Cell adhesion protein Nectin-4 targeted bicyclic peptide fluorescent probe and preparation method thereof

Abstract

The invention discloses a cell adhesion protein Nectin-4 targeted bicyclic peptide fluorescent probe and a preparation method thereof, the probe provided by the invention can be used for accurate detection of recurrent tumors and metastatic lymph nodes, and the probe is applied to rapid pathology and fluorescent surgical navigation in an operation. The method has the advantages of high signal-to-background ratio, high specificity and long imaging time window.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and molecular imaging, and in particular to a bicyclic peptide fluorescent probe targeting the cell adhesion protein Nectin-4 and a preparation method thereof. Background Art

[0002] Nectin-4 is a type I transmembrane protein that primarily forms physical connections between adjacent cells through cell adhesion, thereby participating in biological processes such as intercellular communication and migration. Nectin-4 expression is restricted in normal tissues, but is widely expressed in solid tumor tissues, particularly in tumors such as bladder cancer, head and neck cancer, lung cancer, breast cancer, and ovarian cancer. High nectin4 expression is closely associated with poor prognosis in various tumors. It is an important biological marker for tumor recurrence and metastasis and a popular target for antibody-based drug (ADC) therapeutics. With the approval of the nectin-4-targeted antibody drug ventuinumab for marketing, the development of targeted molecular probes will facilitate the precise diagnosis of tumors with high nectin-4 expression, guide intraoperative tumor resection margins, screen patients suitable for ADC treatment, and dynamically monitor nectin4 expression during treatment. This is of great significance for optimizing precision tumor diagnosis and treatment and developing new combination therapy strategies.

[0003] In the prior art, molecular probes targeting Nectin-4 are mainly radionuclide-labeled bicyclic peptides (CN114133434B), which are suitable for preoperative diagnosis or integrated diagnosis and treatment of tumors. However, there are problems such as radiation hazards of radionuclides, short imaging time window, low tumor signal-to-background ratio, high cost, and unsuitability for intraoperative guidance and real-time imaging. In addition, the patent discloses the cyclic peptide N188 targeting Nectin-4, but its application does not involve the field of optical imaging. In view of the advantages of optical imaging such as high detection sensitivity, real-time imaging capability and low cost, it is suitable for application scenarios such as guiding intraoperative navigation of tumors and rapid postoperative pathological diagnosis. To this end, a fluorescent probe targeting Nectin-4 is to be developed in this field for accurate detection of tumors. Summary of the Invention

[0004] The present invention is achieved through the following technical solutions:

[0005] The present invention provides a bicyclic peptide fluorescent probe targeting the cell adhesion protein Nectin-4, having the general structural formula shown in the following formula (1), namely N188-ICG:

[0006]

[0007] The present invention also provides a method for preparing the bicyclic peptide fluorescent probe targeting the cell adhesion protein Nectin-4, comprising the following steps:

[0008] (1) ICG-NHS and N188 polypeptide were dissolved in DMF solvent, and a mixture containing N188-ICG fluorescent molecular probe was obtained by dehydration condensation reaction between activated carboxyl group and amino group;

[0009] (2) Purification: The mixture was purified by column chromatography to obtain the fluorescent molecular probe N188-ICG.

[0010] Preferably, the molar concentration ratio of ICG-NHS to N188 in step (1) is 1:1.

[0011] Preferably, the dehydration condensation reaction in step (1) is carried out at 24° C. in the dark, and the reaction time is about 4 h.

[0012] The specific probe structure includes: 1) a targeting unit: selected from the cyclic peptide N188 described in CN119060138B; 2) an optical labeling unit: a near-infrared fluorescent dye (such as ICG), covalently coupled to the targeting polypeptide through an amide bond; 3) a linking group: an alkyl chain containing 4-8 carbon atoms, used to optimize the pharmacokinetics of the probe.

[0013] The present invention also provides the use of N188-ICG in preparing a preparation for detecting cell adhesion protein Nectin-4.

[0014] The probe of the present invention can produce NIR-II fluorescence imaging in the 1000-1700nm spectrum; it can be used for the accurate detection of recurrent tumors and metastatic lymph nodes, and the probe is used for rapid intraoperative pathology and fluorescence surgical navigation.

[0015] The present invention has but is not limited to the following beneficial effects:

[0016] First, the present invention provides a fluorescent molecular probe targeting the cell adhesion protein Nectin-4. Utilizing the high-affinity cyclic peptide N188 and the fluorescent dye ICG, which exhibits near-infrared optical signals in the I and II regions, a fluorescent molecular probe was constructed that can effectively target tumors and metastatic lymph nodes with high Nectin-4 expression. This probe exhibits a high signal-to-background ratio, high specificity, and a long imaging time window. Furthermore, it can guide intraoperative visualization of tumor resection margins and rapid intraoperative pathological diagnosis, potentially significantly improving surgical accuracy and safety, reducing recurrence rates, and improving patient quality of life.

[0017] Secondly, the fluorescent molecular probe N188-ICG of the present invention can be produced through a one-step dehydration condensation reaction, and the preparation process is relatively simple and the cost is low.

[0018] Third, ICG is a fluorescent dye that has been approved by the FDA and proven to be safe and non-toxic. It has good biocompatibility and has a fluorescence tailing effect in the near-infrared II band, thus achieving a higher tumor signal-to-background ratio and deeper penetration depth, which can effectively assist in precise tumor resection during surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to clearly illustrate the specific implementation methods of the present invention and certain detection technologies used in the experiments, the implementation methods and the technologies used will be described below, mainly in the form of drawings.

[0020] Figure 1 is the chemical structural formula of the fluorescent probe N188-ICG;

[0021] Figure 2 is the fluorescence spectrum of the fluorescent probe N188-ICG;

[0022] Figure 3 The results of WB detection of Nectin4 expression in tumor cells;

[0023] Figure 4 This is a confocal experiment result demonstrating the specific binding of N188-ICG probe to cells;

[0024] Figure 4 Middle: a is the distribution map of N188-ICG probe; b is the distribution map of DAPI-stained cell nuclei; c is the co-localization map of cell membrane and probe;

[0025] Figure 5 IVIS fluorescence imaging of a mouse subcutaneous tumor model using the N188-ICG probe; Figure 5 Middle: a is the imaging image of the N188-ICG probe targeting group, and b is the imaging image of the N188-ICG probe inhibition group.

[0026] Figure 6 for Figure 5 Quantitative analysis of the results of the line statistical chart;

[0027] Figure 7 This is the imaging effect of N188-ICG intravenously injected into the mouse axillary lymph node metastasis model;

[0028] Figure 8 for Figure 7 Fluorescence imaging of isolated organs and metastatic lymph nodes;

[0029] Figure 9 This is a fluorescence image of an ex vivo specimen from the surgical margin of recurrent nasopharyngeal carcinoma after incubation with N188-ICG probe solution.

[0030] Figure 10 for Figure 9 Pathological verification of corresponding tissue specimens;

[0031] Figure 11 Near-infrared second-zone fluorescence imaging of mouse subcutaneous tumor model using N188-ICG probe.

[0032] Specific implementation methods

[0033] The specific implementation methods of the present invention are explained with the aid of examples. Except that the technology used for detection does not impose any form of limitation on the present invention, some schemes in the described embodiments are part of the embodiments of the invention. The embodiments obtained by ordinary technical operators in this field without creative results are all within the scope of protection of the present invention.

[0034] Example 1

[0035] A method for preparing a cell adhesion protein Nectin-4 targeted fluorescent probe comprises the following steps:

[0036] 1. General peptide synthesis steps:

[0037] 1. Experimental materials, reagents and instruments

[0038] Experimental materials and reagents:

[0039]

[0040]

[0041] Experimental equipment:

[0042] instrument Manufacturer or model Speed-regulating multi-purpose oscillator Jiangsu Jintan Honghua Instrument Factory Dry incubator Jiangsu Taicang Hualida Experimental Equipment Co., Ltd. High performance liquid chromatography (Agilent) Agilent 1100 Mass spectrometer (Waters) WatersZQ2000

[0043] 2. Synthesis steps

[0044] Synthetic linear polypeptide: The specific sequence is shown in CN114133434B.

[0045] 2.1 Solvent treatment

[0046] DMF and methanol were soaked with G3 molecular sieves overnight before use to remove impurities and water.

[0047] 2.2 Full swelling of resin

[0048] Weigh 2.0 g of blank Wang resin into a clean, dry reaction tube, add 15 mL of DMF, and activate at room temperature for about 30 min.

[0049] 2.3 Connect the first amino acid Fmoc-Cys(Trt)-OH

[0050] At room temperature, filter the solvent from the previous step through a sand core. Add 1 mmol of a 5-fold molar excess of the first C-terminal amino acid, a 5-fold molar excess of DMAP, and a 5-fold molar excess of DIC, using DMF as the solvent, and allow to react at room temperature for 3 hours. After the reaction is complete, wash the reaction 4-6 times with 5-6 mL of DMF each time. Then, add an appropriate amount of pyridine and acetic anhydride in a 1:1 volume ratio and allow to react for 30 minutes. After the reaction is complete, wash the reaction 4-6 times with 5-6 mL of DMF each time. (Purpose: To block any active spots on the unloaded resin.)

[0051] 2.4 Removal of Fmoc Protecting Group

[0052] Remove the solvent from the previous step by suction, add 10 mL of 20% piperidine DMF solution to the resin, stir under N2 for 10 minutes, filter out the solution, add 10 mL of 20% piperidine DMF solution, blow and stir under N2 for 5 minutes, and then filter out the solution. Repeat this operation twice, then wash with DMF 4 times and methanol 2 times, 5-6 mL each time.

[0053] 2.5 Ninhydrin detection removal effect

[0054] Take out a small amount of resin, wash it three times with methanol, add one drop each of ninhydrin, KCN, and phenol solution, and heat at 105℃–110℃ for 5 minutes. If it turns dark blue, it is a positive reaction, indicating that the removal is complete and the next step can be carried out. If it is colorless, it means that the protecting group is not completely removed and the above deprotection operation needs to be repeated.

[0055] 2.6 Connecting the second amino acid and removing the Fmoc protecting group

[0056] Weigh a 3-fold molar excess of the second amino acid at the C-terminus, a 3-fold molar excess of HBTU, and a 3-fold molar excess of HOBT into a reaction tube. Add an appropriate amount of DMF solution to completely dissolve it, then add a 10-fold molar excess of (pure) DIEA. React at room temperature for 40 minutes and wash with DMF 4-6 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin detection reagent. It turns colorless. Then add 10 mL of 20% piperidine DMF solution to remove Fmoc. Perform two times, 10 minutes and 5 minutes respectively. Then wash it with DMF 4 times and methanol 2 times, 5-6 mL each time. Take out a small amount of resin and test it with ninhydrin detection reagent. If it turns blue, it can proceed to the next step of the reaction.

[0057] 2.7 Repeat step 2.6 until the last amino acid Fmoc-Cys(Trt)-OH at the N-terminus is synthesized, remove the Fmoc protecting group, and then drain.

[0058] 2.8 Resin shedding and pure product separation detection

[0059] Finally, the product was cut with trifluoroacetic acid cutting solution (95% TFA: 2% TIS: 2% EDT: 1% H2O) for 2 hours, and the reaction solution was filtered to obtain a trifluoroacetic acid solution of the polypeptide. The lysate was blown dry as much as possible with nitrogen, and then precipitated with ether, centrifuged, and then washed with ether 3 to 5 times to obtain a white solid. After dissolving in pure water, it was desalted and purified by HPLC, and the crystals were precipitated after freeze-drying, and a small amount was taken for MS analysis.

[0060] Synthesis of bicyclic peptides:

[0061] Dissolve 1 mmol of a linear peptide in 50 mL of acetonitrile and water (1:1 volume ratio). Add 1.3 eq of TATB as a scaffold and 2 eq of ammonium bicarbonate. Allow to react at room temperature, monitored by HPLC, until the linear peptide reaction is complete. After lyophilization, the bicyclic peptide is purified by HPLC and mass spectrometry.

[0062] 2. Synthesis of N188-ICG fluorescent molecular probe

[0063] 1. Dissolve 0.1 mmol ICG-NHS and 0.1 mmol N188 peptide in 2 mL of anhydrous DMF solvent, add 10 μL of LTEA (triethylamine), and react at room temperature for about 4 hours to obtain a mixture containing the N188-ICG fluorescent molecular probe through a dehydration condensation reaction between the activated carboxyl group and the amino group;

[0064] 2. Monitor by high performance liquid chromatography (HPLC), terminate the reaction after the bicyclic peptide reaction is complete, and identify the final product by HPLC and Mass Spectrometry.

[0065] 3. Preparative HPLC conditions are as follows:

[0066] Instrument: Shimadzu 2010 HPLC semi-preparative liquid chromatograph

[0067] Chromatographic column: 20cm*250cm C18, 5uM

[0068] Mobile phase: A (0.1% TFA, acetonitrile)

[0069] B (0.1% TFA, water)

[0070] Gradient: 0.00-3.00 min 10% A

[0071] 3.00-45.00 10%-75% A

[0072] Detection wavelength: 220nm The chemical structure of N188-ICG fluorescent molecular probe is as follows Figure 1 shown.

[0073] Example 2 Spectral Detection of Fluorescent Molecular Probe N188-ICG

[0074] The fluorescence spectrum of the N188-ICG fluorescent molecular probe at a concentration of 10 μM was detected using a fluorescence spectrophotometer. The results are as follows Figure 2 As shown, the maximum excitation wavelength of the probe is 751 nm, and the maximum emission wavelength is 791 nm.

[0075] Example 3 WB verification of tumor cell lines with high expression of Nectin4

[0076] Western Blot was used to detect the expression of Nectin-4 protein in various nasopharyngeal carcinoma cells (S18, S26, 5-8F, 6-10B). Figure 3 As shown in Figure 3, nectin-4 protein is highly expressed in 5-8F and S18 NPC cells, while its expression is moderate to low in S26 and 6-10B cells. Therefore, the probe's targeting and specificity were verified in vitro and in vivo in the 5-8F cell line, which highly expresses nectin-4.

[0077] Example 4: Targeted Detection of Tumor Cells by N188-ICG Fluorescent Molecular Probe

[0078] N188-ICG was co-incubated with nectin-4 overexpressing cells (5-8F and S18). Fluorescence microscopy showed that the probe specifically bound to the cell membrane. Competitive inhibition experiments (addition of excess free cyclic peptide N188) reduced the fluorescence signal by 80%. The specific protocol is as follows:

[0079] This example provides a method for targeted cell uptake of a probe in vitro, as shown in the figure.

[0080] S18,5-8F was cultured to the logarithmic growth phase and evenly plated in a confocal dish overnight. The experiment was started when the cells grew to 70-80% of the dish bottom. The culture medium in the confocal dish was discarded and rinsed with PBS solution, and repeated three times. In the target group, 200 μL of probe solution (40 μg / mL) was added to each dish and incubated in a cell culture incubator for 0.5 h (37°C, 5% CO2, protected from light). In the block group, the inhibitor N188 solution (70 μg / mL) was first added and incubated for 0.5 h, and then the probe solution of the same concentration was added and incubated for 0.5 h.

[0081] The probe solution in the confocal dish was discarded, and 1 mL of PBS was added to wash the cell surface three times. The PBS waste liquid was then aspirated with a sterile pipette, and the cells were fixed with paraformaldehyde (4%, 1 mL) for 10 min.

[0082] Discard the paraformaldehyde in the confocal dish, add PBS, rinse on a shaker for 5 minutes (protect from light), discard the PBS, repeat 3 times, add 5% BSA to block for 30 minutes, and wash with PBS 3 times.

[0083] Then add 200 μL of 100 ng / ml DAPI solution, discard the solution after 10 minutes, add PBS, place the confocal dish in a light-proof container, rinse on a shaker for 5 minutes, discard the PBS, and repeat 3 times.

[0084] The cells were placed in a Zeiss LSM800 laser confocal microscope for observation. Figure 4 .

[0085] like Figure 4 The results shown in the figure show the specific binding of the probe to S18 and 5-8F cells using laser confocal microscopy. In the targeted group, the probe N188-ICG showed high uptake in S18 and 5-8F cells, with strong fluorescence signals on the cell membrane. In the Block group, which also included the inhibitor N188, the fluorescence signal was significantly reduced. These results demonstrate that the probe N188-ICG can specifically bind to the cell membranes of nasopharyngeal carcinoma cell lines S18 and 5-8F, showing good tumor targeting, and confirming that the probe can specifically detect tumors with high Nectin4 expression.

[0086] Example 5 Construction of nude mouse subcutaneous transplanted tumor and metastasis model

[0087] A nude mouse subcutaneous tumor and metastasis model was established using the 5-8F nasopharyngeal carcinoma cell line and immunodeficient Balb / c nude male mice. Subcutaneous tumor model construction: 5-8F nasopharyngeal carcinoma cells were cultured and centrifuged at 1000 rpm for 5 minutes when the cells showed logarithmic growth. The tumor cells were then washed three times with sterile PBS and diluted appropriately. The cells were counted using a hemocytometer to a concentration of 1×10 6 / mL of tumor cell suspension was subcutaneously inoculated with 100μL of tumor cell suspension on the dorsal right hind limb of each mouse, near the upper edge of the thigh root. After the inoculation is completed, the vitality of the nude mice and the presence of infection at the inoculation site are observed daily, as well as whether the tumor naturally regresses after growth. Imaging experiments for the subcutaneous tumor model begin when the maximum diameter of the tumor reaches 7-8mm. The metastasis model is constructed by observing the appearance of metastatic lymph nodes 4-5 weeks after subcutaneous injection of tumor cells. Ultrasound scanning or magnetic resonance imaging is used for monitoring, and imaging experiments begin when metastatic lymph nodes are clearly formed.

[0088] Example 6: Fluorescence imaging of a nude mouse subcutaneous tumor model using the N188-ICG probe

[0089] This example provides a method and results of in vivo targeted imaging of tumor-bearing nude mice using a probe. The specific steps are as follows:

[0090] Ten to fourteen days after the tumor cell suspension was inoculated, the subcutaneous tumor model of mice was successfully established and 200 μL of 100 μg / mL N188-ICG solution was injected via the retroorbital vein. The Block inhibition group was injected with N188 inhibitor (200 μL, 150 μg / mL) half an hour before the probe solution was injected. After the probe was injected, the mice were anesthetized with isoflurane, and the subcutaneous tumor model mice were subjected to in vivo fluorescence imaging (IVIS Lumina Series III, PerkinElmer, USA) at different time points (6h, 12h, 24h, 36h, 48h, 72h). ROI was delineated, and the fluorescence intensity ratio (TBR) of the tumor and background areas was analyzed to evaluate the optimal time point for imaging the probe in the tumor area.

[0091] like Figure 5 The results showed that the N188-ICG fluorescent molecular probe can specifically target tumor models with high expression of Nectin4. Figure 5 In the targeted group, tumors began to show strong fluorescence 6 hours after probe injection, and tumor imaging contrast reached its peak 24 hours after injection. Over time, N188-ICG significantly concentrated in the tumor region and was excreted through the liver. Clearance from non-target organs was rapid, reducing background signal. Furthermore, the fluorescence signal in the tumor region maintained high imaging contrast for up to 72 hours. Figure 5 b In the inhibition group, the probe fluorescence signal in the tumor area was significantly weakened, and the imaging contrast was significantly lower than that in the targeting group. Figure 6 for Figure 5 Quantitative analysis of the results, as shown in the line chart, showed that 24 hours after probe injection, the TBR value in the targeted group was 2.14, significantly higher than that in the inhibition group (TBR = 1.09). These experiments demonstrate that the probe has good specificity and targeting, and presents an imaging window of up to 72 hours, making it suitable for clinical intraoperative tumor localization and guidance of tumor resection.

[0092] Example 7: Fluorescence imaging of nude mouse metastasis model using N188-ICG probe

[0093] This example provides a method and results of probe targeted imaging in a metastatic lymph node model. The specific steps are as follows:

[0094] Approximately 3-4 weeks after tumor formation, the ipsilateral axillary lymph nodes were observed every two days. Experiments were initiated after ultrasound or magnetic resonance imaging confirmed the formation of metastatic lymph nodes. 200 μL of a 100 μg / mL N188-ICG solution was injected retro-orbitally, and then in vivo fluorescence imaging of the metastatic model mice was performed. Following collection, the mice were euthanized, and organs, tumor tissue, and metastatic lymph nodes were removed to obtain the in vitro mean fluorescence intensity.

[0095] like Figure 7 This is a fluorescence imaging diagram of a mouse metastasis model. The results show that after the probe is injected into the axillary metastatic lymph nodes of tumor-bearing mice, a strong fluorescence signal is evident, indicating that the probe can specifically detect metastatic lymph nodes. After dissecting the tumor and metastatic lymph nodes, ex vivo tissue is obtained for ex vivo imaging ( Figure 8 ) for quantitative analysis of tumor metastasis and background tissue areas. The TBR value for metastatic lymph nodes was 2.36. This demonstrates that the N188-ICG fluorescent molecular probe has excellent imaging effects in tumors with high Nectin4 expression and corresponding metastatic lymph nodes.

[0096] Example 8 Fluorescence imaging of postoperative recurrent nasopharyngeal carcinoma tissue in vitro using N188-ICG probe

[0097] First, the freshly resected human nasopharyngeal carcinoma specimen was cut in half from the coronal plane. The specimen contained normal tissue on the outer edge and tumor tissue on the inner side. The specimen was immersed in a PBS solution containing 5% goat serum and rinsed 3 times for 2 minutes each time. The specimen was placed in a 20μM N188-ICG solution and incubated on ice at low temperature for 30 minutes. Washed 3 times in a PBS solution for 2 minutes each time. The sample was dried with absorbent paper and the fluorescent image of the sample was captured. After imaging, the tissue was fixed with 4% paraformaldehyde, embedded in wax blocks, and sliced ​​for H&E staining to determine whether the tumor area matched the strong signal area collected by fluorescence imaging. The area where the tumor cells are located is shown on the full scan HE staining image ( Figure 10 ) and fluorescence signal area ( Figure 9 ) are consistent.

[0098] Example 9 N188-ICG probe has NIR-II fluorescence imaging capability

[0099] This example provides a method and results of near-infrared second-zone imaging of a probe in a mouse subcutaneous tumor model. The specific steps are as follows:

[0100] After the subcutaneous tumor model of mice was successfully established, 200 μL of 100 μg / mL N188-ICG solution was injected into the retro-orbital vein. 24 h after the probe injection, imaging was performed using a small animal NIR-II fluorescence imaging device (DPM-IVFM-NIR-II, China).

[0101] like Figure 11 As shown in the figure, we observed significant fluorescence signals in the tumor region, with high imaging contrast. This demonstrates that N188-ICG has NIR-II imaging capabilities, making it suitable for existing intraoperative fluorescence navigation equipment and can be used for real-time imaging during surgery to guide surgical margins.

[0102] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Various modifications and variations are readily apparent to those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A bicyclic peptide fluorescent probe targeting the cell adhesion protein Nectin-4, characterized in that: It has the general structural formula shown in the following formula (1):

2. The method for preparing a bicyclic peptide fluorescent probe targeting the cell adhesion protein Nectin-4 according to claim 1, characterized in that The method includes the following steps: (1) ICG-NHS and N188 bicyclic peptide were dissolved in DMF solvent, and a mixture containing N188-ICG fluorescent molecular probe was obtained by dehydration condensation reaction between activated carboxyl group and amino group; (2) Purification: The mixture was purified by column chromatography to obtain the fluorescent molecular probe N188-ICG.

3. The preparation method according to claim 2, characterized in that: The molar concentration ratio of ICG-NHS to N188 in the reaction of step (1) is 1:

1.

4. The preparation method according to claim 2, characterized in that: The dehydration condensation reaction in step (1) is carried out at 24° C. in the dark, and the reaction time is about 4 h.

5. Application of N188-ICG in the preparation and detection of cell adhesion protein Nectin-4.

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