Probes targeting cd137 and uses thereof

Abstract

The present application relates to the technical field of medical detection, and particularly relates to a probe targeting CD137 and application thereof. The present application provides a corresponding molecular probe based on a newly discovered CD137 targeting polypeptide. The preparation method of the molecular probe is simple and easy to implement, the molecular weight is small, the specificity is strong, the cost is low, the practicality is strong, the molecular probe has good biological safety and good application value, and the molecular probe has important significance and application value in atherosclerotic plaque diagnosis and tumor immunotherapy monitoring.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to a probe targeting CD137 and its application. Background Technology

[0002] CD137 (also known as 4-1BB) is a surface glycoprotein of the tumor necrosis factor receptor (TNFR) family, capable of inducing responses in various immune and non-immune cells, including endothelial cells and smooth muscle cells. Human CD137 is a type II membrane glycoprotein found on the cell membrane surface, belonging to the TNFR superfamily. It is expressed not only on activated T lymphocytes but also on monocytes, granulocytes, and dendritic cells (DCs). CD137 is expressed not only in immune cells but also in vascular cells, primarily by activated endothelial cells (ECs) and smooth muscle cells (SMCs). CD137 / CD137L is another important co-stimulatory molecule besides CD28 / B7, mediating co-stimulatory signals generated by the interaction of CD137 and CD137L via the CD28 / B7 pathway, playing different roles in cell-cell interactions, cell adhesion, antigen presentation, T cell co-stimulation, and signal transduction. CD137 and its ligand system exhibit bidirectional signal transduction, capable of transmitting signals to T cells via CD137 receptors and also to cells expressing the ligands. CD137-positive vessels have been found in atherosclerotic lesions, tumors, and vasculitis. It is considered a major participant and regulator of inflammatory responses. Activation of CD137 signaling-induced endothelial dysfunction effectively induces an inflammatory state, playing a crucial role in various pathological conditions.

[0003] 1. CD137 and Atherosclerosis

[0004] Atherosclerosis (AS) is a chronic inflammatory disease of the arterial wall that can persist insidiously for a long time until plaque rupture and thrombus formation, leading to acute coronary syndrome and sudden death. Endothelial dysfunction and morphological damage, including leukocyte adhesion, vasoconstriction, platelet aggregation, oxidative stress, smooth muscle cell proliferation, and thrombus formation, promote endothelial cell damage and induce endothelial cell apoptosis, resulting in local endothelial shedding, plaque erosion, and the development of acute coronary syndrome. Therefore, endothelial dysfunction participates in atherosclerosis through multiple pathways. Persistent inflammation within the plaque is associated with plaque instability and rupture; therefore, detecting the presence of progressive inflammatory responses in plaques is helpful for early diagnosis and risk assessment of AS. In mice and humans, CD137 expression is generally upregulated in atherosclerosis-related vascular cells associated with atherosclerotic plaque lesions. Under in vitro pro-inflammatory environmental stimulation, APOE mice fed a high-fat diet with CD137 knockdown showed a significant reduction in aortic atherosclerotic regions, indicating that CD137 promotes atherosclerosis. Therefore, CD137 can serve as an effective biomarker for studying the progression of ankylosing spondylitis (AS). Olofsson PS et al. demonstrated that CD137 is a co-stimulatory molecule expressed on the membrane surfaces of activated vascular endothelial cells, activated T cells, and monocytes in high-risk plaques, exhibiting high specificity. When ligands bind to it, it promotes further activation and increase of leukocytes within the plaque and the secretion of pro-inflammatory factors, showing potential as a target for tracing and treating high-risk plaques. How to effectively utilize imaging methods to monitor the occurrence and development of AS has become an urgent issue.

[0005] Given the limitations of traditional clinical strategies, there is an urgent need for a more effective and non-invasive method for early intervention in atherosclerosis. How to achieve early diagnosis and effective intervention based on the characteristics of vulnerable plaques to reduce cardiovascular mortality has become a pressing issue. The rapid development of molecular imaging and nanomedicine technologies can fill this gap. These imaging methods can perform in vivo qualitative and quantitative studies of atherosclerotic plaques at the molecular and cellular levels, detecting necrotic cores, inflammation, intraplaque hemorrhage, fibrous cap thickness, and microcalcifications, enabling early detection and monitoring of the development and progression of AS. Based on rationally designed and successfully developed CD137-targeting nanomolecular probes, new hope will be brought to the early diagnosis, prevention, and treatment of cardiovascular diseases such as AS.

[0006] 2. CD137 and Tumor Immunotherapy

[0007] In recent years, the emergence of immunotherapy, represented by PD-1, has fundamentally changed the treatment paradigm for cancer. CD137, a member of the tumor necrosis factor receptor superfamily, is a promising target for enhancing anti-tumor immune responses. CD137 plays an important role in immune responses and tumor immunity, and is highly expressed in various tumors. CD137 helps regulate the activation of many immune cells, including CD4+. + T cells, CD8 + T cells, regulatory T cells (Tregs), dendritic cells, and natural killer cells. In gastric cancer, the immune status of cancer cells is related to the expression status of CD137 / CD137L. If tumor cells express high levels of CD137L, the body's killing effect on tumor cells will be stronger; conversely, if CD137L expression is low, it will lead to tumor immune escape. Significantly elevated levels of CD137 have also been found in the serum of colorectal cancer patients. Subsequent ELISA assays confirmed that CD137 has a specificity of 97% and a sensitivity of 83% in the diagnosis of colorectal cancer, and it is expected to become a new diagnostic biomarker for early colorectal cancer.

[0008] Recent studies have shown that agonist antibodies targeting CD137 can enhance the efficacy of immunotherapy. CD137, as a positive immune checkpoint on human monocytes / macrophages, provides co-stimulatory signals in multiple immune cell subsets. Therefore, co-stimulating activated T cells, natural killer cells, and other immune cells with CD137 antibodies and therapeutic antibodies has become a promising approach to cancer immunotherapy. Several CD137-targeting drugs are under development and clinical trials, and will play an important role in future cancer treatment. However, accurate screening of sensitive populations and real-time dynamic monitoring of efficacy are prerequisites for implementing CD137-based precision medicine. Exploring a method to accurately predict the efficacy of immunotherapy in patients is also an urgent problem to be solved. Currently, the most commonly used predictive method is immunohistochemistry (IHC). However, IHC has fundamental limitations: the heterogeneous spatial expression characteristics of tumors make it insensitive to biomarker detection, and incomplete tumor sampling can lead to false positive or false negative results. In comparison, molecular imaging is playing an increasingly important role in immunotherapy and personalized medicine. The preparation of molecular probes is crucial for molecular imaging. Highly sensitive and specific molecular probes can bind specifically to specific targets within cells and generate specific signals after entering the body. These signals can then be captured in vitro using specific imaging equipment, such as positron emission tomography (PET-CT), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and fluorescence imaging (FL), thereby achieving specific diagnosis and realizing highly specific diagnostics.

[0009] Currently reported immune checkpoint diagnostic reagents are mainly antibody-based. However, antibody diagnostic reagents face challenges such as long in vivo half-life, slow blood clearance, difficulty in recognizing multiple targets simultaneously, difficulty in chemical modification, and poor penetration. In contrast, peptide-based targeted small molecule drugs and diagnostic probes exhibit significant advantages in tumor-targeted drug delivery and cancer diagnosis due to their low cost, small molecular weight, good biocompatibility, strong penetration, lack of immunogenicity, rapid blood clearance, and simple preparation. Summary of the Invention

[0010] With the widespread application of tumor immunotherapy, there is a need to develop non-invasive molecular imaging tools to dynamically monitor clinical immune checkpoints and immune cells in order to assess efficacy and screen suitable patients. Therefore, it is necessary to develop a small molecule probe targeting CD137 to quickly, easily, dynamically, and accurately identify the expression level of CD137 protein on the surface of T cells in tumor tissue, and to see if it effectively activates immune cells. This has significant clinical implications for immunotherapy and prognostic assessment.

[0011] The expression of CD137 is generally upregulated in vascular cells associated with atherosclerotic plaque lesions. It has high specificity and can serve as a target for tracing and treating high-risk plaques, enabling early detection and monitoring of the development and progression of AS.

[0012] The present invention provides a probe targeting CD137 and its application. In order to achieve the above objective, in a first aspect, the present invention provides a CD137 targeting polypeptide, wherein the CD137 targeting polypeptide comprises any of the amino acid sequences shown in SEQ ID NO. 1-3.

[0013] This invention designs and constructs peptide libraries based on hotspot amino acid sites and molecular recognition theory related to CD137 / CD137L interactions. Amino-modified TentaGel resin was used as a solid-phase support, and a single-bead peptide library with a capacity of 10⁵ was synthesized using the Fmoc synthesis strategy. High-throughput screening of the single-bead peptide library was performed using fluorescently labeled magnetic beads and microfluidic chips. Positive peptide beads were identified by MALDI-TOF-MS, yielding a series of active peptides that specifically bind to CD137. Among them, WP1-3 exhibits CD137 affinity.

[0014] SEQ ID NO.1: RKEEFYYWLWFC(WP1);

[0015] SEQ ID NO.2: SVSTYLDASNRAC(WP2);

[0016] SEQ ID NO. 3: ACIEEGQYCPADPYMCA(WP3).

[0017] The present invention further provides a method for preparing the CD137-targeting peptide, comprising:

[0018] The CD137-targeting polypeptide was synthesized using the Fmoc solid-phase peptide synthesis method.

[0019] In a second aspect, the present invention provides a nucleic acid for encoding a CD137-targeting polypeptide as described in SEQ ID NO.1-3.

[0020] Thirdly, the present invention provides a multivalent comprising at least two polypeptides; the polypeptides are selected from the CD137-targeting polypeptides described in SEQ ID NO. 1-3. The multivalent is formed in a linear, D-shaped, cyclic, or mirror-image structure.

[0021] In the multivalent provided by this invention, different polypeptides are linked by covalent linkage, non-covalent linkage, or a mixture of polymers.

[0022] Preferably, the covalent connection is achieved through a linker molecule, which includes one or more of 6-tert-butoxycarbonylhydrazinoic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, or N-hydroxysuccinimide; and / or,

[0023] The polymer includes one or more of polyethylene glycol, polyvinyl alcohol, cyclodextrin, polyamide-amine dendritic polymer, polylactic acid, or polylactic acid-ethanolamine.

[0024] Fourthly, the present invention provides a molecular probe, said molecular probe being obtained by fluorescent labeling and / or radioactive isotope labeling as follows:

[0025] i) The CD137-targeting peptides mentioned above

[0026] ii) As described above, multivalents.

[0027] In the molecular probe provided by this invention, the fluorescent label is one or more of IRDye800CW, Cy7, rhodamine, or indocyanine green, and the radioactive isotope is... 131 I, 177 Lu、 64 Cu、 99 mTc, 18 F or 68 One or more of Ga;

[0028] Preferably, the fluorescent label and the radioisotope are used to label the CD137-targeting polypeptide or multivalent using chelating agents such as DOTA, NOA, DTPA, and HYNIC; they can also be labeled using click chemistry methods such as NHS, EDC, and MAL.

[0029] More preferably, the molecular probe further includes iron oxide nanoparticles Fe3O4.

[0030] The present invention further provides the application of the CD137-targeting polypeptide, or the multivalent, or the molecular probe in the specific binding of CD137.

[0031] Fifthly, the present invention also provides the application of the above-mentioned CD137 targeting polypeptide, or the above-mentioned nucleic acid, or the above-mentioned multivalent, or the above-mentioned molecular probe in the preparation of disease detection kits.

[0032] And the application of the above-mentioned CD137-targeting polypeptide, or the above-mentioned nucleic acid, or the above-mentioned multivalent, or the above-mentioned molecular probe in the preparation of drugs;

[0033] Preferably, the drug is used for the prevention, diagnosis, or treatment of diseases related to CD137-positive vascular endothelial cells, T cells, and monocytes;

[0034] Preferably, the drug further includes an imaging agent and an agent capable of killing cancer cells; the imaging agent is one or more of a radionuclide, a radionuclide label, an iron oxide magnetic resonance contrast agent, or a molecular imaging agent. Further, the molecular probe or the multivalent is conjugated to or mixed with the imaging agent.

[0035] More preferably, the drug further includes a pharmaceutically acceptable carrier, such as a PLGA polymer, a Dendrimer dendritic macromolecule, a hydrogel, micelles, liposomes, or inorganic nanoparticles.

[0036] In the above-described applications provided by this invention, the disease includes any one of tumors, atherosclerosis, rheumatoid arthritis, and coronary heart disease. The tumor is multiple myeloma, glioma, non-small cell lung cancer, melanoma, kidney cancer, prostate cancer, Hodgkin's lymphoma, colorectal cancer, pancreatic cancer, liver cancer, stomach cancer, and / or breast cancer.

[0037] In the applications provided by this invention, the drug is used for the prevention, diagnosis, or treatment of CD137-positive atherosclerotic plaques and tumors.

[0038] In a sixth aspect, the present invention provides a drug or disease detection kit, wherein the drug comprises the above-mentioned CD137 targeting polypeptide, or the above-mentioned nucleic acid, or the above-mentioned multivalent, or the above-mentioned molecular probe.

[0039] The beneficial effects of the present invention are at least as follows:

[0040] (1) This invention has discovered a polypeptide with the property of targeting CD137 positive T cells or vascular endothelial cells, which can be used as a molecular probe to monitor patients receiving immunotherapy and evaluate the efficacy; it can also be used as a targeted polypeptide to be conjugated or mixed with immunotherapies for targeted therapy and imaging of various tumors; and the polypeptide can be optimized to be used as a polypeptide drug to activate the CD137-CD137L signaling pathway and activate T cells to promote immunotherapy.

[0041] (2) This invention further provides a molecular probe based on the discovered CD137-targeting peptide, which can overcome the shortcomings of monoclonal antibody-based molecular probes, such as large molecular weight, easy inactivation, slow tissue penetration, and slow blood clearance, while maintaining good targeting. The molecular probe provided by this invention can be used for commonly used clinical PET detection, which is beneficial for subsequent clinical translation. It can comprehensively display the CD137 expression level on T cells in the tumor area, and monitor the dynamic changes of CD137 level throughout the treatment process, which has good research prospects and clinical guidance significance.

[0042] (3) The molecular probe preparation method provided by the present invention is simple and easy to implement, low in cost, highly practical, and has good biosafety and high application value. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0044] Figure 1 This is a schematic diagram showing the results of detecting the affinity of peptides WP1, WP2 and WP3 for human CD137 protein using the surface plasmon resonance (SPRi) method provided in Example 2 of the present invention; where (a) is the detection result of WP1, (b) is the detection result of WP2 and (c) is the detection result of WP3.

[0045] Figure 2 This is a schematic diagram of the cellular level confocal imaging detection results of Rhodamine B-labeled WP1, WP2 and WP3 peptides and CD137-positive cell line HUVEC provided in Example 3 of the present invention.

[0046] Figure 3 Provided for Embodiment 4 of the present invention 68 Ga-DOTA-WP1 is used for PET imaging of mouse tumors.

[0047] Figure 4 The image shows the characterization of the WP3@Fe3O4 / ICG dual-mode probe provided in Embodiment 5 of the present invention. In the image, WP3@Fe3O4 / ICG is displayed as CD137@Fe3O4 / ICG.

[0048] Figure 5 The expression level of CD137 in human tissues was analyzed, where a represents vulnerable plaques, b represents stable plaques, and c represents normal arterial tissue.

[0049] Figure 6 Quantitative analysis of near-infrared fluorescence imaging of plaques by the WP3@Fe3O4 / ICG probe in an ApoE- / - mouse model of vulnerable atherosclerotic plaques. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0051] The N-methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), ninhydrin, vitamin C, phenol, tetramethylurea hexafluorophosphate (HBTU), hexahydropyridine, triisopropylsilane (TIS), ethylenedithiol (EDT), N,N-dimethylformamide (DMF), anhydrous diethyl ether, resin, methanol, various Fmoc protected amino acids, CD137 recombinant protein, MB-Streptavidin (streptavidin magnetic beads), Streptavidin-HRP (streptavidin-labeled horseradish peroxidase), polypeptide synthesis tubes, shaker, vacuum water pump, rotary evaporator, and laser confocal microscope (ZEISS LSM710) involved in the embodiments of this invention were all obtained commercially.

[0052] Unless otherwise specified, all instruments and reagents used in the following examples are commercially available.

[0053] Example 1

[0054] This embodiment provides the construction and screening process of a CD137-targeting peptide screening system, as detailed below:

[0055] 1. Design and synthesis of "one bead, one substance" polypeptide libraries

[0056] The target sites of CD137-CD137L were simulated using DOCK 4.0, and peptide libraries were designed by partially replacing the selected sites.

[0057] The peptide library was synthesized using the Fmoc solid-phase synthesis strategy, in which amino acids were randomly coupled one by one to the solid-phase TentaGel resin by a mixed splitting method (loading amount 0.35 mmol / g). During the coupling process, the amino acids protected by HBTU and Fmoc were dissolved in DMF containing 0.4 mol / L NMM, and the coupling time for each time was >2 h.

[0058] The deprotection process was then carried out: the Fmoc group was removed using a DMF solution containing 20% ​​hexahydropyridine, and the deprotection time was >1 hour each time.

[0059] Finally, the side-chain protecting groups were removed under the action of strong acid (92.5% TFA, 2.5% H2O, 2.5% TIS and 2.5% EDT), that is, the side-chain protecting groups were removed by reacting at room temperature for 2 hours. The pyrolysis solution was removed by filtration, and the solution was washed and shrunken with methanol multiple times. It was then vacuum dried for later use.

[0060] 2. High-throughput microarray chip screening and in-situ mass spectrometry identification of peptide libraries

[0061] The CD137 protein was labeled with biotin and purified using a Zeba spinning column. After blocking the non-specific sites of the peptide library for 2 hours, the biotinylated protein was diluted 1:1000 and added to the peptide library, and incubated at 37°C for 2 hours.

[0062] After gently washing three times with PB, 1 mL of streptavidin-labeled magnetic beads was added to the peptide library and incubated at 37°C for 2 h. The peptide resin was then transferred to a 1 mm diameter PTFE tube, and the flow rate was set to 600 μL / min.

[0063] Finally, positive peptide resin was collected onto a high-throughput microarray chip, and lysis buffer (30 mg / mL cyanogen bromide) was injected into the channel for overnight lysis. The cover was gently removed, and the microarray chip was introduced into a mass spectrometer for point-by-point secondary mass spectrometry sequencing. Finally, the peptide sequence was obtained by despectrometry using Mascot software.

[0064] 3. Synthesis of CD137-targeting peptides

[0065] Peptides were synthesized using the Fmoc solid-phase peptide synthesis method: 200 mg of Wang resin was weighed, and 200 mg of amino acids were added sequentially for the following reactions: the protected amino acids were coupled one by one to the solid-phase resin, and then the peptide chain was cleaved from the resin under strong acid while removing the side chain protecting groups; after the reaction was completed, FITC and an equal amount of HBTU were added for coupling; after coupling was completed, the side chain protecting groups were removed under strong acid (92.5% TFA, 2.5% H2O, 2.5% TIS and 2.5% EDT) and reacted at room temperature for 2 h to remove the side chain protecting groups. The lysate was filtered to remove the lysate, and the peptides were washed and condensed multiple times with methanol to obtain the products WP1, WP2 and WP3 peptides. The crude products were identified by ESI mass spectrometry and purified by HPLC for subsequent experiments.

[0066] Mass spectrometry and liquid chromatography results of WP1, WP2 and WP3 indicate that the peptide probe has high purity and is correctly synthesized for further research.

[0067] Example 2

[0068] In this embodiment, the affinity interactions between WP1, WP2, and WP3 and CD137 protein were detected using the surface plasmon resonance (SPRi) method. The specific procedure is as follows:

[0069] 1 mg / mL of WP1, WP2 and WP3 peptides and 1×PBS buffer were spotted onto the chip and incubated overnight at 4°C under humid conditions; then washed with 10×PBS for 10 min; then washed with 1×PBS for 10 min; and finally washed twice with deionized water for 10 min each time.

[0070] The chip was immersed in 1×PBS containing 5% milk and incubated overnight at 4°C. It was then washed with 10×PBS for 10 min, followed by another 1×PBS wash for 10 min. Finally, it was washed twice with deionized water for 10 min each time, dried with nitrogen, and then mounted on the instrument (Plexera). HT surface plasmon resonance imaging system.

[0071] The mobile phase was sequentially passed through 1×PBS, 2×PBS, 0.78 μg / mL, 1.56 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL and 25 μg / mL human CD137 to purify the protein, and the SPRi signal was recorded and analyzed.

[0072] Depend on Figure 1 The results show that the SPRi signals of WP1, WP2, and WP3 gradually increase with increasing protein concentration, indicating that the WP1, WP2, and WP3 peptides prepared in Example 1 of this invention have a strong binding ability to CD137, with an affinity constant K. DThey are 4.31×10 -7 M, 8.09×10 -7 M and 8.16×10 -8 M has an affinity close to that of an antibody, but stronger than that of the previously reported CD137-targeting peptide (GGACIEEGQYCF), whose affinity constant is 1.37 × 10⁻⁶. -7 M indicates that the polypeptide provided by this invention can better target CD137-positive vascular endothelial cells and T cells as a probe for application research in related diseases.

[0073] Example 3

[0074] This embodiment detects the binding of WP1, WP2, and WP3 peptides to CD137-highly-expressing HUVEC cells using imaging techniques. The specific procedure is as follows:

[0075] Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM medium containing 10% fetal bovine serum at a concentration of 1×10⁻⁶. 5 Cells were cultured in round glass-bottomed culture dishes (35 mm) at a concentration of 1 μmol / L in a 37°C, 5% CO2 incubator for 24 h. The culture medium was then discarded. Three fluorescent peptides, WP1, WP2, and WP3, labeled with 1 μmol / L Hoechst 33342 and 50 μmol / L Rhodamine B, were added to each of the three cell types. After incubation at 4°C in the dark for 15 min, the cells were washed three times with pre-cooled 1×PBS. Fluorescence distribution in the cells was detected using a laser scanning confocal microscope (ZEISSLSM 710).

[0076] The results are as follows Figure 2 As shown, strong red fluorescence was observed on the HUVEC cell membranes with fluorescently labeled WP1, WP2, and WP3 peptides, indicating that the peptides bind to the cell membranes of positive cells. Furthermore, the specificity of these peptides is positively correlated with the expression level of the target protein, suggesting they could serve as targeting molecules for the diagnosis and detection of CD137-related diseases. This further validates... Figure 1 The reliability of SPRi data.

[0077] Example 4

[0078] This embodiment provides 68 The preparation method of Ga-DOTA-WP1 peptide and its application in in vivo tumor PET imaging specifically includes the following steps:

[0079] 1. Dissolve WP1 prepared in Example 2 in deionized water, rinse the germanium gallium (68Ge / 68Ga) generator (JSC Isotope) into the EP tube with 5 mL of 0.1 mol / L high-purity hydrochloric acid solution, collect 1 mL of the solution with the highest radioactivity content, and add 100 μL of 1.25 mol / L sodium acetate to adjust the pH of the mixture to 3.5-4.5;

[0080] 2. Add 30 μg of precursor WP1 to the mixture and mix thoroughly. Heat to 100℃ and maintain for 10 min. After the reaction is complete, cool the reaction solution to room temperature, add 4 mL of sterile water for injection, and filter through a sterile filter membrane (0.22 μm, 13 mm) into a sterile product bottle.

[0081] 3. Radioactivity detection utilizes a dedicated HPLC radioactivity detector. High-performance liquid chromatography (Waters Corporation, USA, 515 pump), a UV detector (486 type), and a radioactivity detector (EG&G BERTHOLD Corporation, USA) are used for detection. 68 The radiochemical purity of Ga-DOTA-WP1 is >99%, making it suitable as a PET probe for in vivo tumor imaging.

[0082] 4. H929 cells were subcutaneously injected into the hind legs of 6-8 week old male mice. When the tumor volume reached 100 mm, 3 Around 1 week after PD-1 antibody treatment, 10 MBq of [a specific drug / antibody] was injected via the tail vein. 68 Ga-DOTA-WP1 peptide probe. PET images were performed using an Inveon micro-PET / CT scanner (Siemens, Germany) at 30, 40, 50, 60, and 80 minutes post-injection. Figure 3 The results showed that when the prepared polypeptide imaging agent WP1 was injected via the tail vein into tumor-bearing mice with high CD137 expression, PET imaging revealed significant radioactivity accumulation in the tumor within 30 minutes of injection, with high uptake observed from 30 to 80 minutes. In contrast, no significant radioactivity accumulation was observed in the untreated tumor model. This demonstrates that the molecular probe of the present invention has CD137 targeting specificity and can achieve highly sensitive imaging of the activation and proliferation of immune cells during tumor immunotherapy.

[0083] Example 5

[0084] This embodiment provides a method for preparing the WP3@Fe3O4 / ICG dual-modal molecular imaging probe and its in vivo imaging detection in mice with atherosclerotic plaques, specifically including the following steps:

[0085] 1. First, 30 nm oil-phase Fe3O4 nanoparticles were synthesized via high-temperature thermal decomposition and dissolved in tetrahydrofuran to a concentration of 1 mg / mL. 150 mg of DP-PEG-Mal was weighed and added to 10 mL of the solution. The mixture was heated to 60 °C in a metal bath and shaken for 12 h. The nanoparticles were then precipitated with cyclohexane, washed three times with cyclohexane, and dried under vacuum at room temperature to remove n-hexane. The dried nanoparticles were dissolved in deionized water and ultrafiltered using a 100 kDa ultrafiltration tube. The nanoparticles were then washed three times with deionized water to remove unmodified PEG. The DP-PEG-Mal-modified Fe3O4 nanoparticles were dissolved at a concentration of 3 mg / mL in Tris-HCl buffer (1 mM, pH 7.4). Then, the CD137 targeting peptide (WP3) was added, and the mixture was stirred at 37 °C for 20 minutes. ICG-SH dye was added, and the mixture was stirred at 4°C for 48 h. Finally, it was centrifuged in a 100 kDa ultrafiltration tube and washed three times with PBS buffer (0.01 M, pH 7.4) to remove uncoupled CD137 peptide and ICG-SH, yielding the bimodal molecular probe WP3@Fe3O4 / ICG, shown in the figure as CD137@Fe3O4 / ICG. Its morphology and particle size were subsequently characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Figure 4 The obtained modal molecular probe WP3@Fe3O4 / ICG is shown to be spherical particles with good dispersibility and uniformity, with a particle size of about 50 nm.

[0086] 2. Analyze the expression of CD137 in vulnerable atherosclerotic plaques in humans. CD137 expression in atherosclerotic tissues of humans and mice was investigated. Human intima samples after carotid endarterectomy were paraffin-embedded and sectioned, then immunohistochemically stained with CD137 antibody for analysis. Figure 5 As shown, CD137 expression in vulnerable plaques was significantly higher than in stable plaques and normal arterial tissue. This invention also observed increased CD137 expression in vulnerable plaques in the mouse aorta and branches.

[0087] 3. The WP3@Fe3O4 / ICG dual-modal molecular imaging probe was used for high-sensitivity imaging of carotid artery plaques in mice. Eight-week-old male (approximately 20g) C57BL / 6J ApoE- / - mice were purchased from Beijing HFK Biotechnology Co., Ltd. (Beijing, China). Mice were fed a high-fat diet containing 15% fat and 1.25% cholesterol for 6 months before the experiment, while the control group was fed a standard laboratory diet. Each mouse was injected intravenously with 200 μL (2 μM) of the WP3@Fe3O4 / ICG probe, and anesthetized with isoflurane 30 min later. The mice were placed in a multispectral small animal in vivo imaging system IVIS Spectrum (PerkinElmer); the excitation wavelength was set to 805±10 nm; the emission wavelength to 835±17.5 nm; and the exposure time to 50 ms. Fluorescence detection and quantitative analysis were performed. Figure 6 As shown, the fluorescence signal intensity of plaques in the aorta and carotid arch of the high-fat feeding group was significantly higher than that of the control group, especially 1 hour after injection. The signal intensity of the plaque-bearing group in the high-fat feeding group was 1.1 × 10⁻⁶. 8 p / sec / cm 2 The signal gradually decreased over time, and essentially disappeared after 24 hours. This demonstrates that the molecular probe of this invention can target CD137 in plaque-inflamed tissue to achieve highly sensitive in vivo imaging of arterial plaques. The method of this invention can non-invasively identify high-risk atherosclerotic vulnerable plaques by targeting CD137.

[0088] In summary, this invention provides a polypeptide probe with the characteristic of targeting CD137-positive cells. Therefore, in practical applications, this targeted polypeptide probe can be used for tumor immunotherapy imaging to achieve non-invasive and visual detection of the immunotherapy response marker CD137. Furthermore, the development of this multimodal magnetic nanoprobe CD137@Fe3O4 / ICG can be used to detect vulnerable atherosclerotic plaques.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A CD137-targeting polypeptide, characterized in that, The amino acid sequence of the CD137-targeting polypeptide is shown in SEQ ID NO.

3.

2. A nucleic acid, characterized in that, The nucleic acid is used to encode the CD137-targeting polypeptide as described in claim 1.

3. A molecular probe, characterized in that, The molecular probe is obtained by fluorescent labeling and / or radioisotope labeling of the CD137-targeting peptide of claim 1.

4. The molecular probe according to claim 3, characterized in that, The fluorescent label is one or more of IRDye800CW, Cy7, rhodamine, or indocyanine green, and the radioactive isotope is... 131 I, 177 Lu、 64 Cu、 99 mTc, 18 F or 68 One or more of Ga.

5. The molecular probe according to claim 4, characterized in that, The fluorescent label and the radioisotope are used to label the CD137-targeting peptide using chelating agents DOTA, NOTA, DTPA, and HYNIC.

6. The use of the CD137-targeting polypeptide of claim 1, the nucleic acid of claim 2, or the molecular probe of any one of claims 3-5 in the preparation of a drug, characterized in that, The drug is used for the diagnosis of diseases related to CD137-positive vascular endothelial cells, T cells, and monocytes.

7. The application according to claim 6, characterized in that, The drug is used for the diagnosis of multiple myeloma or atherosclerosis.

8. The application according to claim 6 or 7, characterized in that, The drug also includes imaging agents.

9. The application according to claim 8, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

10. The use of the CD137-targeting polypeptide of claim 1, the nucleic acid of claim 2, or the molecular probe of any one of claims 3-5 in the preparation of a disease detection kit, characterized in that, The disease detection kit is used to detect diseases associated with CD137-positive vascular endothelial cells, T cells, and monocytes.

11. The application according to claim 10, characterized in that, The disease detection kit is used to detect multiple myeloma or atherosclerosis.

12. A test kit, characterized in that, The detection kit includes the CD137 targeting peptide of claim 1, or the nucleic acid of claim 2, or the molecular probe of any one of claims 3-5; The test kit is used to detect diseases associated with CD137-positive vascular endothelial cells, T cells, and monocytes.

13. The detection kit according to claim 12, characterized in that, The test kit is used to detect multiple myeloma or atherosclerosis.

14. A diagnostic drug, characterized in that, The diagnostic drug includes the CD137-targeting polypeptide of claim 1, or the nucleic acid of claim 2, or the molecular probe of any one of claims 3-5.

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