer nuclear diagnostic and therapeutic integrated 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 nuclear diagnostic and therapeutic integrated probe as described herein and the additional pharmaceutically active agent can be administered simultaneously, separately, or sequentially.

[0040] 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.

[0041] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" of the Nectin-4 targeted nucleic acid aptamer andraconazole diagnostic and therapeutic 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 other concurrently administered treatments.

[0042] Reagent test kit

[0043] The present invention also provides a kit comprising the Nectin-4 targeted nucleic acid aptamer nuclide diagnosis and treatment integrated probe as described herein.

[0044] 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.

[0045] The kit may further 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, and the like.

[0046] Diagnostic and therapeutic applications

[0047] In the Nectin-4 targeted nucleic acid aptamer radionuclide diagnosis and treatment integrated 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, radiotherapy 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.

[0048] 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 with abnormally high nectin-4 expression. The present invention utilizes a malignant bladder cancer tumor-bearing mouse model to validate the molecular radioactive imaging and therapeutic efficacy of a targeted nucleic acid aptamer-based integrated radionuclide diagnostic and therapeutic probe.

[0049] Beneficial effects of the present invention:

[0050] The sequence of the nucleic acid aptamer used in the present invention can be synthesized in a solid phase using a DNA synthesizer; the sequence can be customized; and 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 noninvasive molecular imaging of malignant bladder cancer. 177 The Lu]DOTA-Apt probe can be used for radiotherapy of malignant bladder cancer. The probe preparation method disclosed in this invention can be universally applied to the creation of molecular probes targeting other molecular targets and different radionuclides. It has broad application prospects and very high clinical translational value in the diagnosis and precision treatment of tumor tissues in the future. It has the advantages of simple preparation process, low cost, high specificity, high stability, high imaging signal-to-multiplication ratio, and easy clinical translation. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of coupling the nucleic acid aptamer of Example 1 of the present invention with nuclide chelating ligands (NOTA and DOTA);

[0052] Figure 2 is a high performance liquid chromatogram of the nucleic acid aptamer of Example 1 of the present invention coupled with the NOTA ligand;

[0053] Figure 3 is a high performance liquid chromatogram of the nucleic acid aptamer of Example 1 of the present invention coupled with the DOTA ligand;

[0054] Figure 4 is a high-resolution mass spectrum of the nucleic acid aptamer of Example 1 of the present invention coupled with the NOTA ligand;

[0055] Figure 5 This is a high-resolution mass spectrum of the nucleic acid aptamer of Example 1 of the present invention coupled with the DOTA ligand;

[0056] Figure 6 The embodiment of the present invention 3 68 Ga-labeled Apt probes N4A-6, N4A-7T, and N4A-8T ([ 68 PET / CT images of mice bearing orthotopic bladder tumors two hours after intravesical instillation of Ga]NOTA-Apt.

[0057] Figure 7 The weight change curve of the in situ cell line HT1376 tumor-bearing mice (Nude) in Example 4 of the present invention, the volume ratio of the bladder (tumor-bearing and healthy) to the healthy bladder at the end of bladder perfusion radionuclide radiotherapy, and the ratio of the bladder weight (tumor-bearing and healthy) to the corresponding mouse weight;

[0058] Figure 8The weight change curve of MB49 tumor-bearing mice (C57) in situ cell line according to Example 4 of the present invention, the volume ratio of the bladder (tumor-bearing and healthy) to the healthy bladder at the end of radionuclide radiotherapy, and the ratio of the bladder weight (tumor-bearing and healthy) to the corresponding mouse weight;

[0059] Figure 9 These are digital images of in vitro tumors in tumor-bearing mice of different groups of mice in Example 4 of the present invention 14 days after intravesical radiotherapy. DETAILED DESCRIPTION

[0060] 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.

[0061] 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 receptor.

[0062] In the embodiment of the present invention, a Nectin-4 targeted nucleic acid aptamer nuclide diagnosis and treatment integrated probe is specifically disclosed. 68 Ga]NOTA-Apt and [ 177 Lu] DOTA-Apt, wherein 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 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 in positron emission tomography (PET). 177 Lu (T1 / 2=6.65day) is a metal nuclide used in radiotherapy.

[0063] Table 1

[0064]

[0065] In the nucleic acid aptamer nuclide diagnosis and treatment integrated probe, the Nectin-4 targeting nucleic acid aptamer N4A-6 was obtained by screening the ligand evolution technology of exponential enrichment of Nectin-4 protein, specifically from a large number of (10 12 ~10 15Specific 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 N4A-7T and N4A-8T were simulated using the same screening method described above to obtain conformational information for two other specific nectin-4-targeting nucleic acid sequences. 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.

[0066] The binding affinity (Kd) of the nucleic acid aptamer N4A-6 and the nectin-4 protein was tested, and the measured Kd value was: Kd = 1.51e-07M.

[0067] In a specific embodiment, the preparation process of the Nectin-4 targeted nucleic acid aptamer nuclide diagnostic and therapeutic integrated probe is described in detail.

[0068] In another embodiment, the process of constructing a bladder cancer mouse model with positive Nectin-4 expression is described in detail. The constructed malignant bladder cancer mouse model is used to verify the radioactive imaging and therapeutic efficacy of the Nectin-4-targeted nucleic acid aptamer radionuclide diagnostic and therapeutic integrated probe molecule.

[0069] In another embodiment, Nectin-4 targeted nucleic acid aptamer nuclide diagnosis and treatment integration ([ 68 Molecular imaging of bladder cancer using Ga]NOTA-Apt) PET / CT molecular probe.

[0070] In another embodiment, the specific nuclide diagnosis and treatment integration based on aptamers is described in detail ([ 177 Lu]DOTA-Apt) PET / CT molecular probe for the internal radiotherapy of bladder cancer.

[0071] In the following examples, HT376 cells were obtained from ATCC, MB49 cells were obtained from Millipore, and mice were purchased from Unilever; all reagents used were commercially available or prepared using existing methods.

[0072] In the following examples, the nucleic acid sequences used were synthesized by solid phase synthesis.

[0073] Example 1

[0074] A method for preparing an integrated diagnostic and therapeutic probe based on a nectin-4 targeted nucleic acid aptamer comprises the following steps:

[0075] (1) Preparation of 5-terminal amino-modified aptamers: 6-amino-1-hexanol was condensed with a 5-terminal phosphorylated nucleic acid chain using conventional methods to obtain 5-terminal amino-modified aptamers.

[0076] (2) If Figure 1 As shown in the schematic diagram of coupling a short single-stranded nucleic acid with NOTA or DOTA, NOTA or DOTA with NHS is dissolved in DMSO, and the dissolved NOTA-NHS or DOTA-NHS is added to the 5-terminal amino-modified nucleic acid aptamer (carbonate dissolved), and the reaction is carried out at room temperature for 120 minutes. It is then purified by HPLC and the molecular weight of the product is confirmed by high-resolution mass spectrometry. Figure 2 As shown in the HPLC chromatogram of the aptamers (N4A-6, N4A-7T, and N4A-8T) after NOTA ligand coupling, the main peak at the retention time is the main product NOTA-aptamer. Figure 3 As shown in the HPLC chromatogram of the nucleic acid aptamers (N4A-6, N4A-7T, and N4A-8T in order) after DOTA ligand coupling, the main peak at the retention time is the main product DOTA-nucleic acid aptamer. Figure 4 As shown in the high-resolution mass spectra of the aptamers (N4A-6, N4A-7T, and N4A-8T) after NOTA ligand coupling, the main peak is the main product NOTA-aptamer. Figure 5 The high-resolution mass spectra of the aptamers (N4A-6, N4A-7T, and N4A-8T, in order) after DOTA ligand coupling are shown, and the main peak shown is the main product DOTA-aptamer;

[0077] (3) Take 1 ml 68 Ga or 177 A 0.1 M hydrochloric acid solution of Lu with a radioactive dose of 6 mCi was added to it, and 0.125 ml of a 1 M sodium acetate solution was added to adjust the pH to about 4-4.5, and the reaction solution was formed after standing for 5 minutes;

[0078] (4) The reaction solution obtained in step (3) was mixed with the NOTA-aptamer or DOTA-aptamer solution obtained in step (2), heated to 95 degrees Celsius and shaken for 15 minutes, and then PBS was used as the mobile phase to purify and separate the free 68 Ga or 177Lu ions, and finally radioactive thin layer chromatography (Radio-TLC, Eckert & Ziegler Radiopharma Inc) was used to determine the concentration of each probe ([ 68 Ga]NOTA-Apt or [ 177 The radiochemical purity (RCP) of the probes was greater than 99%.

[0079] Example 2

[0080] Establishment of a nectin-4-positive orthotopic bladder cancer mouse model. The specific steps include:

[0081] (1) Using bladder cancer cell lines HT1376 and MB49, which were confirmed to be Nectin-4 positive by Western blot, they were pre-cultured;

[0082] (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 Matrigel (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;

[0083] (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).

[0084] Example 3

[0085] A Nectin-4 targeted nucleic acid aptamer radionuclide diagnosis and treatment integrated probe ([ 68 Ga]NOTA-Apt) PET / CT molecular imaging of bladder cancer. Specific steps include:

[0086] (1) Each tumor-bearing mouse was instilled with approximately 3.7 MBq [ 68 Ga]NOTA-Apt molecular probe (3–6 per group);

[0087] (2) Two hours after perfusion, the tumor-bearing nude mice were anesthetized with isoflurane mixed with oxygen (concentration of 3%), 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 the image reconstruction was completed using the IRIS system's own software. Figure 6As shown, 68 Ga-labeled aptamer ([ 68 PET / CT integrated diagnosis and treatment images of tumor-bearing mice two hours after bladder instillation of the Ga]NOTA-Apt (Apt is N4A-6, N4A-7T, N4A-8T) molecular probe, showing one mouse in each group, and each group shows a separate PET / CT fusion image (left), PET image (middle) and CT image (right), as can be seen [ 68 The Ga]NOTA-Apt molecular probe can accurately molecularly image in situ bladder cancer tumors, namely HT1376 and MB49 tumors. Figure 6 The Library is the probe of the injection control group ([ 68 Comparison with the Ga]NOTA-Library (Ga]NOTA-Library) revealed that the PET / CT integrated diagnostic and therapeutic image showed very low enrichment of the control probe in the bladder region. The control probe used a random sequence (a library with consistent length but random sequence was prepared by selecting a specific number of 76 bases during DNA synthesis without specifying base types and randomly combining them during synthesis). The control probe was prepared using the method described in Example 1.

[0088] Example 4

[0089] A Nectin-4 targeted nucleic acid aptamer for integrated diagnosis and treatment ([ 177 Radiotherapy using a Lu]DOTA-Apt molecular probe in a tumor-bearing mouse model. This involves the following steps:

[0090] (1) In the treatment group, each tumor-bearing nude mouse was instilled with approximately 3.7 MBq via the bladder. 177 Lu]DOTA-Apt molecular probe (5 in each group), the control group (PBS) was perfused with an equal volume of normal saline, the 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 with about 3.7 MBq of the control probe; the control group 2 (177Lu) was perfused with an equal volume of 177 Lu; the healthy group (Health) was set as healthy Balb / c nude mice or C57 black mice, and the same volume of PBS was perfused; the base sequence of the control sequence (Library) used in the control group 1 was a random sequence (the specific number of bases was selected as 76 during DNA synthesis, and the base type was not specified. The library with the same length but random sequence was obtained by random combination during the synthesis process). The control group probe ([ 177 The preparation method of Lu]DOTA-Library is as described in Example 1;

[0091] (2) The body weight of the mice was recorded every two days after intravesical radiotherapy. The observation was continued and the experiment was terminated when the weight of the mice decreased by more than 20%. In this experiment, all mice were euthanized on the 14th day of radiotherapy. Figure 7 and Figure 8 In the middle, the left graphs are all curves of changes in the weight of mice in each experimental group; the middle graphs are all volume ratios of the bladder volume of orthotopic tumor-bearing mice removed on the 14th day of radiotherapy compared with the bladder volume of healthy mice; the right graphs are all weight ratios of the bladder weight of orthotopic tumor-bearing mice removed on the 14th day of radiotherapy compared with the bladder weight of healthy mice. The experiment found that in the HT1376 and MB49 xenograft tumor models, the volume and weight ratio (B / W) of the normal saline control group was much greater than that of healthy mice, and the volume and weight ratio (B / W) of control group 1 and control group 2 was also much greater than that of healthy mice, and did not play an inhibitory effect on tumor growth; the B / W ratio of the bladder tumor of the treatment group using N4A-7T nucleic acid aptamer and the control group (PBS) did not show significant differences in the HT1376 xenograft tumor model, but the volume ratio of the bladder tumor to the control group (PBS) showed a significant difference in the HT1376 xenograft tumor model. There was a significant difference in the volume ratio and B / W ratio of the bladder tumor between the treatment group and the control group (PBS) in the MB49 orthotopic xenograft tumor model; the volume ratio and B / W ratio of the bladder tumor between the treatment group and the control group (PBS) were significantly different in both tumor models, 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 tumor in each treatment group were reduced, indicating that the nucleic acid aptamer probe prepared by the present invention has an effective therapeutic effect in bladder cancer. Figure 7 、 Figure 8 After the treatment, the mice in each group were euthanized to terminate the experiment. Finally, the tumor volumes of the mice in each treatment group (N4A-6, N4A-7T, N4A-8T) were smaller than those in the control group and control group 1 and control group 2. Figure 9 As shown. Furthermore, the body weights of all mice in this experiment fluctuated within a small range, and no serious side effects were observed. This experiment validated the ability of the described Nectin-4-targeted nucleic acid aptamer radionuclide diagnostic and therapeutic probe to inhibit tumor growth, and no significant physiological toxicity was observed.

[0092] 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 nuclide diagnosis and treatment integrated probe, characterized in that: The integrated diagnosis and treatment probe includes a Nectin-4 targeting nucleic acid aptamer, a nuclide chelating ligand, and a labeling nuclide; 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 nuclide diagnosis and treatment integrated probe according to claim 1, characterized in that: The nuclide 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 targeted nucleic acid aptamer nuclide diagnosis and treatment integrated probe according to claim 1, characterized in that: The labeling nuclide 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, and Sc-44; or Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-6 0, any one of 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, and Yb-177.

4. The Nectin-4 targeted nucleic acid aptamer nuclide diagnosis and treatment integrated probe according to claim 3, characterized in that: The labeling nuclide is Ga-68 or Lu-177.

5. A method for preparing a Nectin-4 targeted nucleic acid aptamer nuclide diagnosis and treatment integrated probe according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. A nuclide chelating ligand with NHS is coupled with a nectin-4 targeting nucleic acid aptamer with an amino group modified at the end to obtain a nuclide chelating ligand-nucleic acid aptamer conjugate; S2, dissolving the labeling nuclide in a hydrochloric acid solution, and then adding a pH adjuster to adjust the pH to obtain a solution containing the nuclide; S3. Mixing the nuclide chelating ligand-nucleic acid aptamer conjugate prepared in step S1 with the nuclide-containing solution prepared in step S2, and purifying the mixture after reaction to obtain the integrated diagnosis and treatment probe.

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

5.

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

8. Use of the Nectin-4 targeted nucleic acid aptamer nuclide diagnostic and therapeutic integrated probe according to any one of claims 1 to 4 in the preparation of a kit for visually detecting the expression of Nectin-4.

9. Use of the Nectin-4 targeted nucleic acid aptamer nuclide diagnostic and therapeutic integrated probe according to any one of claims 1 to 4 in the preparation of a kit for diagnosing or 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 nuclide diagnostic and therapeutic integrated probe according to any one of claims 1 to 4 in the preparation of a pharmaceutical composition for treating Nectin-4 related tumors; The Nectin-4 related tumor is a bladder tumor with abnormally high expression of Nectin-4 protein.

11. Use of the Nectin-4 targeted nucleic acid aptamer radionuclide diagnostic and therapeutic integrated probe according to any one of claims 1 to 4 in the preparation of a pharmaceutical composition for Nectin-4 related tumor targeted radiotherapy; The Nectin-4 related tumor is a bladder tumor with abnormally high expression of Nectin-4 protein.

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