A PEG-G5.NH2-FITC-DOTA(Gd)-Monalizumab / IPH4301 nanoprobe and its application

Abstract

The present invention relates to a PEG-G5.NH2-FITC-DOTA(Gd)-Monalizumab / IPH4301 nanoprobe and an application thereof. The prepared nanoprobe is characterized, the binding ability of the nanomaterial and NK-92MI cells is analyzed by flow cytometry, MRI is used to image the NK-92MI cells labeled with the nanoprobe in vitro and the T1-weighted (T1WI) signal changes are analyzed, Western blot is used to detect the expression of apoptosis-related proteins in breast cancer MDA-MB-231 cells after co-culture, and Elisa The expression level of IFN-γ in the culture system was detected by the PEG-G5.NH2-FITC-DOTA(Gd)-Monalizumab / IPH4301 nanoprobe of the present invention was found to be able to bind to NK-92MI cells and to be imaged in vitro using 3.0TMRI. After being modified with Monalizumab and IPH4301 double antibodies, the nanoprobe promoted the release of IFN-γ by NK-92MI cells and enhanced its ability to kill MDA-MB-231 cells, providing an experimental basis for in vivo imaging and immunotherapy.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a PEG-G5.NH2-FITC-DOTA(Gd)-Monalizumab IPH4301 nanoprobe and application thereof. Background Art

[0002] Tumor immunotherapy is a biological treatment method that mobilizes the body's own immune system to kill and eliminate tumor cells. Tumor immunotherapy is widely used to block the signal pathway by using co-inhibitory molecules or ligand antagonists and other drugs to relieve the expression of programmed cell death 1 receptor (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in tumor patients. In addition, monoclonal antibody therapy targeting PD-1 / PD-L1 (programmed death-ligand 1) has made significant clinical progress in the treatment of various solid tumors. Recent studies have shown that natural killer cells (NKs), as the main effector cells in the innate immune system, play an important role in controlling the occurrence and development of tumors and are a very potential research object in tumor immunotherapy. NK cells perform immune surveillance on malignant transformed cells and maintain the body's homeostasis through the regulatory receptors related to killing function expressed on their surface, including activating receptors and inhibitory receptors. Among them, the activated receptor NKG2D and its corresponding ligand constitute a stress-induced danger detection system, which can directly respond to cancerous cells and play a decisive role in tumor immune surveillance.

[0003] NK cells are a type of effector lymphocyte that plays a vital role in the body's resistance to tumors. NK cells do not require prior sensitization and can promote immune responses by secreting cytokines and quickly dissolve target cells. [1] Clinical studies and animal experiments have found that NK cell dysfunction or NK cell damage can lead to an increased incidence of various cancers. [2] There are many receptors on the surface of NK cells, which can transmit various activation signals or inhibitory signals. The balance between them is strictly regulated to regulate related immune functions and maintain balance. [3]Molecular imaging visualizes cells or subcellular units at the molecular and anatomical levels through tissue-intrinsic properties or the use of specific molecular probes. It not only displays the physiological and pathophysiological processes of cells or subcellular units, but also characterizes and quantifies these processes through molecular imaging, thus achieving integrated tumor diagnosis and treatment. Therefore, it is extremely important to construct a probe that can bind to NK cells and can be used for in vivo imaging and immunotherapy.

[0004] Chinese patent CN111991564A, published on August 26, 2020, discloses a targeted nanoprobe bound to NK92 cells, and its preparation method and application. A Fe3O4-PEG-CD56 / Avastin@Ce6 nanoprobe targeting NK92 cells was synthesized, which is a superparamagnetic Fe3O4 nanoparticle surface covalently linked to an anti-CD56 antibody targeting NK cells and an anti-tumor drug bevacizumab, and then physically adsorbed photosensitizer Ce6 on the surface. The probe can be used for fluorescence and magnetic resonance dual-modal imaging, has good biocompatibility and stability, can be used for long-term monitoring of living bodies, and exhibits excellent anti-tumor effects and biosafety both in vivo and in vitro, laying the foundation for the study of the mechanism of action of NK cells and immunotherapy. However, there is no report on a PEG-G5.NH2-FITC-DOTA(Gd)-MonalizumabIPH4301 nanoprobe and its application of the present invention. Summary of the invention

[0005] The first object of the present invention is to provide a probe to address the deficiencies in the prior art.

[0006] The second object of the present invention is to provide uses of the probe.

[0007] To achieve the above first purpose, the technical solution adopted by the present invention is:

[0008] A PEG-G5.NH2-FITC-DOTA(Gd)-Monalizumab / IPH4301 nanoprobe is prepared by the following steps:

[0009] a) G5.NH2 was dissolved in 1 ml of DMSO, and DOTA-NHS was dissolved in 4 ml of ultrapure water. The mixture was added dropwise to the G5.NH2 solution under stirring. After reacting for 24 hours under magnetic stirring at room temperature, Gd(NO3)3·6H2O dissolved in 1 ml of water was added. The mixture was stirred and reacted for 24 hours at room temperature to chelate Gd(III) ions. The mixed reactants were dialyzed in distilled water with a cellulose dialysis membrane for 1 day to obtain a G5.NH2-DOTA(Gd) solution;

[0010] b) PEG 2K-COOH was dissolved in 5 ml of DMSO, EDC dissolved in 2 ml of DMSO was added dropwise, and the reaction was carried out under magnetic stirring at room temperature for half an hour, and NHS-sulfo dissolved in 2 ml of DMSO was added, and the activation was continued under stirring at room temperature for 3 hours;

[0011] c) The activated PEG 2K -COOH was added dropwise to G5.NH2-DOTA(Gd) obtained in step a), and the mixture was stirred under magnetic stirring at room temperature for 24 h. The mixed reactants were dialyzed in distilled water for 1 day using a cellulose dialysis membrane to obtain PEG-G5.NH2-DOTA(Gd);

[0012] d) FITC was dissolved in 1 ml of DMSO and added dropwise to the PEG-G5.NH2-DOTA(Gd) obtained above. The two were then mixed and reacted with magnetic stirring at room temperature for 24 hours. The mixed reactants were dialyzed in distilled water with a cellulose dialysis membrane for 24 hours to obtain targeted nanoprobes (NPs) PEG-G5.NH2-DOTA(Gd)-FITC.

[0013] e) SMCC was dissolved in 1 ml of DMF, and then diluted to 1 mg / ml by adding 0.1 M phosphate buffer. The above mixture was added to Monalizumab and IPH4301 antibody, respectively, and reacted at room temperature for 1 hour, and then the obtained product was purified by ultrafiltration tube.

[0014] f) Using Traut's reagent to introduce thiol groups on the surface of the targeted nanoprobe, 10 times the number of primary amine groups of Traut's reagent was dissolved in 0.1M phosphate buffer. The reaction was continued for 1 hour under the conditions of pH = 8.0 and N2, and the product was purified by ultrafiltration tube. The activated Monalizumab and IPH4301 monoclonal antibodies were reacted with the modified NPs at a molar ratio of 1:100 to obtain the final product NPS-Monalizumab / IPH4301.

[0015] As a preferred example, the amounts of G5.NH2, DOTA-NHS and Gd(NO3)3·6H2O added in the preparation step a) are 28.8 mg, 15.24 mg and 27.1 mg respectively.

[0016] As a preferred example, in the preparation step b), the amounts of PEG2K-COOH, EDC and NHS-sulfo added are 50 mg, 2.9 mg and 3.3 mg respectively.

[0017] As a preferred example, the amount of FITC added in the preparation step d) is 2.4 mg.

[0018] As a preferred example, it is characterized in that the phosphate buffer in the preparation steps e) and f) has a pH of 7.6 and contains 5 mM EDTA.

[0019] As a preferred example, the specification of the ultrafiltration tube in the preparation step e) is MW=30000, and the specification of the ultrafiltration tube in the preparation step f) is MW=3000.

[0020] As a preferred example, the nanoprobe can bind to NK-92MI cells

[0021] To achieve the above second purpose, the technical solution adopted by the present invention is:

[0022] Use of any of the above-mentioned nanoprobes in the preparation of cell imaging probes.

[0023] Use of any of the above-mentioned nanoprobes in the preparation of anti-tumor drugs.

[0024] The present invention has the advantages that:

[0025] 1. This study designed a nanoprobe PEG-G5.NH2-FITC-DOTA(Gd)-Monalizumab / IPH4301 that binds to NK-92MI cells. The experimental results showed that different probe concentrations in the experiment had no significant effect on the growth activity of NK-92MI cells, indicating that the prepared targeted probe has low cytotoxicity.

[0026] 2. NPs-Monalizumab / IPH4301 has limited ability to kill tumor cells at the cellular level and promotes apoptosis of target cells.

[0027] 3. The nanoprobe PEG-G5.NH2-FITC-DOTA(Gd)-Monalizumab / IPH4301 that can mark immune cells successfully synthesized by the present invention can perform MRI imaging at the cellular level and kill tumor cells, providing a basis for subsequent research on its use in in vivo experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Attached Figure 1 This is the transmission electron microscopy image and particle size distribution diagram of NPs-Monalizumab / IPH4301.

[0029] Attached Figure 2 Flow cytometry was used to quantitatively analyze the uptake of NPs-Monalizumab / IPH4301 by NK-92MI cells.

[0030] Attached Figure 3MR imaging of NK-92MI labeled with NPs-Monalizumab / IPH4301, *p<0.05, **p<0.01, ***p<0.001.

[0031] Attached Figure 4 Western Blot was used to detect the expression of Cleaved-caspase3, Bax, Bcl-2 and other proteins, *p<0.05, **p<0.01, ***p<0.001.

[0032] Attached Figure 5 Elisa was used to detect the content of IFN-γ, *p<0.05, **p<0.01, ***p<0.001 DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0034] Example 1 Preparation of PEG-G5.NH2-FITC-DOTA(Gd)-Monalizumab / IPH4301 Nanoprobe

[0035] 1. Materials and Methods

[0036] 1.1 Materials

[0037] Reagents: fifth-generation polyamidoamine dendrimer (G5.NH2, Dendritech, USA); fluorescein isothiocyanate (FITC, Aladdin); 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, Aladdin); N-hydroxysuccinimide (NHS-sulfo, Aladdin); gadolinium nitrate hexahydrate (Gd(NO3)3·6H2O, J&K Technology); carboxyl-polyethylene glycol (PEG2K-COOH, Xi'an Ruixi Biotechnology Co., Ltd.); 2,2',2"-( 10-(2-(2,5-dioxopyrrolidine-1-oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-tri)triacetic acid (DOTA-NHS-ester, Xi'an Ruixi Biotechnology Co., Ltd.); 2-Iminothiolane hydrochloride (Traut'sreagent, Xi'an Ruixi Biotechnology Co., Ltd.); Monalizumab (Wuhan Costan Biotechnology Co., Ltd.); ADCC-enhanced anti-MICA / B antibody (IPH4301, Wuhan Keshen Biotechnology Co., Ltd. Stan Biotechnology Co., Ltd.); ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA, Sigma-Aldrich); dimethyl sulfoxide (DMSO, Sigma-Aldrich); N,N-dimethylformamide (DMF, Sigma-Aldrich); 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC, Shanghai MacLean Biochemical Technology Co., Ltd.); α-MEM medium (Gibco); fetal bovine serum (FBS, Gibco) ); horse serum (Gibco); penicillin-streptomycin solution (New Saimei Biotechnology Co., Ltd.); trypsin (New Saimei Biotechnology Co., Ltd.); RIPA lysis buffer (Biyuntian); protease phosphatase inhibitors (Biyuntian); protein loading buffer (Biyuntian); Cleaved-caspase3 antibody (Affinity); Bax antibody (Affinity); precast gel (Tannon); Bcl-2 antibody (Affinity); IFN-γ Elisa kit (Lianke Company).

[0038] Instruments: UV-visible spectrophotometer (PerkinElmer, USA), JEOL2010F transmission electron microscope (TEM, JEOL Ltd.), flow cytometer (BD), and microplate reader (BioTek).

[0039] 1.2 Synthesis of targeted nanoprobes

[0040] G5.NH2 (28.8 mg) was dissolved in 1 ml of DMSO, and DOTA-NHS (15.24 mg) was dissolved in 4 ml of ultrapure water. The mixture was added dropwise to the G5.NH2 solution with stirring. After reacting with magnetic stirring at room temperature for 24 hours, Gd(NO3)3·6H2O (27.1 mg) dissolved in 1 ml of water was added to the above reaction system. The mixture was stirred continuously at room temperature for 24 hours to chelate Gd(III) ions. The mixed reactants were dialyzed in distilled water with a cellulose dialysis membrane for 1 day to obtain a G5.NH2-DOTA(Gd) solution.

[0041] PEG2K-COOH (50 mg) was dissolved in 5 ml of DMSO, EDC (2.9 mg) dissolved in 2 ml of DMSO was added dropwise, and the mixture was stirred under magnetic stirring for half an hour at room temperature. Subsequently, NHS-sulfo (3.3 mg) dissolved in 2 ml of DMSO was added thereto, and the mixture was stirred and activated for 3 hours at room temperature.

[0042] The activated PEG2K-COOH was added dropwise to the G5.NH2-DOTA(Gd) obtained by the above reaction, and the mixture was stirred under magnetic stirring at room temperature for 24 hours. The mixed reactants were dialyzed in distilled water for 1 day using a cellulose dialysis membrane to obtain PEG-G5.NH2-DOTA(Gd).

[0043] FITC (2.4 mg) was dissolved in 1 ml of DMSO and added dropwise to the PEG-G5.NH2-DOTA(Gd) obtained above. The two were then mixed and reacted with magnetic stirring at room temperature for 24 hours. The mixed reactants were dialyzed in distilled water for 1 day using a cellulose dialysis membrane to obtain targeted nanoprobes (NPs) PEG-G5.NH2-DOTA(Gd)-FITC.

[0044] 1.3 Synthesis of Monalizumab and IPH4301 Antibody-Modified NPs

[0045] SMCC was dissolved in 1 ml of DMF, and then 0.1 M phosphate buffer (pH 7.6, containing 5 mM EDTA) was added to dilute to 1 mg / ml. The above mixture was added to Monalizumab and IPH4301 antibodies respectively, and reacted at room temperature for 1 hour, and then the obtained product was purified using an ultrafiltration tube (MW = 30000). Traut's reagent was used to introduce thiol groups on the surface of the targeted nanoprobe, and Traut's reagent with 10 times the number of primary amine groups was dissolved in 0.1 M phosphate buffer. The reaction was continued for 1 hour under the conditions of pH = 8.0 and N2, and the product was purified by an ultrafiltration tube (MW = 3000). The activated Monalizumab and IPH4301 monoclonal antibodies were reacted with the modified NPs at a molar ratio of 1:100 to obtain the final product NPS-Monalizumab / IPH4301.

[0046] 1.4 Material morphology

[0047] The morphology of the nanomaterials was observed under TEM and their particle size was measured, which were then analyzed using ImageJ software.

[0048] 1.5 Cell culture and probe uptake

[0049] The MDA-MB-231 cells used in this experiment were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the NK-92MI cells were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. NK92MI cells were cultured in α-MEM medium containing 12.5% ​​FBS and 12.5% ​​horse serum, and were placed in an incubator at 37°C and 5% CO2 for routine culture. MDA-MB-231 cells were cultured in L15 medium containing 10% FBS, and were placed in an incubator at 37°C for routine culture. NK-92MI cells in the logarithmic growth phase were taken and cultured at 2×10 5 / well were plated in a 24-well plate, and 1 ml of the corresponding culture medium was added and cultured for 24 h at 37 ° C and 5% CO2. In order to determine whether NPs-Monalizumab / IPH4301 can bind to NK-92MI cells, they were collected after 8 h, washed three times with PBS, and then quantitatively detected by flow cytometry, with 10,000 cells collected for each sample.

[0050] 1.6 In vivo and in vitro MR imaging analysis

[0051] NK-92MI cells were co-cultured with 0, 5, 10, and 15 μM NPs-Monalizumab / IPH4301 for 8 h, and then dissolved in 0.5 mL of 1% agarose in an EP tube for MRI scanning after centrifugation. 3.0 T Philips MRI scanning parameters: T1 weighted (T1WI), echo time = 8.0 ms, repetition time = 609.0 ms, slice thickness 3 mm, field of view 130 mm × 130 mm, number of excitations 1. After the scan, the region of interest (ROI) of the same cross section and size was selected for each sample.

[0052] 1.7 Protein extraction and Western blot detection

[0053] 10μM NPs, NPs-Monalizumab / IPH4301 were co-cultured with NK-92MI cells for 8h, and then the treated NK-92MI cells were co-cultured with breast cancer MDA-MB-231 cells at a ratio of 10:1. After 24 hours of co-culture, the culture medium and suspended cells were aspirated, and then protein lysis buffer, protease inhibitors and phosphatase inhibitors were added, and the cells were placed on ice for 30min to promote full lysis. The liquid was then transferred to an EP tube and centrifuged at 12000r / min. The supernatant was taken to detect the protein concentration by the BCA method, and the protein loading buffer was added according to the proportion and boiled at 100℃ for 15 minutes, and finally stored in a -80℃ refrigerator. Take 30μg protein supernatant sample, separate by 20% polyacrylamide gel electrophoresis and wet transfer to 0.45μm PVDF membrane. After transfer, block with 5% skim milk at room temperature for 2h, add 1:1000 Cleaved-caspase3, Bax, Bcl-2 primary antibody in 4℃ incubator overnight, and then rinse with TBST buffer. Add 1:10000 HRP-labeled goat anti-rabbit secondary antibody incubator at room temperature for 1h, and then rinse with TBST buffer. Finally, develop with ECL chemiluminescent solution, and measure the expression of Cleaved-caspase3, Bax, and Bcl-2.

[0054] 1.8 Cytokine Elisa assay

[0055] Take the culture medium after NK-92MI cells and MDA-MB-231 cells are co-cultured, then centrifuge at 300g and take the supernatant. According to the IFN-γElisa kit elisa method, the specific steps are as follows: take out the ELISA plate, incubate for 2 hours according to the concentration gradient and sample wells (100ul each), wash the plate 6 times and pat dry. Then add 100ul of enzyme-labeled antigen to each well, incubate for 1 hour, wash the plate 6 times and pat dry, add substrate solution to the well, and add stop solution after 30 minutes of reaction. Read the absorbance value of each well by an ELISA reader.

[0056] 2 Statistical analysis

[0057] Graphpad prism9 software was used for statistical analysis. The measured data that met the normal distribution were expressed as mean ± standard deviation. One-way analysis of variance was used for comparison among multiple groups, and P<0.05 was considered statistically significant.

[0058] 3 Results

[0059] 3.1 Morphology and particle size of PEG-G5.NH2-FITC-DOTA(Gd)-Monalizumab / IPH4301

[0060] TEM shows that the microstructure of all nanoparticles is similar to a spherical structure ( Figure 1 ), the average diameter of NPs-Monalizumab / IPH4301 nanoparticles detected by ImageJ software was 102.28±19.42nm. 3.2 Cell endocytosis and cell suspension MR imaging analysis

[0061] The results of flow cytometry showed that the signal intensity of 0, 5, 10, 15, and 20 μg / ml NPs-Monalizumab / IPH4301 solution in the FL1 (FITC) channel increased with increasing concentration. This result showed that as the concentration increased, the number of nanoprobes bound to NK-92MI cells increased ( Figure 2 ). The T1WI scan of NK-92MI cells labeled with NPs-Monalizumab / IPH4301 is shown in Figure X. As the concentration of the nanoprobe increases, the corresponding T1WI signal value gradually increases. The inter-group comparison shows that the results are different ( Figure 3 , p<0.05).

[0062] 3.3 Western blot to detect changes in apoptosis-related proteins

[0063] Western Blot results showed that the expression of Cleaved-caspase3 and Bax proteins in MDA-MB-231 cells co-cultured with NK cells treated with NPs-Monalizumab / IPH4301 was significantly upregulated, and the expression of Bcl-2 protein was significantly downregulated, and the difference was statistically significant ( Figure 4 , p<0.05), indicating that the tumor-killing ability of NK cells treated with NPs-Monalizumab / IPH4301 increased, promoting tumor cell apoptosis.

[0064] 2.4 Cytokine Elisa assay

[0065] The results of cytokine Elisa assay showed that the content of cytokine IFN-γ in the co-culture system of NK cells and MDA-MB-231 cells treated with NPs-Monalizumab / IPH4301 was much higher than that in the control group, and the difference was statistically significant ( Figure 5 , p<0.05), indicating that NPs-Monalizumab / IPH4301 promoted the release of IFN-γ from NK-92MI cells, suggesting that NPs-Monalizumab / IPH4301 promoted the cytotoxic effect mediated by NK-92 cells.

[0066] In summary, the results show that different probe concentrations have no significant effect on the growth activity of NK-92MI cells, suggesting that the prepared targeted probe has low cytotoxicity. As the probe concentration increases, the binding of the probe to NK-92MI cells gradually increases. NK-92MI cells labeled with probes of different concentrations were resuspended in agarose for magnetic resonance scanning, and the results showed that the MRI signal intensity gradually increased, suggesting its targeted molecular targeted imaging capability. Subsequently, after the co-culture experiment of MDA-MB-231 cells and NK-92MI cells, it was found that NPs-Monalizumab / IPH4301 had limited ability to kill tumor cells at the cellular level and promoted target cell apoptosis. The nanoprobe of the present invention can be used for MRI imaging and killing tumor cells at the cellular level, providing a basis for subsequent research on its use in vivo experiments.

[0067] References

[0068] [1]WU SY, FU T, JIANG YZ, et al. Natural killer cells in cancerbiology and therapy[J]. Mol Cancer, 2020, 19(1):120.

[0069] [2]XIE G,DONGH,LIANGY,etal.CAR-NKcells:Apromisingcellularimmunotherapyforcancer[J].EBioMedicine,2020,59:102975.

[0070] [3]MYERS JA,MILLER J S. Exploring the NK cell platform for cancerimmunotherapy[J]. Nat Rev ClinOncol, 2021,18(2):85-100.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A PEG-G5.NH2-FITC-DOTA(Gd)-Monalizumab / IPH4301 nanoprobe, characterized in that: The nanoprobe is prepared by the following steps: a) G5.NH2 was dissolved in 1 ml of DMSO, and DOTA-NHS was dissolved in 4 ml of ultrapure water. The mixture was added dropwise to the G5.NH2 solution under stirring. After reacting for 24 hours under magnetic stirring at room temperature, Gd(NO3)3·6H2O dissolved in 1 ml of water was added. The mixture was stirred and reacted for 24 hours at room temperature to chelate Gd(III) ions. The mixed reactants were dialyzed in distilled water with a cellulose dialysis membrane for 1 day to obtain a G5.NH2-DOTA(Gd) solution; b) PEG 2K -COOH was dissolved in 5 ml of DMSO, EDC dissolved in 2 ml of DMSO was added dropwise, and the reaction was carried out under magnetic stirring at room temperature for half an hour, and NHS-sulfo dissolved in 2 ml of ultrapure water was added, and the activation was continued by stirring at room temperature for 3 hours; c) adding the activated PEG2K-COOH dropwise to the G5.NH2-DOTA(Gd) obtained in step a), and reacting under magnetic stirring at room temperature for 24 hours. The mixed reactants were dialyzed in distilled water with a cellulose dialysis membrane for 1 day to obtain PEG-G5.NH2-DOTA(Gd); d) FITC was dissolved in 1 ml of DMSO, and added dropwise to the PEG-G5.NH2-DOTA(Gd) obtained above, and then the two were mixed and placed under magnetic stirring at room temperature for 24 hours, and the mixed reactants were dialyzed in distilled water for 24 hours using a cellulose dialysis membrane to obtain targeted nanoprobes (NPs) PEG-G5.NH2-DOTA(Gd)-FITC; e) Dissolve SMCC in 1 ml of DMF, then add 0.1 M phosphate buffer to dilute to 1 mg / ml. Add the above mixture to Monalizumab and IPH4301 antibody, respectively, react at room temperature for 1 hour, and then purify the obtained product using an ultrafiltration tube; f) Using Traut's reagent to introduce thiol groups on the surface of the targeted nanoprobe, 10 times the number of primary amine groups of Traut's reagent was dissolved in 0.1M phosphate buffer. The reaction was continued for 1 hour under the conditions of pH = 8.0 and N2, and the product was purified by ultrafiltration tube. The activated Monalizumab and IPH4301 monoclonal antibodies were reacted with the modified NPs at a molar ratio of 1:100 to obtain the final product NPS-Monalizumab / IPH4301.

2. The nanoprobe according to claim 1, characterized in that The amounts of G5.NH2, DOTA-NHS and Gd(NO3)3·6H2O added in the preparation step a) are 28.8 mg, 15.24 mg and 27.1 mg respectively.

3. The nanoprobe according to claim 1, characterized in that: In the preparation step b), the amounts of PEG2K-COOH, EDC and NHS-sulfo added are 50 mg, 2.9 mg and 3.3 mg respectively.

4. The nanoprobe according to claim 1, characterized in that: The amount of FITC added in the preparation step d) is 2.4 mg.

5. The nanoprobe according to claim 1, characterized in that: The phosphate buffer in the preparation steps e) and f) has a pH of 7.6 and contains 5 mM EDTA.

6. The nanoprobe according to claim 1, characterized in that: The specification of the ultrafiltration tube in the preparation step e) is MW=30000, and the specification of the ultrafiltration tube in the preparation step f) is MW=3000.

7. The nanoprobe according to claim 1, characterized in that: The nanoprobe can be combined with NK-92MI cells.

8. Use of the nanoprobe according to any one of claims 1 to 7 in the preparation of cell imaging probes.

9. Use of the nanoprobe according to any one of claims 1 to 7 in the preparation of anti-tumor drugs.

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