Nuclide ligand targeting CEACAM6, PET probe and preparation method and application thereof
By designing nuclide ligands and PET probes targeting CEACAM6, the problem of difficult to distinguish between benign and malignant lung nodules in the prior art is solved, and accurate diagnosis and targeted imaging of early LUAD are achieved.
Patent Information
- Application Number
- CN202411809991.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-10
- Publication Date
- 2025-03-18
AI Technical Summary
The prior art is difficult to accurately distinguish between benign and malignant pulmonary nodules, especially in the diagnosis of early lung adenocarcinoma, and a real-time, non-invasive and effective method is lacking.
A nuclide ligand targeting CEACAM6 was designed, and the targeting molecules were modified by a bifunctional chelating agent, and combined with radionuclide labeling was prepared to target CEACAM6.
The PET probe is able to efficiently target CEACAM6, significantly distinguishing between CEACAM6-positive and negative tumors, providing accurate diagnosis of early LUAD, avoiding misdiagnosis and overtreatment.
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Figure CN119633143A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a probe for medical imaging, in particular to a nuclide ligand targeting CEACAM6, a PET probe and a preparation method and application thereof. The probe is suitable for distinguishing benign and malignant lung nodules, and is particularly suitable for diagnosing early lung adenocarcinoma (LUAD). Background Art
[0002] Although low-dose CT (LDCT) screening can effectively reduce the mortality of lung adenocarcinoma, how to evaluate the malignant potential of uncertain lung nodules remains a difficult problem. Existing studies have shown that in the evaluation of benign and malignant single pulmonary nodules (SPNs), 18 F-FDG PET has a clear advantage in SPNs larger than 8 mm. However, pulmonary infectious diseases can 18 The diagnostic accuracy of F-FDG PET has been recently studied. 68 Ga-FAPI is considered superior to 18 Although F-FDG can be detected by the lung, its accumulation in non-neoplastic lung diseases may also affect the diagnosis. Therefore, there is currently no real-time, non-invasive, and effective method to accurately distinguish benign from malignant lung nodules. Summary of the invention
[0003] The technical problem to be solved by the present invention is: to solve the above-mentioned deficiencies in the prior art, thereby providing a radionuclide ligand targeting CEACAM6, a PET probe, and a preparation method and application thereof.
[0004] The technical solution adopted by the present invention to solve its technical problem is:
[0005] The first aspect of the present invention provides a radionuclide ligand targeting CEACAM6, which is obtained by modifying a targeting molecule with a bifunctional chelator, wherein the targeting molecule is a radionuclide aptamer, an antibody or a polypeptide capable of targeting CEACAM6.
[0006] Preferably, the targeting molecule is Glu-Trp-Gln-Arg-Leu-Lys-Trp-Phe-Asp-His or Gly-Arg-Gln-Tyr-Asp-Tyr-Trp-Gly-Gln-Gly.
[0007] Preferably, the bifunctional chelator and the polypeptide further contain a linker, wherein the linker is Wherein: n is an integer from 1 to 8.
[0008] Preferably, the bifunctional chelating agent is a macrocyclic ligand or an acyclic ligand, and the macrocyclic ligand is preferably 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane-1,4-diacetic acid (NODA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonic acid) (NOTP) or a derivative thereof.
[0009] Preferably, the configuration of each amino acid in the polypeptide targeting CEACAM6 is independently selected from D-form or L-form.
[0010] Preferably, each amino acid in the polypeptide targeting CEACAM6 can be independently selected from substituted amino acids, and the substitution includes chloro, fluoro, bromo, or methyl, nitro, or methoxy substitution.
[0011] A second aspect of the present invention provides a PET probe targeting CEACAM6, which is obtained by labeling a radionuclide ligand with a radionuclide.
[0012] Preferably, the radionuclide is preferably 99 mTc, 68 Ga, 64 Cu, 67 Ga, 90 Y. 111 In, 177 Lu, 125 One of I.
[0013] The third aspect of the present invention provides a method for preparing a PET probe targeting CEACAM6, comprising the following steps: adding a radioactive nuclide to a reaction bottle containing a nuclide ligand targeting CEACAM6, adjusting the pH of the reaction solution to acidity, then heating the mixture to react, purifying the complex, eluting it in an organic solvent with a strong acid solution, and finally recombining the product in physiological saline.
[0014] A fourth aspect of the present invention provides a use of a PET probe targeting CEACAM6 in preparing a reagent for detecting CEACAM6 expression levels and diagnosing CEACAM6-related diseases, wherein the CEACAM6-related disease is lung nodules or lung adenocarcinoma.
[0015] The beneficial effects of the present invention are:
[0016] The present invention confirms that CEACAM6 can be used as a biomarker to distinguish between lung nodules and LUAD, and is highly expressed in in situ malignant lung nodules and LUAD patients. The expression level of CEACAM6 protein is significantly correlated with the progression of malignant lung nodules. These results indicate that CEACAM6 can be used as a suitable target for the non-invasive diagnosis of malignant lung nodules based on PET in the early stage of LUAD. In addition, the present invention utilizes this property of CEACAM6 to design a series of targeting ligands, and obtains good PET tracers through radioactive labeling. In vivo PET imaging experiments successfully identified a PET tracer that can non-invasively and specifically target CEACAM6. 68 Ga-NODA-P2, 68 Ga-NODA-P3 can be used as a potential PET tracer for accurate diagnosis of early malignant lung nodules in LUAD, avoiding misdiagnosis and overtreatment caused by the inability of LDCT screening to accurately assess the malignant risk of nodules, especially 68 The Ga-NODA-P3 probe showed highly specific imaging capabilities in the CEACAM6-positive A549 xenograft tumor model, and was able to significantly distinguish between CEACAM6-positive and -negative tumors. It also had a relatively high imaging target and rapid metabolic clearance, which was conducive to rapid imaging diagnosis. It would help in the accurate identification of malignant lung nodules in the early stages of LUAD and benefit the treatment and prognosis of patients. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The technical solution of the present application is further described below in conjunction with the accompanying drawings and embodiments.
[0018] Figure 1 CEACAM6 is significantly overexpressed in LUAD and significantly correlated with overall survival. (A, B) Analysis of CEACAM6 mRNA expression in LUAD and correlation with different tumor stages based on the GEPIA database Protein expression of CEACAM6 in LUAD patients (n=103) compared with normal controls. (C) Survival analysis of LUAD patients with low tumor mutation burden and different CEACAM6 mRNA expression levels by Kaplan-Meier Plotter's pan-cancer RNA-seq dataset. (D) Flow cytometric analysis of CEACAM6 protein expression on the cell surface of LUAD tumor cells (A549) and other tumor cells (Huh7). All data are expressed as mean±SEM, *p<0.05, **p<0.01.
[0019] Figure 2The expression of CEACAM6 was significantly correlated with the progression of malignant pulmonary nodules. (A) Experimental process of urethane-induced LUAD mouse model. (B) Small animal CT analysis of the development and progression of malignant pulmonary nodules at different times after urethane treatment. Nodules are clearly marked with circles. (C) Pathological progression of urethane-induced malignant pulmonary nodules and expression of CEACAM6 protein were observed by H&E staining and immunohistochemical analysis. In addition, the total positive area of CEACAM6 was quantified in the immunohistochemical images. The calibration bar size is 100 μm. All data are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.
[0020] Figure 3 68 Ga-NODA-P2 and 68 Ga-NODA-P3 showed an appropriate target-to-species uptake ratio in PET imaging. (A) CEACAM6-positive xenograft tumors in mice 68 Comprehensive PET analysis and representative images of Ga-NODA-Peptides (P1-P6). (B) Injection 68 ROI analysis of target uptake ratio of CEACAM6-positive xenograft tumor mice after Ga-NODA-peptides (P1-P6). All data are expressed as mean±SEM, n=4 per group.
[0021] Figure 4 68 Ga-NODA-P3 showed good PET imaging performance. Molecular docking analysis of hydrogen bond counts between P2 and CEACAM6 (A), molecular docking analysis of hydrogen bond counts between P3 and CEACAM6 (C). Uptake of Ga-NODA-P3 in A549 and Huh7 tumor-bearing mice 68 Representative PET imaging and ROI analysis of Ga-NODA-P2 (B) and uptake in A549 and Huh7 tumor-bearing mice 68 Representative PET imaging and ROI analysis of Ga-NODA-P3 (D). All data are presented as mean ± SEM. **p < 0.01, ns means no statistical significance, n = 4 for each group.
[0022] Figure 5 68 Ga-NODA-P3 was specifically taken up in CEACAM6-positive tumor-bearing mice. (A) 68 Representative PET images of mice bearing A549 and Huh7 xenografts after Ga-NODA-P3. (B) Uptake of 3D-positive cells in mice bearing A549 and Huh7 xenografts, respectively. 68 ROI analysis of Ga-NODA-P3. (C) 68Pharmacokinetic analysis of Ga-NODA-P3 in A549 tumor-bearing mice. (D) 0.5 h after injection, 68 Biodistribution of Ga-NODA-P3 in A549 and Huh7 tumor-bearing mice. (E) Immunohistochemical analysis of CEACAM6 in A549 and Huh7 tumor-bearing mice. The calibration bar size is 50 μm. All data are expressed as mean ± SEM, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 4 per group. DETAILED DESCRIPTION
[0023] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application may be combined with each other.
[0024] The technical solution of the present application will be described in detail below with reference to the accompanying drawings and in combination with embodiments.
[0025] The sources of raw materials and related instruments are as follows:
[0026] p-SCN-benzyl-NODA-GA was purchased from Chematech, France. 68 Ga by 68 Ge / 68 The Ga generator (ITG, Germany) used 0.05 M hydrochloric acid solution to generate the radioactivity. The radioactivity was measured using a γ-counter (Packard Multi-Prias, PerkinElmer, USA), and PET imaging was performed using an Inveon dedicated PET scanner (Siemens, Germany). Peptide coupling purification was performed using a Waters high performance liquid chromatography (HPLC) system equipped with a Waters 2998 photodiode array detector (PDA) and a C18 preparative HPLC column (5 μm, 250×10 mm, Phenomenex): the initial mobile phase was 95% solvent A (0.1% trifluoroacetic acid in water) and 5% solvent B (0.1% trifluoroacetic acid in acetonitrile), which transitioned to 35% solvent A and 65% solvent B within 35 minutes at a flow rate of 5 mL / min; purity analysis was performed using reversed-phase high performance liquid chromatography (RP-HPLC) on a Waters Breeze system, which used a Waters 2487 dual-wavelength absorbance detector and a PerkinElmer Radiomatic 610TR flow scintillation analyzer using a Luna C18 chromatographic column (5 μm, 250×4.6 mm, Phenomenex), flow rate of 1 mL / min, solvent A was 0.1% trifluoroacetic acid in water, solvent B was 0.1% trifluoroacetic acid in acetonitrile, at 0 min, the gradient started from 95% solvent A and decreased to 35% solvent A after 15 min.
[0027] Example 1 Bioinformatics Analysis of CEACAM6 in LUAD Patients
[0028] In this example, the GEPIA (Gene Expression Profiling Interactive Analysis) database was used to study the CEACAM6 mRNA level in LUAD patients, and a total of 483 LUAD patients and 347 controls were included. The mRNA level was converted to log2 (TPM+1) for differential analysis and staging analysis. Log2FC is defined as median (tumor) vs median (normal). In order to perform tumor staging analysis and increase the number of cases, the data of all patients with non-small cell lung cancer (NSCLC), including lung adenocarcinoma (LUAD), were analyzed. The pathological stage was mainly divided into 4 categories (Ⅰ, Ⅱ, Ⅲ, Ⅳ) to analyze the expression differences of CEACAM6 in different disease stages. In order to explore the changes in protein expression in LUAD patients, the proteomics database of 103 LUAD patients in previous studies was analyzed, and the abundance of CEACAM6 between LUAD and normal controls was compared. In addition, the Kaplan-Meier database analyzed the correlation between CEACAM6 expression and overall survival of LUAD patients. In short, LUAD patients were divided into CEACAM6 high expression group and low expression group according to the upper quartile. Subsequently, patients with low tumor mutation burden (who had a poorer prognosis in terms of immune response, further highlighting the significance of this gene) were selected for survival analysis of overall survival. Finally, the median survival time between the different CEACAM6 expression groups was determined.
[0029] To verify the specificity of CEACAM6 in LUAD, we analyzed the RNA-Seq dataset of GEPIA and the proteomics dataset of previous LUAD patient studies. Differential analysis showed that both RNA and protein levels of CEACAM6 were significantly increased in LUAD patients and significantly correlated with the tumor stage of non-small cell lung cancer ( Figure 1 A, B). Kaplan-Meier Plotter of pan-cancer RNA-seq dataset showed that CEACAM6 overexpression was significantly associated with poor prognosis in LUAD patients with low tumor mutation burden. The median survival of patients with high or low CEACAM6 expression was 41.17 months and 50.93 months, respectively ( Figure 1 C).
[0030] Example 2 Cell culture and flow cytometry
[0031] Huh7 human hepatoma cells and A549 human LUAD cells were obtained from the American Type Culture Collection (ATCC) through the Shanghai Stem Cell Bank. The two cell lines were cultured in an incubator at 37°C and 5% CO2 under standard conditions using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Flow cytometry was used to detect the expression of CEACAM6 on the surface of A549 and Huh7 cells. Rabbit monoclonal anti-human CEACAM6 antibody (1:1000, Abcam) and coralite488-conjugated donkey anti-rabbit IgG (H+L) (1:250, Proteintech) were incubated in cold phosphate-buffered saline (PBS) at a concentration of 5×10 5 cells / mL. BD FACSCelesta TM Flow cytometric analysis was performed using a flow cytometer (BD Biosciences), and mean fluorescence intensity was evaluated using FlowJo software.
[0032] Flow cytometry results showed that CEACAM6 expression was significantly increased in LUAD cell line (A549) compared with liver cancer cell line (Huh7), which also proved that CEACAM6 expression is specific in LUAD tumors ( Figure 1 D).
[0033] Example 3 Animal Model and Monitoring of CEACAM6 Expression in LUAD Progression
[0034] Objective: To establish a subcutaneous xenograft model. Female nude mice (Cavens Animal Co. Ltd.) aged about 6 weeks were subcutaneously implanted with 5×10 6 When the tumor volume reaches 100-300mm 3 When CEACAM6 was expressed, a tumor model was established for diagnosis. To monitor the expression of CEACAM6 during the progression of LUAD, a urethane-induced LUAD orthotopic animal model was established. Briefly, 8-week-old A / J mice were injected intraperitoneally with urethane (100 mg / 100 g, Sigma Aldrich) or the same volume of the corresponding drug carrier once. The changes in body weight and overall health of all mice were then monitored every other day. All animal experiments were performed in accordance with the Guide for Experimental Animals and approved by the Ethics Committee of Jiangsu Institute of Nuclear Medicine in compliance with national laws and regulations (JSINM-2024-110 and JSINM-2023-079).
[0035] The protocol for monitoring CEACAM6 expression during LUAD progression included lung nodule detection based on small animal CT images, pathological evaluation to determine whether the nodule was malignant and LUAD, and immunohistochemical detection of CEACAM6 expression within the nodule. Shortly after, at 8 and 16 weeks after injection, urethane-induced mice (n=4) were anesthetized with isoflurane for small animal CT imaging. Each mouse was placed in the scanner and a series of high-resolution images were acquired over a specified range of angles to generate a three-dimensional reconstruction of the lung nodule and surrounding tissue. Imaging parameters included an x-ray source voltage of 45 keV, a current of 0.5 mA, and an average scan time of 30 minutes to obtain 60 images to ensure optimal contrast and resolution. After small animal CT screening, mice were euthanized with isoflurane and lung tissue was obtained. The excised lungs were then fixed in buffered formalin for 24 hours, dehydrated, and embedded in paraffin. For histological analysis, 5 μm thick sections were prepared from the lungs of four mice and H&E stained according to standard protocols. Afterwards, immunohistochemical analysis of lung tumor tissues was performed using rabbit anti-CEACAM6 recombinant monoclonal antibody (1:500, Absin) and DAB detection kit (Gene Tech) to detect the expression of CEACAM6. The morphological characteristics and staining patterns were observed using an Olympus microscope, and image analysis was performed using ImageJ software. Finally, the expression of CEACAM6 was calculated as the mean value of IOD to further analyze the differences in urethane-induced orthotopic LUAD mice at different time points.
[0036] In situ malignant pulmonary nodule animal model was induced by intraperitoneal injection of urethane ( Figure 2 A). Computed tomography (CT) showed that certain lung nodules were found in urethane-treated mice at 8 and 16 weeks after injection compared with untreated controls ( Figure 2 B). Pathological examination confirmed that the lung nodules were malignant and determined their pathological type to be LUAD. Immunohistochemistry results showed that the expression level of CEACAM6 was significantly increased in the malignant nodules of mice 8 weeks after injection, while it was almost undetectable in the lungs of normal mice. In addition, compared with mice exposed to urethane for 8 weeks, the expression of CEACAM6 in the malignant nodules of the lungs of mice exposed to urethane for 16 weeks was significantly increased, indicating that CEACAM6 was positively correlated with the disease progression of malignant lung nodules ( Figure 2 C). These results confirm that CEACAM6 is a specific biomarker for LUAD. The differential expression of CEACAM6 in different tumor stages and its positive correlation with the degree of malignant progression of lung nodules confirm its important role in LUAD disease progression. In addition, this result also indicates that A549 and Huh7 cell lines are suitable for screening CEACAM6-targeted tracers in xenograft tumor models.
[0037] Example 4 Design of CEACAM6 targeting peptide
[0038] In this study, Alphfold2 was used to generate the three-dimensional structure of CEACAM6 interacting with CEACAM8, HopQ, and 2A3, and the top three-dimensional structure was selected for subsequent peptide design. Subsequently, Rosetta protein modeling software (Rosettapeptiderived) was used to generate truncated linear peptides of 10-25 amino acids from the region where the ligand binds to CEACAM6 based on the three-dimensional structure derived from Alphfold2. Among them, the linear peptide with the largest CEACAM6 ligand binding energy was retained. Finally, the obtained peptides were subjected to amino acid substitution mutations using MOE (Molecular Operating Environment) software to obtain candidate CEACAM6 targeting peptides with excellent binding energy and root mean square deviation (RMSD). Then, a total of 6 peptides were obtained, named P1-P6, among which P1 was: Tyr-Thr-Leu-Gln-Val-Ile-Lys-Ser-Asp-Leu-Val-Asn-Glu-Glu-Ala-Thr-Gly-Gln-Phe-His-Val-Tyr,
[0039] P2 is: Glu-Trp-Gln-Arg-Leu-Lys-Trp-Phe-Asp-His,
[0040] P3 is: Gly-Arg-Gln-Tyr-Asp-Tyr-Trp-Gly-Gln-Gly,
[0041] P4 is: Ala-Thr-Gly-His-Leu-Leu-Asp-Thr-Val-His,
[0042] P5 is: His-Ser-Asp-Ile-Pro-Asn-Gly-Gln-Thr-Asn-Gln-Gly-Val-Gln-Ala, P6 is Glu-Gly-Asp-Val-Arg-Phe-Gln-Asn-Gln-Gly-Arg-Gln-Gln-Cys.
[0043] Example 5 Synthesis and Radiolabeling of Non-Radioactive Precursor NODA-Peptide and In Vivo Screening
[0044] p-scn-benzyl-NODA-Ga was added to dimethyl sulfoxide (DMSO) containing the peptide and diisopropylethylamine (DIPEA), with the molar ratio of peptide to p-scn-benzyl-NODA-Ga being 1:2 to 1:3, and reacted at 40°C for 8 hours. The resulting mixture was purified by HPLC using the linear gradient described previously. The desired product was collected and lyophilized to obtain a white powder with a purity greater than 95%. For labeling 68 Ga, fresh 68 The Ga portion was added to the reaction bottle containing NODA-Peptide. Sodium acetate buffer (1.0 M) was added and the pH of the reaction solution was adjusted to 3.0-4.0. The mixture was then heated and stirred at 60°C for 10 minutes. After dilution with deionized water, the complex was purified using a BOND ELUT C18 cartridge (Varian). The labeled peptide was eluted with 200 μL 10.0 mM HCl in ethanol. The final product was reconstituted in saline and filtered through a 0.22 μm Millipore filter into a sterile vial.
[0045]
[0046] In vivo screening of radiolabeled peptides was performed using A549 tumor-bearing mice and positron emission tomography (PET) imaging. Briefly, tumor-bearing mice were anesthetized with isoflurane and placed in a PET scanner. Approximately 3.7 MBq of 68 100 μL of normal saline with Ga-NODA-Peptide. Static PET images were obtained at 10 minutes, 30 minutes, 60 minutes and 120 minutes after injection. The tissue uptake of the tracer was quantified using the region of interest (ROI) technique, and the results were expressed as the percentage of the radiotracer in the ROI relative to the total injected radioactive dose (%ID / g). For pharmacokinetic studies, blood was collected from the tail vein of A549 tumor-bearing mice (n=4) at different time points (5, 10, 15, 20, 25, 30, 45, 60, 90 and 120 minutes). Each blood sample was weighed and the radioactivity was measured with a γ counter. Pharmacokinetic parameters were analyzed using DAS2.1 software.
[0047] In this study, LUAD xenograft tumor models were constructed using overexpressed CEACAM6 cells (A549) for PET screening. ROI analysis after PET scanning showed that P2 and P3 peptides had good clarity between tumor and muscle ( Figure 3 A) At 60 minutes, 68 Ga-NODA-P2 and 68 The target / background binding ratios (TBR) of Ga-NODA-P3 reached 15.6±6.9 and 6.6±1.4, respectively, and its uptake in the liver was relatively low ( Figure 3 B). These results indicate that P2 and P3 are more suitable as targeting peptides for CEACAM6.
[0048] After obtaining the possible CEACAM6-targeting peptide, the imaging performance of the radiotracer needs to be further studied. First, molecular docking revealed that the interaction between P2 and CEACAM6 is mediated by 6 hydrogen bonds formed by 3 pairs of amino acids ( Figure 4 A), while the interaction between P3 and CEACAM6 is mediated by 9 hydrogen bonds formed by 7 pairs of amino acids ( Figure 4 C). This finding suggests that P3 has a stronger affinity for CEACAM6. Secondly, using CEACAM6-negative cells (Huh7) as negative control, a xenograft tumor model was constructed to explore 68 Ga-NODA-P2 and 68 In addition, PET imaging and ROI analysis showed that after intraperitoneal injection 68 10 minutes after Ga-NODA-P2 (2.03±0.89% ID / g vs 0.83±0.18% ID / g), 68 The tumor uptake of Ga-NODA-P2 in A549 xenografts was significantly higher than that in Huh7 xenografts, and the tumor uptake of Ga-NODA-P2 in A549 xenografts was significantly higher than that in Huh7 xenografts. 68 There was no significant difference in tumor uptake between A549 xenografts and HUH7 xenografts 30 min after Ga-NODA-P2 (0.56±0.39% ID / g vs 0.62±0.12% ID / g). Figure 4 B). A549 xenografts showed a significantly higher level of cytotoxicity at 10 min (3.08 ± 0.27% ID / g vs 0.83 ± 0.21% ID / g) and 30 min (1.84 ± 0.23% ID / g vs 0.54 ± 0.10% ID / g) compared to Huh7 xenografts. 68 Tumor uptake of Ga-NODA-P3 was significantly higher ( Figure 4 D) PET imaging results further confirmed that 68 Compared with Ga-NODA-P2, 68 Ga-NODA-P3 has better affinity, imaging performance and good targeting.
[0049] Example 6 68 PET imaging, biodistribution and data analysis of Ga-NODA-P3 in tumor-bearing mice
[0050] A549 and Huh7 tumor-bearing mice (n=4) were injected with 3.7 MBq 68Ga-NODA-P3 saline 200 μL. At each time point, mice were anesthetized and scanned in the prone position. In the blocking study, A549 tumor mice received unlabeled NODA-P3 (10 mg / kg body weight) and 3.7 MBq 68 Combined injection of Ga-NODA-P3. 68 A549 and Huh7 tumor-bearing mice (n=4) were sacrificed 30 minutes after Ga-NODA-P3 to observe its biodistribution in vivo. Tumors and other tissues (including blood, heart, lung, liver, spleen, kidney, stomach, colon, intestine, bone, muscle and brain) were collected and analyzed for radioactivity using a γ-counter.
[0051] GraphPad Prism software was used for statistical analysis. All data are expressed as mean ± SEM. T test was used for comparison between two groups, and one-way analysis of variance or two-way repeated measures analysis of variance with Dunnet T test was used for multiple comparisons. P < 0.05 was considered a significant difference between groups.
[0052] Compare 68 Imaging performance of Ga-NODA-P3 in CEACAM6-negative Huh7 cells. PET imaging shows specific uptake of Ga-NODA-P3 in A549 xenograft tumors at multiple time points after tail vein administration. 68 Ga-NODA-P3( Figure 5 A) ROI analysis at 10, 30, 90 and 120 minutes showed that uptake 68 The expression of Ga-NODA-P3 in A549 tumor xenografts was 3.08±0.27, 1.84±0.02, 0.93±0.19, 0.62±0.06% ID / g, which were significantly higher than those in Huh7 xenografts (0.83±0.21, 0.54±0.10, 0.43±0.05, 0.31±0.09% ID / g) and unlabeled NODA-P3 pre-blocked A549 tumor xenografts (1.21±0.52, 0.52±0.14, 0.34±0.20, 0.08±0.06% ID / g), respectively. Figure 5 B). In addition, in vivo pharmacokinetic studies have shown that 68 The blood half-life of Ga-NODA-P3 is 32.06 min, indicating rapid clearance from the body ( Figure 5 C). In vitro biodistribution also showed that 68 The uptake of Ga-NODA-P3 was dominant in the tumor, reaching 2.67±0.42% ID / g ( Figure 5D). At 30 min, Huh7 tumor uptake of the tracer was 0.72 ± 0.20% ID / g, significantly lower than A549. Co-injection of unlabeled NODA-P3 significantly reduced 68 The uptake of Ga-NODA-P3 in A549 tumors was 0.73±0.03% ID / g at 30 min after tracer injection. Immunohistochemistry results showed that the expression of CEACAM6 protein in A549 xenografts was significantly higher than that in Huh7 xenografts ( Figure 5 E). At 30 min, high levels of radioactivity were also observed in the kidney (6.85 ± 1.08% ID / g), indicating that it was mainly retained in the kidney. The above results suggest that 68 Ga-NODA-P3 specifically targets CEACAM6 and is suitable for PET imaging of CEACAM6-positive LUAD or malignant lung nodules.
[0053] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Based on the concept of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of simplicity.
[0054] The embodiments of the present disclosure are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure should be included in the scope of protection of the present disclosure.
Claims
1. A radionuclide ligand targeting CEACAM6, characterized in that: The targeting molecule is obtained by modifying the targeting molecule with a bifunctional chelator, wherein the targeting molecule is a nuclide aptamer, an antibody or a polypeptide capable of targeting CEACAM6, and the targeting molecule is preferably Glu-Trp-Gln-Arg-Leu-Lys-Trp-Phe-Asp-His or Gly-Arg-Gln-Tyr-Asp-Tyr-Trp-Gly-Gln-Gly.
2. The nuclear ligand targeting CEACAM6 according to claim 1, characterized in that: The bifunctional chelating agent and the polypeptide further contain a linker, wherein the linker is Wherein: n is an integer from 1 to 8.
3. The nuclear ligand targeting CEACAM6 according to claim 1 or 2, characterized in that: The bifunctional chelating agent is a macrocyclic ligand or an acyclic ligand, and the macrocyclic ligand is preferably 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane-1,4-diacetic acid (NODA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonic acid) (NOTP) or a derivative thereof.
4. The radionuclide ligand targeting CEACAM6 according to any one of claims 1 to 3, characterized in that: The configuration of each amino acid in the polypeptide targeting CEACAM6 is independently selected from the D-form or the L-form.
5. The nuclear ligand targeting CEACAM6 according to any one of claims 1 to 4, characterized in that: Each amino acid in the polypeptide targeting CEACAM6 can be independently selected from substituted amino acids, and the substitution includes chloro, fluoro, bromo, or methyl, nitro, or methoxy substitution.
6. Use of the radionuclide ligand targeting CEACAM6 according to any one of claims 1 to 5 in the preparation of a reagent for detecting the expression level of CEACAM6, diagnosing or treating a CEACAM6-related disease, wherein the CEACAM6-related disease is lung nodule or lung adenocarcinoma.
7. A PET probe targeting CEACAM6, characterized in that: The method is obtained by labeling the nuclide ligand according to any one of claims 1 to 5 with a radioactive nuclide.
8. The PET probe targeting CEACAM6 according to claim 7, characterized in that: The radionuclide is 99 mTc, 68 Ga, 64 Cu, 67 Ga, 90 Y. 111 In, 177 Lu, 125 One of I.
9. A method for preparing a PET probe targeting CEACAM6 according to claim 7 or 8, characterized in that: The method comprises the following steps: adding a radioactive nuclide into a reaction bottle containing a nuclide ligand targeting CEACAM6, adjusting the pH of the reaction solution to acidic, then heating the mixture to react, purifying the complex, eluting with a strong acid solution in an organic solvent, and finally recombining the product in physiological saline.
10. Use of the PET probe targeting CEACAM6 according to claim 7 or 8 in preparing a reagent for detecting the expression level of CEACAM6, diagnosing or treating a CEACAM6-related disease, wherein the CEACAM6-related disease is lung nodule or lung adenocarcinoma.