A met dimer targeting radionuclide probe and a preparation method and application thereof

Abstract

The application discloses a MET dimer-targeted radionuclide probe and a preparation method and application thereof, and relates to the technical field of biological medicine. The radionuclide probe is obtained by hybridization of T-dimer and a T3 modified chain; the T3 modified chain is modified with polyethylene glycol at a 3' end and is labeled with a radionuclide; the T-dimer is obtained by connecting two single aptamer chains Apt1 and Apt2 through a disulfide bond, and the connecting sites of the disulfide bond are the 32nd base of Apt1 and the 37th base of Apt2. The radionuclide probe provided by the application can evaluate the abnormal activation state of MET in the body by targeting MET dimers, so that early monitoring of molecular targeted therapy drug resistance is realized, and guidance is provided for clinical medication decision-making of tumor patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a radionuclide probe targeting MET dimer, its preparation method, and its application. Background Technology

[0002] Non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer. In recent years, molecular targeted therapy has become a pillar of systemic treatment for NSCLC; however, drug resistance remains an unavoidable clinical and scientific challenge in the treatment of NSCLC. Abnormal activation of mesenchymal-epithelial transforming factor (MET) is one of the main mechanisms of acquired resistance to targeted therapy in NSCLC patients, and dynamic monitoring of its activation status is crucial for personalized treatment. In NSCLC, the main forms of MET dysfunction include MET exon 14 skipping mutations, MET gene amplification, and protein overexpression. However, existing in vitro diagnostic techniques such as immunohistochemistry, fluorescence in situ hybridization, and next-generation sequencing can only detect specific types of alterations and cannot provide a comprehensive assessment of MET activation status. Furthermore, in vitro detection techniques have limitations such as poor accessibility to NSCLC samples, difficulty in overcoming the spatiotemporal heterogeneity of tumors, and the inability to achieve dynamic tracking throughout the entire disease course. Therefore, there is an urgent need to develop a new strategy to achieve non-invasive, dynamic monitoring of MET activation status, thereby accurately guiding subsequent medication decisions for drug-resistant NSCLC patients.

[0003] Oligomerization is an essential process for the activation of cell membrane surface receptor kinases and the exertion of their biological effects. When a ligand binds to a cell membrane receptor, it induces receptor oligomerization, forming dimers or multimers, which then activate downstream signal transduction molecules through protein-protein interactions. Studies have shown that protein dimerization can regulate protein function in multiple dimensions by affecting ligand binding, receptor membrane localization and internalization, and the selective activation of downstream signals. MET is an important member of the receptor tyrosine kinase family. Structural analysis revealed that the key site of HGF-mediated dimerization (the unmutated HGF / MET binding site) directly inhibits downstream MET phosphorylation, further emphasizing that the dimer is a key active form of the MET protein, suggesting that the MET dimer is a potential biomarker for assessing MET activation status and monitoring targeted therapy resistance. Although some experimental techniques, such as immunoprecipitation, protein fragment complementation assays, mass spectrometry, X-ray crystallography, and cryo-electron microscopy, have been successfully used for protein-protein interaction and dimerization detection, these traditional methods usually require protein separation before analysis, leading to the loss of crucial biological background information. In recent years, several research teams have successfully revealed protein oligomerization in situ using adjacent linkage techniques. These methods utilize the principle of the adjacent effect, where receptor dimerization brings two target ligand molecules closer together, thereby increasing the effective local concentration of the target molecule and improving the efficiency of subsequent enzymatic ligation or hybridization reactions. However, these established strategies are mainly limited to cell or tumor biopsies, such as CN116904556A and CN117741147A, which limits their ability to longitudinally and dynamically monitor the protein activation state.

[0004] Nuclear medicine imaging technology, by introducing radionuclide probes that can bind to specific molecular targets, provides a powerful tool for non-invasive, longitudinal monitoring of biomolecular events in vivo. However, due to the difficulty in effectively distinguishing between protein monomers and dimers, there is currently a lack of effective strategies for directly visualizing protein dimers. Summary of the Invention

[0005] The purpose of this invention is to provide a MET dimer-targeting radionuclide probe, its preparation method, and its application, to address the problems existing in the prior art. This probe can assess the abnormal activation state of MET in vivo by targeting the MET dimer, thereby enabling early monitoring of resistance to molecularly targeted therapy in non-small cell lung cancer and providing guidance for clinical medication decisions in cancer patients.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a radionuclide probe that targets MET dimer, wherein the radionuclide probe is obtained by hybridization of T-dimer and T3 modified strand;

[0008] The 3' end of the T3 modified chain is modified with polyethylene glycol and labeled with a radionuclide;

[0009] The T-dimer is obtained by connecting two separate aptamer chains, Apt1 and Apt2, via disulfide bonds, with the disulfide bond connection sites being the 32nd base of Apt1 and the 37th base of Apt2.

[0010] The nucleotide sequence of the T3 modified strand is shown in SEQ ID NO.1;

[0011] The nucleotide sequence of Apt1 is shown in SEQ ID NO.2;

[0012] The nucleotide sequence of Apt2 is shown in SEQ ID NO.3.

[0013] Furthermore, the polyethylene glycol is PEG. 36 .

[0014] Furthermore, the radionuclide is labeled at the 3' end of the polyethylene glycol using a metal chelating agent.

[0015] Further, the metal chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid, 1,4,7-triazacyclononane-N,N',N''-triacetic acid, or deferoxamine.

[0016] Furthermore, the radioactive nuclide is 68 Ga、 64 Cu or 89 Zr.

[0017] The present invention also provides a method for preparing the above-mentioned radionuclide probe, comprising the step of performing a hybridization reaction between the T-dimer and the T3 modified chain to obtain the radionuclide probe.

[0018] Furthermore, the molar ratio of the T-dimer to the T3 modified chain is 1:(1.1-1.5).

[0019] Furthermore, the hybridization reaction is carried out at a temperature of 37°C for a time of 30-60 minutes.

[0020] The present invention also provides the application of the above-mentioned radionuclide probe in the preparation of tumor molecular targeted therapy drug resistance monitoring products, wherein the tumor has MET overactivation characteristics.

[0021] The present invention also provides a tumor molecular targeted therapy drug resistance monitoring product, comprising the above-mentioned radionuclide probe.

[0022] The present invention discloses the following technical effects:

[0023] This invention develops a radionuclide probe that actively targets MET dimers rather than protein monomers. It is rapidly cleared from normal tissues but accumulates at tumor sites, thus enabling in vivo diagnostics. This probe can assess abnormal MET activation in vivo by targeting MET dimers, thereby facilitating early monitoring of EGFR-targeted therapy resistance and providing guidance for clinical medication decisions in non-small cell lung cancer patients.

[0024] Unlike strategies for detecting protein dimers in vitro, in vivo imaging probes must consider two key issues: probe stability in vivo and tumor imaging contrast. The probe disclosed in this invention is a pre-hybridized assembled probe, mainly composed of two DNA strands: a MET-targeting aptamer double-stranded T-dimer and a radiolabeled T3-modified strand. PEG modification at the T3 strand ends further improves biocompatibility and ensures its stability in vivo. Furthermore, to achieve specific uptake at the tumor site, this invention introduces a GSH-responsive element—a disulfide bond—into the probe's targeting region, thereby achieving specific activation of the probe at the tumor site, reducing uptake by non-target tissues, and further improving the tumor background signal ratio in imaging. Regarding the disulfide bond connection sites, this invention has explored different methods to ensure that the T-dimer and T3 can effectively hybridize when the disulfide bond is present, while simultaneously restricting the recognition of the MET protein by the targeting region; and that after the disulfide bond breaks, the targeting region regains its MET recognition activity. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 A schematic diagram of probe assembly;

[0027] Figure 2 This is a schematic diagram illustrating the working principle of the probe.

[0028] Figure 3 Image of nucleic acid electrophoresis on agarose gel;

[0029] Figure 4 For DOTA-T3P(A) HPLC detection results and 68 Graph of radioactive HPLC detection results of Ga-DOTA-T3P(B);

[0030] Figure 5 for 68Agarose gel electrophoresis image of Ga-DOTA-T3P and T-dimer hybridization; the framed area indicates the probe METLAB.

[0031] Figure 6 for 68 iTLC analysis images of Ga-METLAB probe after co-incubation with 50% FBS for different times;

[0032] Figure 7 for 68 Agarose gel electrophoresis images of Ga-METLAB probes after co-incubation with 50% FBS for different times;

[0033] Figure 8 The graphs show the analysis of MET expression levels in H1975 and H661 cell lines; where A is an immunofluorescence graph and B is a flow cytometry graph; the scale bar is 50 μm.

[0034] Figure 9 The image shows fluorescence microscopy images of H1975 / H661 cells after co-incubation with the fluorescent probe FAM-METLAB and different treatments; the scale bar is 50 μm.

[0035] Figure 10 Radioactive probe 68 A statistical graph showing the proportion of cells that bind to radioactive signals after co-incubation of Ga-METLAB with H1975(A) or H661(B) cells treated with different methods; where %AD represents the percentage of the injected dose.

[0036] Figure 11 For MET aptamer sequences and 68 Figure showing receptor competitive binding analysis of Ga-METLAB on H1975 cells;

[0037] Figure 12 for 68 Figure showing the change in mouse body weight within 2 weeks after administration of Ga-METLAB probe;

[0038] Figure 13 for 68 The results of the detection of hemoglobin (A), red blood cells (B), white blood cells (C), platelets (D), creatinine (E), blood urea nitrogen (F), alanine aminotransferase (G) and aspartate aminotransferase (H) in mice 2 weeks after administration of Ga-METLAB probe are shown in the figure.

[0039] Figure 14 for 68 H&E staining images of major organs in mice 2 weeks after administration of Ga-METLAB probe; scale bar is 100 μm.

[0040] Figure 15 for68 PET / MRI imaging results at different time points after Ga-METLAB probe injection; where signal intensity is expressed as the percentage of injected dose per cubic centimeter of tissue (%ID / cc).

[0041] Figure 16 for 68 Biodistribution map of Ga-METLAB probe at different time points after injection; results are expressed as percentage of injected dose per gram of tissue (%ID / g).

[0042] Figure 17 for 68 Statistical graph of the ratio of tumor uptake to probe uptake in blood at different time points after Ga-METLAB probe injection;

[0043] Figure 18 for 68 Statistical graph of the ratio of probe uptake in tumor and muscle at different time points after Ga-METLAB probe injection;

[0044] Figure 19 PET / MRI images of three tumor models: H1975, A549, and H661;

[0045] Figure 20 Immunohistochemical staining images of H&E, MET, and phosphorylated MET in three types of tumors; the scale bar is 50 μm.

[0046] Figure 21 A schematic diagram illustrating the construction of the MET dimerization blocking model;

[0047] Figure 22 Immunoblot analysis of MET, phosphorylated MET, AKT, and phosphorylated AKT proteins;

[0048] Figure 23 Immunohistochemical staining images of MET and phosphorylated MET; the scale bar is 50 μm.

[0049] Figure 24 PET / MRI images of mice with a MET dimerization blockade model;

[0050] Figure 25 A graph showing the quantitative analysis of tumor uptake values ​​in the MET dimerization blocking model; the results are expressed as the percentage of injected dose per cubic centimeter of tissue (%ID / cc).

[0051] Figure 26 Figure showing the cytotoxicity analysis of different concentrations of HGF mimicry on H1975 cells;

[0052] Figure 27The figure shows the effect of different concentrations of HGF mimics on the tumor-killing effect of osimertinib; where Osi represents osimertinib.

[0053] Figure 28 A schematic diagram illustrating the construction of a drug-resistant tumor model; where HGF-m represents an HGF mimic.

[0054] Figure 29 Tumor growth curves for different treatment groups;

[0055] Figure 30 Immunohistochemical staining images of MET, phosphorylated MET, and phosphorylated AKT in H1975 tumors after treatment with HGF mimics; the scale bar is 50 μm.

[0056] Figure 31 Immunoblot analysis of MET, phosphorylated MET, AKT, and phosphorylated AKT proteins in H1975 tumor after treatment with HGF mimics.

[0057] Figure 32 This is a PET / MRI image of H1975 tumor-bearing mice after treatment with HGF mimicry. Detailed Implementation

[0058] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0059] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0060] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0061] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0062] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0063] This invention develops a radioactive nucleic acid probe targeting MET dimers. The probe's composition and working principle are as follows:

[0064] This probe is mainly assembled from two DNA strands, including a MET-targeting aptamer double-stranded T-dimer and a radiolabeled T3-modified strand. The T-dimer consists of two separate aptamer strands (Apt1 and Apt2) linked by disulfide bonds modified in the target region, each containing three functional regions: a MET-targeting region, a complementary region, and a T-strand trapping region. Polyethylene glycol (PEG) is modified at the 3' end of the T3 strand to prolong the probe's metabolic half-life, followed by modification with a metal chelating agent (such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid, abbreviated as DOTA), and finally radiolabeling (such as...). 68 The labeling of Ga. The T-dimer and T3 modified chain are assembled through complementary base pairing to obtain a "locked" probe ( Figure 1 When the probe reaches the tumor, the high concentration of glutathione (GSH) in the tumor microenvironment causes disulfide bonds to break, restoring the original structure of the target region. The probe then enters an "unlocked" state, allowing it to target and bind to the MET protein. However, when the probe binds only to protein monomers, the base stacking force between the two aptamer chains is insufficient to maintain the T-structure, leading to probe disintegration. Conversely, when the probe binds to dimers, the ortho-position effect replaces the disulfide bond, stabilizing the probe's intact structure and enabling specific imaging of the dimer. Figure 2 ).

[0065] The metal chelating agent is not limited to DOTA, but can also be other bifunctional chelating agents such as 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), deferoxamine (DFO), etc.; the radionuclide is also not limited to 68 Ga can also be... 64 Cu、 89 Zr and other diagnostic radionuclides.

[0066] During the research, it was found that if the disulfide bond is attached to the end of the T-dimer targeting region (base 1 of Apt1 and base 69 of Apt2), the hybridization efficiency is significantly reduced. However, if the disulfide bond attachment site is too close to the complementary region (base 38 of Apt1 and base 31 of Apt2), it will be difficult to limit the MET recognition ability of the T-dimer targeting region, and the probe cannot achieve specific binding at the tumor site. Through optimization, the disulfide bond attachment site was finally determined to be base 32 of Apt1 and base 37 of Apt2.

[0067] Example 1

[0068] This invention provides a nuclide probe targeting MET dimers, the assembly schematic of which is shown in the figure. Figure 1 The sequences involved are shown in Table 1. All nucleic acid chains were synthesized using conventional methods, such as solid-phase synthesis.

[0069] DOTA-T3P is made by sequentially modifying PEG at the 3' end of the T3 chain. 36 T-dimer was obtained by combining it with the metal chelating agent DOTA. It consists of two separate aptamer chains, Apt1 and Apt2, connected by disulfide bonds at the 32nd base of Apt1 and the 37th base of Apt2.

[0070] Table 1 Nucleic Acid Sequences

[0071]

[0072] Note: The underlined areas indicate disulfide bond connection sites.

[0073] Probe hybridization and GSH responsiveness verification: 500 nM T3 strand and T-dimer were mixed at a 1:1 molar ratio and added to annealing buffer (1M TE buffer at pH 8.0 with 20 mM MgCl2 added). The mixture was incubated at 37°C for 30 minutes to complete the in vitro assembly of the probe, yielding the METLAB probe. Subsequently, 5.0 mM GSH was added to the system and the mixture was incubated at 500 rpm at 37°C for 2 hours. Afterward, all samples were analyzed by agarose gel electrophoresis. The results are shown below. Figure 3 The connection of disulfide bonds effectively promoted the hybridization of the T3 chain and T-dimer, while the treatment with GSH induced the breaking of disulfide bonds, triggering the disassembly and assembly of the probe, proving that the designed probe met the expected characteristics.

[0074] Probe radionuclide labeling and assembly: 10 μL of DOTA-T3P (2 nmol) was added to 200 μL of 2.5M HEPES buffer (pH 7.5), followed by 300 μL of... 68GaCl3 solution (6 mCi) was reacted at 80 °C for 15 min, and then purified by NAP-5 column chromatography to obtain... 68 Ga-DOTA-T3P purity was verified by radioactive high-performance liquid chromatography (HPLC). It was then processed at a molar ratio of 1:1.2. 68 T-dimer was added to Ga-DOTA-T3P, followed by annealing buffer, and the mixture was incubated at 37°C for 30 minutes to obtain the probe. 68 Ga-METLAB was used, and the successful assembly of the probe was verified by agarose gel electrophoresis. Results are as follows: Figures 4-5 As shown, after purification 68 The radiochemical purity of Ga-DOTA-T3P is ≥99%, and the hybridization efficiency is ≥90%.

[0075] Since the stability of nucleic acid probes is a key concern, in vitro stability verification was performed in this embodiment. The probe was co-incubated with 50% fetal bovine serum (FBS) at room temperature for 30, 60, 120, 180, and 240 minutes, respectively, followed by radioactive thin-layer chromatography (iTLC) and agarose gel electrophoresis to verify the integrity of the probe. The results are as follows: Figures 6-7 As shown, the nuclide probe underwent slight degradation after co-incubation with serum, but a clear target band was still visible after 4 hours.

[0076] Example 2

[0077] A radionuclide probe targeting MET dimers is prepared as follows:

[0078] Add 10 μL of DOTA-T3P (2 nmol) to 200 μL of 2.5M HEPES buffer (pH 7.5), followed by 300 μL of... 68 GaCl3 solution (approximately 6 mCi) was reacted at 80°C for 15 min, and then purified by NAP-5 column chromatography to obtain... 68 Ga-DOTA-T3P purity was verified by radioactive high-performance liquid chromatography (HPLC). It was then processed at a molar ratio of 1:1.1. 68 T-dimer was added to Ga-DOTA-T3P, followed by annealing buffer, and the mixture was incubated at 37°C for 40 minutes to obtain the probe. 68 Ga-METLAB. DOTA-T3P and T-dimer are the same as in Example 1.

[0079] Example 3

[0080] A radionuclide probe targeting MET dimers is prepared as follows:

[0081] Add 10 μL of DOTA-T3P (2 nmol) to 200 μL of 2.5M HEPES buffer (pH 7.5), followed by 300 μL of... 68 GaCl3 solution (approximately 6 mCi) was reacted at 80°C for 15 min, and then purified by NAP-5 column chromatography to obtain... 68 Ga-DOTA-T3P purity was verified by radioactive high-performance liquid chromatography (HPLC). It was then processed at a molar ratio of 1:1.5. 68 T-dimer was added to Ga-DOTA-T3P, followed by annealing buffer, and the mixture was incubated at 37°C for 60 minutes to obtain the probe. 68 Ga-METLAB. DOTA-T3P and T-dimer are the same as in Example 1.

[0082] Example 4

[0083] A radionuclide probe targeting MET dimers is prepared as follows:

[0084] Add 10 μL of NOTA-T3P (2 nmol) to 200 μL of 2.5M HEPES buffer (pH 7.5), followed by 300 μL of... 64 CuCl2 solution (approximately 6 mCi) was reacted at 80°C for 15 min, and then purified by NAP-5 column chromatography to obtain... 64 Cu-NOTA-T3P purity was verified by radioactive high-performance liquid chromatography (HPLC). It was then processed at a molar ratio of 1:1.2. 64 T-dimer was added to Cu-NOTA-T3P, followed by annealing buffer, and the mixture was incubated at 37°C for 30 minutes to obtain the probe. 64 Cu-METLAB. The T-dimer is the same as in Example 1; NOTA-T3P is formed by sequentially modifying the 3' end of the T3 chain with PEG. 36 It was obtained with the metal chelating agent NOA.

[0085] Example 5

[0086] A radionuclide probe targeting MET dimers is prepared as follows:

[0087] 10 μL of DFO-T3P (2 nmol) was added to 1000 μL of 0.25M HEPES buffer (pH 7.2), followed by 100 μL of zirconium oxalate (…). 89 The Zr solution was reacted at 80°C for 15 min, and then purified by NAP-5 column chromatography to obtain... 89 The purity of Zr-DFO-T3P was verified by radioactive high-performance liquid chromatography (HPLC). It was then processed at a molar ratio of 1:1.2. 89T-dimer was added to Zr-DFO-T3P, followed by annealing buffer, and the mixture was incubated at 37°C for 30 minutes to obtain the probe. 89 Zr-METLAB. The T-dimer is the same as in Example 1; DFO-T3P is formed by sequentially modifying the 3' end of the T3 chain with PEG. 36 It was obtained with the metal chelating agent DFO.

[0088] Example 6

[0089] First, to visualize MET protein dimerization at the cellular level, a fluorescent probe, FAM-METLAB, was constructed. The assembly method was the same as for the radioactive probe, specifically, the signal group from Example 1 was changed from... 68 Ga is replaced with the FAM fluorescent group.

[0090] Based on the differences in MET expression levels, non-small cell lung cancer cell lines H1975 and H661 were selected as positive and negative models, respectively. These cells were seeded in 24-well plates containing cell slides, and after adhesion, the serum-free medium was replaced and incubated overnight. 100 ng / mL HGF recombinant protein was added to the culture medium of the experimental group cells, and they were cultured for 30 minutes to induce dimer formation. The culture medium was discarded, the cells were washed, fixed, blocked, and then 400 nM of the probe was added. Simultaneously, 5.0 mM MGSSH was added to the staining buffer of the experimental group cells. After incubation at 37℃ for 2 hours, DAPI staining was performed, and images were observed and acquired using a fluorescence microscope. The results are shown below. Figures 8-9 As shown.

[0091] Theoretically, binding to the ligand HGF should induce MET protein dimerization in H1975 cells. However, when FAM-METLAB was co-incubated with HGF-stimulated cells, very little probe binding was detected on the cell surface because the presence of disulfide bonds restricted conformational changes in the targeting aptamer sequence, inhibiting MET recognition in the target region. Upon addition of GSH, the disulfide bonds broke, the target region regained its original conformation, and the probe could specifically recognize and bind to MET. Importantly, only when binding to dimerized MET did the ortho-position effect of the complementary region stabilize the probe's intact structure, at which point a significant fluorescent signal could be observed on the cell surface. Conversely, on the surface of unstimulated H1975 cells, the probe hardly bound due to the lack of dimerization-induced ortho-position effect. Under the same conditions, almost no fluorescence was detected in MET-negative H661 cells.

[0092] Similarly, this embodiment validated the radionuclide probe at the cellular level. 68The targeting specificity of Ga-METLAB was first demonstrated through cell binding assays. H1975 or H661 cells were seeded in 24-well plates. After adhesion, the medium was replaced with serum-free medium, and the cells were incubated overnight. Then, 100 ng / mL of recombinant HGF protein was added, and the cells were cultured for 30 minutes to induce dimer formation. After washing, fixation, and blocking, the probe was added. 68 Ga-METLAB, 1 μCi per well; 5.0 mM GSH added to the staining buffer of the experimental group; excess MET targeting aptamer added to the blocking group. Incubate at 37°C for 2 hours, wash cells with 0.1 M NaOH, transfer to radioimmunoassay tubes, and measure cell binding radioactivity per well using a γ-counter. Results are as follows: Figure 10 As shown, in the presence of GSH, this radiotracer can specifically recognize MET dimers on the surface of HGF-stimulated H1975 cells, and this binding can be inhibited by an excess of MET-targeting aptamers, while no specifically bound radioactive signal was detected in H661 cells.

[0093] In addition, to further verify the targeting specificity of the radionuclide probe, a competitive binding experiment was conducted in this embodiment. Cell pretreatment was the same as in the binding experiment; after sealing, 1 μCi of the radionuclide probe was added to each well. 68 Ga-METLAB and 5.0 mM GSH were added, along with different concentrations of MET-targeting aptamers: 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 Mole. Incubate at 37°C for 2 hours, wash cells with 0.1 M NaOH, transfer to radioimmunoassay tubes, and measure cell binding radioactivity per well using a γ-counter. Results are as follows. Figure 11 As shown, the MET aptamer has a strong inhibitory effect on the binding of radioactive tracers, and its IC50 value is [missing information]. 50 The value is 13.8 nM.

[0094] In summary, these results indicate that the probe 68 Ga-METLAB selectively targets MET dimers on the cell surface via receptor-mediated binding.

[0095] Example 7

[0096] This embodiment primarily evaluates the probe. 68 In vivo metabolic characteristics of Ga-METLAB.

[0097] First, safety was assessed. Healthy female Kunming mice aged 5-6 weeks were selected and injected via the tail vein with an imaging dose 200 times greater than the recommended dose. 68The mice were euthanized after 15 days using Ga-METLAB. A 100 μL blood sample was collected, anticoagulated with dipotassium ethylenediaminetetraacetate (EDTA-K2), and then subjected to complete blood cell count analysis. The remaining sample was allowed to stand for 1 hour, then centrifuged to prepare serum, which was then analyzed using an automated biochemical analyzer. Simultaneously, major organs were collected from the mice, and tissue sections were prepared for H&E staining. Results are as follows: Figures 12-14 As shown, excessive 68 Ga-METLAB did not cause significant changes in body weight, blood routine indicators, blood biochemical indicators, or major organ histology in mice, indicating that... 68 Safety of Ga-METLAB in vivo studies.

[0098] Then proceed 68 In vivo imaging validation of Ga-METLAB. First, an H1975 tumor-bearing mouse model was constructed. Five- to six-week-old BALB / c nude mice were selected, and 10 mg of Ga-METLAB was injected into the right axilla. 6 100 μL of H1975 cells were seeded. The tumor volume was increased to 200-300 mm². 3 Tail vein injection 68 Ga-METLAB, at a dose of 7.4 MBq / animal, was used for PET / MRI imaging at 0.5, 1, and 2 hours after probe injection. Results are as follows: Figure 15 As shown, 68 The tumor uptake value of Ga-METLAB peaked 0.5 hours after injection and was then rapidly cleared. After 2 hours, almost no probe signal remained at the tumor site.

[0099] Biodistribution experiments were conducted simultaneously. The probe was injected via the tail vein into an H1975 tumor-bearing mouse model at a dose of 3.7 MBq / mouse. Mice were sacrificed at 0.5, 1, and 2 hours post-injection, and blood, tumors, major organs, and tissues were collected and weighed. Radioactivity in the tissues was measured using a γ-counter. Results are expressed as percentage of injected dose per gram of tissue (%ID / g). Results are as follows... Figures 16-18 As shown, the probe is primarily excreted via the kidneys and bladder, with the highest tumor uptake value observed 0.5 hours post-injection (%ID / g: 2.458±0.203). Furthermore, the ratios of tumor to blood and tumor to muscle showed little difference between 0.5 and 1 hour post-injection, but a slight decrease was observed at 2 hours.

[0100] In summary, the nuclide probe provided by this invention... 68 Ga-METLAB exhibits excellent in vivo imaging performance.

[0101] Example 8

[0102] In this embodiment, three non-small cell lung cancer mouse models, H1975, A549, and H661, were first constructed, with tumor volumes reaching 200-300 mm. 3 At that time, injection via tail vein 68 Ga-METLAB, at a dose of 7.4 MBq / animal, was used for PET / MRI imaging 0.5 hours after probe injection. Results are as follows: Figure 19 As shown, 0.5 hours after probe injection, the H1975 tumor was clearly visible, while the radioactive signals of the A549 and H661 tumors were weaker. Mice were subsequently sacrificed, and tumors were collected for paraffin sectioning and H&E and immunohistochemical staining. The results are as follows. Figure 20 As shown, the MET levels in the three tumor types H1975, A549, and H661 were high, intermediate, and low, respectively, while phosphorylated MET was only observed as a positive signal in H1975, with almost no positive phosphorylated MET in A549 and H661. This indicates that... 68 Ga-METLAB PET imaging can specifically assess MET activation levels, rather than total MET protein.

[0103] To further verify the above observations, this embodiment constructs a MET dimerization blocking model. For example... Figure 21 As shown, in the H1975 tumor-bearing mouse model, anti-HGF antibody was injected intraperitoneally to block the binding of HGF to MET, at a dose of 1 mg / kg, administered every other day for three times. On the second day after the end of administration, a probe was injected via the tail vein for PET / MRI imaging. After imaging, tumors were collected; a portion was prepared into paraffin sections for immunohistochemical staining with MET and phosphorylated MET, and another portion was used for protein immunoblotting. The results are as follows: Figures 22-25 As shown, the combined results of immunohistochemical and Western blot analyses indicate that in vivo application of anti-HGF antibodies effectively inhibited MET autophosphorylation and activation of the HGF / MET downstream signaling pathway PI3K / AKT. Imaging results showed that the probe signal at the tumor site was weakened after blocking MET dimerization with anti-HGF antibodies. Quantitative results further indicated that in the dimerization blocking model... 68 Ga-METLAB uptake in tumors was reduced by approximately 50% compared to the control group (%ID / cc: 1.223±0.117 vs 0.663±0.021).

[0104] In summary, based on the nuclide probes disclosed in this invention... 68 Ga-METLAB PET imaging enables in vivo visualization of MET dimers, thus allowing for non-invasive assessment of MET activation status.

[0105] Example 9

[0106] This embodiment first constructed an H1975 tumor model resistant to osimertinib (a third-generation EGFR kinase inhibitor). The HGF mimic ANG3777 has been shown to stimulate MET activation in vitro and in vivo. This embodiment first analyzed the effect of HGF mimic pretreatment on the anticancer effect of osimertinib at the cellular level. H1975 cells were seeded in 96-well plates and treated with different concentrations of HGF mimics for 48 hours: 0 μM, 0.1 μM, 1.0 μM, and 10 μM. After 24 hours, 0.1 μM osimertinib was added for another 24 hours, and cell viability was analyzed according to the CCK-8 assay kit instructions. The results are as follows: Figures 26-27 As shown, HGF mimics have no cytotoxic effects, but pretreatment of H1975 tumor cells with them significantly weakens the cell-killing effect of osimertinib.

[0107] Subsequently, as Figure 28 As shown, an osimertinib-resistant H1975 tumor model was constructed. The H1975 tumor volume was increased to 150-200 mm². 3 At that time, an HGF mimic was injected intratumorally at a dose of 2 mg / kg, administered daily. Three days later, osimertinib treatment was started at a dose of 2 mg / kg, administered daily by gavage, with continuous monitoring of tumor volume changes. Results were as follows... Figure 29 As shown, osimertinib significantly inhibited the growth of H1975 tumors, and HGF mimicry pretreatment partially reversed the antitumor effect of osimertinib. These results further indicate that MET can induce EGFR-targeted therapy resistance through a bypass activation mechanism. In this embodiment, the HGF mimicry pretreatment was performed on day 3 after the initial treatment. 68 Ga-METLAB PET imaging was performed, followed by tumor collection for subsequent ex vivo analysis. Results are as follows: Figures 30-32 As shown, immunohistochemical and protein immunoblotting analyses together indicate that HGF mimicry pretreatment did not alter the total MET protein expression level, but significantly enhanced MET autophosphorylation and AKT phosphorylation. In vivo imaging results show that the application of HGF mimicry significantly enhanced the uptake of the radiotracer in tumors. These experimental results collectively indicate that... 68 Ga-METLAB PET imaging technology can detect EGFR-targeted therapy resistance early by assessing abnormal activation of the MET pathway.

[0108] In summary, this invention provides a radionuclide probe that actively targets MET dimers rather than protein monomers. It is rapidly cleared in normal tissues but accumulates at tumor sites, thus enabling in vivo diagnostics. This probe can assess abnormal MET activation in vivo by targeting MET dimers, thereby achieving early monitoring of EGFR-targeted therapy resistance and providing guidance for clinical medication decisions in non-small cell lung cancer patients.

[0109] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A radionuclide probe targeting MET dimers, characterized in that, The radionuclide probe was obtained by hybridization of T-dimer and T3 modified strands; The 3' end of the T3 modified chain is modified with polyethylene glycol and labeled with a radionuclide; The T-dimer is obtained by connecting two separate aptamer chains, Apt1 and Apt2, via disulfide bonds, with the disulfide bond connection sites being the 32nd base of Apt1 and the 37th base of Apt2. The nucleotide sequence of the T3 modified strand is shown in SEQ ID NO.1; The nucleotide sequence of Apt1 is shown in SEQ ID NO.2; The nucleotide sequence of Apt2 is shown in SEQ ID NO.3; The radionuclide is labeled at the 3' end of the polyethylene glycol using a metal chelating agent.

2. The radionuclide probe according to claim 1, characterized in that, The polyethylene glycol is PEG. 36 .

3. The radionuclide probe according to claim 1, characterized in that, The metal chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid, 1,4,7-triazacyclononane-N,N',N''-triacetic acid, or deferoxamine.

4. The radionuclide probe according to claim 1, characterized in that, The radioactive nuclide is 68 Ga、 64 Cu or 89 Zr.

5. A method for preparing a radionuclide probe as described in any one of claims 1-4, characterized in that, The method includes the step of hybridizing the T-dimer and the T3 modified chain to obtain the radionuclide probe.

6. The preparation method according to claim 5, characterized in that, The molar ratio of the T-dimer to the T3 modified chain is 1:(1.1-1.5).

7. The preparation method according to claim 5, characterized in that, The hybridization reaction was carried out at a temperature of 37°C for 30-60 minutes.

8. The application of a radionuclide probe as described in any one of claims 1-4 in the preparation of a monitoring product for drug resistance to EGFR-targeted therapy in non-small cell lung cancer, characterized in that, The non-small cell lung cancer exhibits MET dimerization characteristics.

9. A product for monitoring resistance to EGFR-targeted therapy in non-small cell lung cancer, characterized in that, Includes the radionuclide probes according to any one of claims 1-4.

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