A dual-modal magnetic particle fluorescence probe and its uses

Abstract

The present application provides a dual-modal magnetic nanoparticle fluorescence probe, which comprises a CLDN18.2 monoclonal antibody, a near-infrared fluorescent molecule, and magnetic nanoparticles. The present application also provides a preparation method and uses of the dual-modal magnetic nanoparticle fluorescence probe. The dual-modal magnetic nanoparticle fluorescence probe can be used for the treatment and monitoring of tumors with high CLDN18.2 expression levels, and has a higher degree of enrichment in tumors, especially gastric cancer tissues, and can stably exist for a longer period of time.

Description

Technical Field

[0001] This application relates to the field of tumor plasmids and detection, and particularly to a dual-modal magnetic particle fluorescence probe for treating or detecting tumors and its uses. Background Art

[0002] Gastric cancer is a type of cancer with relatively high incidence and mortality rates. Due to the lack of typical symptoms in the early stage of gastric cancer patients, 70% of gastric cancer patients are already in the advanced stage when they seek medical treatment. Among patients with advanced or metastatic gastric cancer or adenocarcinoma of the gastroesophageal junction, the median overall survival is no more than 10 months. Although human epidermal growth factor receptor 2 (HER-2) targeted therapy and immune checkpoint inhibitors have brought benefits to specific populations, the current population that benefits from the discovered targets is limited. It is imperative to search for other targets in advanced gastric cancer. Claudin18 splice variant 2 (CLDN18.2) is a tetraspanin expressed at tight junctions, which is strictly confined to the basal cells of gastric mucosal epithelium in normal gastric tissue and can be stably expressed during tumorigenesis, having the translational value of diagnosis and precision therapy. Currently, there is no disclosure of the visualization and treatment of nanomaterials related to this molecule. Summary of the Invention

[0003] Through immunohistochemical analysis, it was found in this application that CLDN18.2 has a high expression level in gastric cancer and is negatively correlated with effector T cells and programmed death ligand, suggesting that the overexpression of CLDN18.2 is related to the immunosuppressive state. Therefore, a novel magnetic particle-fluorescence dual-modal probe targeting CLDN18.2 was constructed and its characterization was detected. The targeting and safety of the probe were verified in gastric cancer cell lines overexpressing CLDN18.2. Subsequently, animal experiments were carried out using tumor-bearing mice overexpressing CLDN18.2, and it was monitored by fluorescence and magnetic particle imaging techniques that the probe can specifically target tumors overexpressing CLDN18.2 and has almost no toxic side effects on the body within a certain dose range. Using this probe can achieve image-guided precise magnetic hyperthermia treatment, activate the tumor immune microenvironment that can be manipulated under spatio-temporal conditions, and ultimately enhance the efficacy of immunotherapy.

[0004] The specific technical solutions of this application are as follows:

[0005] 1. A dual-modal magnetic particle fluorescence probe, which comprises a CLDN18.2 monoclonal antibody, a near-infrared fluorescent molecule, and magnetic nanoparticles.

[0006] 2. The dual-modal magnetic particle fluorescence probe according to item 1, wherein the near-infrared fluorescent molecule and the CLDN18.2 monoclonal antibody are respectively connected to the magnetic nanoparticles.

[0007] 3. The dual-modal magnetic particle fluorescence probe according to item 1 or 2, wherein the near-infrared fluorescent molecule is connected to the magnetic nanoparticles through an amide bond.

[0008] 4. The dual-modal magnetic particle fluorescence probe according to any one of items 1 to 3, wherein the CLDN18.2 monoclonal antibody is connected to the magnetic nanoparticles through an amide bond.

[0009] 5. The dual-modal magnetic particle fluorescence probe according to any one of items 1 to 4, wherein the near-infrared fluorescent molecule is indocyanine green ICG.

[0010] 6. The dual-modal magnetic particle fluorescence probe according to any one of items 1 to 5, wherein the magnetic nanoparticles are carboxyl-modified superparamagnetic nanoparticles;

[0011] Preferably, the saturation magnetization intensity of the magnetic nanoparticles is above 65 emu / g;

[0012] Preferably, the diameter of the magnetic nanoparticles is 1 to 100 nm, more preferably 10 to 40 nm;

[0013] Preferably, the magnetic nanoparticles are MagBeads, Perimag, Synomag or Vivotrax superparamagnetic nanoparticles;

[0014] Preferably, the magnetic nanoparticles are magnetite magnetic nanoparticles or iron oxide magnetic nanoparticles;

[0015] More preferably, the magnetic nanoparticles are Mag3200.

[0016] 7. The dual-modal magnetic particle fluorescence probe according to any one of items 1 to 6, wherein the CLDN18.2 monoclonal antibody is a full-length antibody.

[0017] 8. The dual-modal magnetic particle fluorescence probe according to item 7, wherein the full-length antibody is IgG1.

[0018] 9. The dual-modal magnetic particle fluorescence probe according to any one of items 1 to 8, wherein the CLDN18.2 monoclonal antibody is a humanized antibody, a murine antibody or a chimeric antibody.

[0019] 10. The dual-modal magnetic particle fluorescence probe according to any one of items 1 to 9, wherein the CLDN18.2 is human CLDN18.2 or murine CLDN18.2.

[0020] 11. The dual-modal magnetic particle fluorescence probe according to any one of items 1 to 10, wherein the CLDN18.2 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2.

[0021] 12. The dual-modal magnetic particle fluorescence probe according to any one of items 1 to 11, wherein the mass ratio of the magnetic nanoparticles, the CLDN18.2 monoclonal antibody, and the near-infrared fluorescent molecule is 1: 0.02 to 1: 0.001 to 0.05.

[0022] 13. A method for constructing the dual-modal magnetic particle fluorescence probe according to any one of items 1 to 12, comprising the following steps:

[0023] Co-incubating magnetic nanoparticles with a carboxyl terminus with the CLDN18.2 monoclonal antibody; and

[0024] In a dimethylformamide solvent, co-incubating magnetic nanoparticles with a carboxyl terminus with a near-infrared fluorescent molecule having an N-hydroxysuccinimide group.

[0025] 14. The method according to item 13, wherein the pH during the co-incubation of the magnetic nanoparticles with the near-infrared fluorescent molecule is 4 to 6.

[0026] 15. The method according to item 13 or 14, wherein:

[0027] The mass ratio of the magnetic nanoparticles with a carboxyl terminus to the CLDN18.2 monoclonal antibody is 1: 0.02 to 1;

[0028] The mass ratio of the magnetic nanoparticles with a carboxyl terminus to the near-infrared fluorescent molecule having an N-hydroxysuccinimide group is 1: 0.001 to 0.05; or

[0029] The mass ratio of the magnetic nanoparticles with a carboxyl terminus, the CLDN18.2 monoclonal antibody, and the near-infrared fluorescent molecule having an N-hydroxysuccinimide group is 1: 0.02 to 1: 0.001 to 0.05.

[0030] 16. Use of the dual-modal magnetic particle fluorescence probe according to any one of items 1 to 12 for the preparation of a drug for treating tumors.

[0031] 17. Use of the dual-modal magnetic particle fluorescence probe according to any one of items 1 to 12 for the preparation of a reagent for detecting tumors.

[0032] 18. The use according to item 16 or 17, wherein the tumor is gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, breast cancer or lung cancer.

[0033] 19. The use according to item 16 or 17, wherein the dual-modal magnetic particle fluorescence probe is administered to a subject in need by intravenous injection or intratumoral injection.

[0034] 20. The use according to item 16, wherein magnetic hyperthermia is used to treat the tumor by applying an alternating magnetic field to the tumor site enriched with the dual-modal magnetic particle fluorescence probe to generate a thermal effect.

[0035] 21. The use according to item 20, wherein the magnetic hyperthermia is administered simultaneously with an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0036] 22. The use according to item 17, wherein the detection uses fluorescence imaging technology and magnetic particle imaging technology.

[0037] Advantages of the invention

[0038] In response to the major need to improve the efficiency of gastric cancer diagnosis and treatment, this application proposes to use MPI imaging, fluorescence imaging, and magnetic hyperthermia as the main technical means, targeting the key molecule CLDN18.2 in the process of gastric cancer evolution. MPI and fluorescence imaging are used to visually label the migration process of tumor cells of the key molecule, and the basic characteristics of the functional changes of the key molecule are studied. Alternating electric field magnetic hyperthermia treatment is used to provide a new treatment option for people with low response rates to gastric cancer immunotherapy.

[0039] The preclinical MPI imaging technology adopted in this application has the advantages of high spatial resolution and high temporal resolution. The imaging depth is not limited, the anatomical structure and background tissue are not shown, and it can directly detect nanoparticle tracers at any time and space in the body, enabling in-depth understanding of diseases from the organ, cell, and molecular levels. The adoption of the above technologies can ensure rapid, real-time, and dynamic monitoring of the evolution process of gastric cancer peritoneal metastasis while ensuring the accuracy of quantifying the functions of key molecules during the evolution process. Brief description of the drawings

[0040] Figure 1A It shows that CLDN18.2 is highly expressed in immunohistochemically stained tumors.

[0041] Figure 1B From left to right are immunohistochemical staining diagrams of gastric cancer tissues in the CLDN18.2 negative group (-), CLDN18.2 weakly positive group (+), CLDN18.2 positive group (++), and CLDN18.2 strongly positive group (+++). Among them, the lower diagram of each immunohistochemical staining diagram is an enlarged 16-fold effect diagram of the box in the upper diagram; Figure 1CThe figure shows the proportion of patients in each group of CLDN18.2 negative group (-), CLDN18.2 weakly positive group (+), CLDN18.2 positive group (++), and CLDN18.2 strongly positive group (+++).

[0042] Figure 1D Show the relationship between CLDN18.2 expression and prognosis in stage I-IIIa gastric cancer in the TCGA database; Figure 1E Show the relationship between CLDN18.2 expression and prognosis in stage I-II gastric cancer in the PKUCH immunohistochemistry data.

[0043] Figure 1F (a)-(i) respectively show the correlations between immunohistochemical CLDN18.2 and molecules such as Lauren classification, differentiation, TNM stage, PD-L1, CD3, HER2, ABCC2, ERCC1, LYZ, etc.

[0044] Figure 1G and Figure 1H respectively show the TIDE and TIMER scores of CLDN18 in gastric cancer in the TCGA database. Among them, Figure 1H for each type of cell, the left side represents the CLDN18.2 low-expression group, and the right side represents the CLDN18.2 high-expression group.

[0045] Figure 2A Show the schematic synthesis diagram and chemical structure diagram of the bimodal probe targeting CLDN18.2.

[0046] Figure 2B Show that the particle size of the probe detected by DLS is about 100 nm.

[0047] Figure 2C Show that the surface potential of the probe detected by Zeta is about -20 mV.

[0048] Figure 2D Show that the peak of ICG is detected by UV at 750 - 800 nm.

[0049] Figure 2E and Figure 2F respectively show the particle size distribution of the probe detected by TEM before and after coupling.

[0050] Figure 2G Show that the saturation magnetization intensity of the probe detected by VSM is 14.88 emu / g, and the probe has coercivity and superparamagnetism.

[0051] Figure 2H (a) and (b) respectively show the concentration-signal curve and fluorescence imaging diagram of the fluorescence imaging of the probe.

[0052] Figure 2I(a) and (b) show the concentration-signal curve and magnetic particle imaging diagram of the probe, respectively.

[0053] Figure 3A The expression of CLDN18.2 in gastric cancer and normal gastric tissue cell lines is shown.

[0054] Figure 3B , Figure 3C , Figure 3D The overexpression results of CLDN18.2 in AGS and MFC cell lines detected by Western blot are shown, where "+" represents overexpression and "-" represents no overexpression.

[0055] Figure 3E and Figure 3F Shows the overexpression efficiency of CLDN18.2 in AGS and MFC cell lines detected by flow cytometry; Figure 3E The curves on the left, middle, and right represent the AGS blank control group, AGS-AF488 group, and AGS CLDN18.2 - AF488 group; Figure 3F The curves on the left, middle, and right represent the MFC blank control group, MFC-AF488 group, and MFC CLDN18.2 -AF488 group.

[0056] Figure 3G and Figure 3H show Cell viability after 48 h of incubation with the dual-modality targeting probe 0-50 μ / ml.

[0057] Figure 3I , Figure 3J , Figure 3K , Figure 3L Results showing the targeting and uptake capabilities of the probes detected by confocal laser scanning.

[0058] Figure 4A Display 615 mouse MFC CLDN18.2 Fluorescence images of the MFC tumor-bearing model 48 hours after tail vein injection of dual-modality CLDN18.2 targeting probe and IgG control probe.

[0059] Figure 4B Shown are fluorescence imaging images of in vitro organs and tumors from 615 mice 48 hours after targeted and control group probes.

[0060] Figure 4C Display 615 mouse MFC CLDN18.2 Fluorescence intensity of dual-modality CLDN18.2 targeting probe and IgG control probe in the tail vein of MFC tumor-bearing model within 48 hours; Among them, for each time point, the left side represents MFC CLDN18.2+SPIO@1D5 group, with MFC indicated in the middle CLDN18.2 +SPIO@IgG group, with MFC + SPIO@1D5 group indicated on the right.

[0061] Figure 4D Showing the ex vivo fluorescence imaging of tumors in 615 mice after treatment with targeted and control probes for 48 h; Figure 4E Showing the ex vivo fluorescence intensity of tumors in 615 mice after treatment with targeted and control probes for 48 h.

[0062] Figure 4F Showing the expression level of CLDN18.2 in the PDX model;

[0063] Figure 4G Showing the fluorescence images of the PDX model after intravenous injection of dual - modal CLDN18.2 - targeted probe and IgG control probe via the tail vein for 4 h; Figure 4H 、 Figure 4I Showing the curve of the signal - to - background ratio of the PDX model after intravenous injection of dual - modal CLDN18.2 - targeted probe and IgG control probe via the tail vein over time; in the attached drawings of this application, P1 represents the adjacent cancer area with low expression of CLDN18.2 on the left side of the mouse, and P2 represents the tumor area with high expression of CLDN18.2 on the right side of the mouse.

[0064] Figure 4J 、 Figure 4K Showing the ex vivo fluorescence imaging of organs and tumors in the PDX model after treatment with targeted and control probes for 4 h respectively.

[0065] Figure 4L of (a) and (b) and Figure 4M Showing the ex vivo fluorescence imaging and fluorescence intensity of tumors in the PDX model after treatment with targeted and control probes for 4 h.

[0066] Figure 5A Showing 615 mouse MFC CLDN18.2 and MPI images within 72 h after intratumoral injection of magnetic nanoparticle probes in the MFC tumor - bearing model.

[0067] Figure 5B Showing 615 mouse MFC CLDN18.2 and the quantitative analysis of MPI signals within 72 h after intratumoral injection of magnetic nanoparticle probes in the MFC tumor - bearing model; among them, for the bar chart at each time point, the left side represents MFC CLDN18.2 +SPIO@1D5 group, with MFC indicated in the middle CLDN18.2 +SPIO@IgG group, with MFC + SPIO@1D5 group indicated on the right.

[0068] Figure 5C 、 Figure 5DOptical 3D-CT imaging and magnetic resonance imaging at 12 hours after intratumoral injection of each group of probes were shown respectively.

[0069] Figure 5E MPI imaging of ex vivo organs and tumors after treatment with targeted and control probes for 72 h was shown; Figure 5F MPI signals of ex vivo organs and tumors after treatment with targeted and control probes for 72 h were shown.

[0070] Figure 5G Four-modal imaging of H&E, immunohistochemistry (IHC), Prussian blue staining and Odyssey fluorescence of tumors after intratumoral injection of probes for 72 h was shown.

[0071] Figure 6A Showing MFC of 615 mice CLDN18.2 Schematic diagram of magnetic nanoparticle-mediated magnetothermal combined immunotherapy for tumor-bearing model.

[0072] Figure 6B 、 Figure 6C Ultraviolet thermograms during magnetic nanoprobe and magnetothermal therapy of tumor-bearing mice were shown respectively.

[0073] Figure 6D Photographs of tumors of mice in the control group, simple magnetothermal group, simple PD1 antibody group and combined drug group were shown 21 days after modeling.

[0074] Figure 6E Weights of tumors of each group were shown at 21 days after modeling; Figure 6F Volume changes of tumors of each group were shown from 7 to 21 days after modeling; Figure 6G - 6J Curves of tumor volume changes of each mouse in the untreated group, simple magnetothermal group, simple PD1 antibody group and combined drug group were shown respectively.

[0075] Figure 6K Schematic diagram of magnetic nanoparticle-mediated magnetothermal therapy for PDX model with high expression of CLDN18.2 was shown.

[0076] Figure 6L Photographs of tumors of mice with and without magnetic field were shown at 21 days after modeling, with untreated PDX on the left and tumors injected with SPIO@1D5 on the right for each mouse.

[0077] Figure 6M Weights of tumors of each group were shown at 21 days after modeling; Figure 6N - 6Q Curves of tumor volume changes of each mouse in the untreated group, simple magnetic field group, simple SPIO@1D5 administration group and magnetic field + SPIO@1D5 group were shown respectively; Figure 6R Weight changes of tumors of each group were shown from 5 to 21 days after modeling. Specific implementation scheme

[0078] Gastric cancer is a common malignant tumor globally. Due to the lack of typical symptoms in the early stage of gastric cancer, most patients are already in the advanced stage when they seek medical treatment. The expression rate of the immune therapy benefit index PD-L1 in the population is relatively low, and there is an urgent need to break through in exploring new drug treatment targets and treatment methods. CLDN18.2 is a common specific marker in gastric tissue, which is easily exposed in tumors and thus has the potential for translational diagnosis and treatment. There are currently no reports on the visualization and treatment of nanoprobes related to this molecule. In this application, for the population with high expression of CLDN18.2 in gastric cancer, a dual-modal probe composed of superparamagnetic nanoparticles conjugated with CLDN18.2 monoclonal antibody and indocyanine green was designed, and its good targeting and biosafety were verified at the CDX and PDX levels. By injecting the probe, mild magnetic hyperthermia treatment guided by magnetic particle imaging technology combined with immune checkpoint inhibitors can significantly and stably inhibit tumor growth, reverse the immune microenvironment inhibition state of some patients who are ineffective in immunotherapy. The mechanism of this treatment method is that the magnetic nanoparticle probe induces immunogenic death of tumor cells through magnetic hyperthermia, thereby recruiting and promoting the maturation and activation of dendritic cells, turning "cold tumors" into "hot tumors". Through the novel dual-modal targeting probe, a magnetic nanofluorescent multimodal imaging magnetic hyperthermia system based on CLDN18.2 navigation was constructed, and a treatment method of image-guided magnetic hyperthermia was used to achieve spatiotemporally controllable enhancement of immune checkpoint therapy for advanced gastric cancer, providing a new treatment option for the population with low response rate to immunotherapy for advanced gastric cancer.

[0079] The following further describes the present application in detail in conjunction with specific embodiments. The embodiments given are for the purpose of being able to understand the present application more thoroughly and being able to convey the scope of the present application completely to those skilled in the art.

[0080] On the one hand, the present application provides a dual-modal magnetic particle fluorescent probe, which comprises a CLDN18.2 monoclonal antibody, a near-infrared fluorescent molecule, and magnetic nanoparticles.

[0081] The dual-modal magnetic particle fluorescent probe of the present application can visualize gastric cancer positive for CLDN18.2, reveal the occurrence and development law of gastric cancer, and at the same time, the precise magnetic hyperthermia treatment of the targeting probe provides a new treatment plan for the combination of nanomaterials and immunotherapy for patients positive for CLDN18.2 and negative for PDL1.

[0082] In this article, the term "Monoclonal Antibody (mAb)" refers to a homogeneous group of antibodies, that is, except for possible naturally occurring mutations and / or post-translational modifications (such as isomerization, amidation) that may be present in small amounts, each antibody constituting the group is the same. Monoclonal antibodies are highly specific and target a single antigen or epitope. "Monoclonal" indicates the characteristic that the antibody is obtained from a substantially homogeneous group of antibodies and should not be construed as limiting the structure, source, or preparation method of the antibody. In some embodiments, monoclonal antibodies are prepared by hybridoma method, phage display method, yeast display method, recombinant DNA method, single cell screening or single cell sequencing method.

[0083] In this article, the term "near-infrared fluorescent molecule" is a commonly used fluorescent probe in living organisms, which is divided into near-infrared region I (NIR-I, 650 - 900 nm) and near-infrared region II (NIR-II, 1000 - 1700 nm) fluorescent dyes. It absorbs energy in the near-infrared spectral range and releases fluorescence in the visible light range. Because of its longer wavelength, it can reduce the interference of biological fluorescence on detection, and can greatly improve the detection limit and sensitivity. Also because of its advantages such as invasiveness, real-time, high resolution, etc., NIR fluorescent probes are widely used in research fields such as biomacromolecule labeling and tumor research.

[0084] Magnetic nanoparticles (MNP) are a kind of magnetic material at the nanoscale (1 - 100 nm), which have quantum size effect, surface effect, small size effect, macroscopic quantum tunneling effect, etc., and have good magnetic guidance, biocompatibility and biodegradability, etc., and can bind a variety of biofunctional molecules.

[0085] In some embodiments, the near-infrared fluorescent molecule and the CLDN18.2 monoclonal antibody are respectively connected to the magnetic nanoparticles.

[0086] In some embodiments, the amino terminus of the CLDN18.2 monoclonal antibody is randomly coupled to the carboxyl terminus of the magnetic nanoparticles to form a stable amide bond; the near-infrared fluorescent molecule is covalently coupled to other carboxyl groups of the magnetic nanoparticles to form an amide bond.

[0087] In some embodiments, the near-infrared fluorescent molecule is indocyanine green, and the indocyanine green is indocyanine green modified with N-hydroxysuccinimide group, that is, ICG-NHS.

[0088] Indocyanine green (ICG) is a near-infrared region I fluorescent dye and is a dye approved for in vivo use by the US Food and Drug Administration (FDA). Its excitation and emission wavelengths are around 785 nm and 810 nm respectively, which are longer than those of Cy series dyes such as Cy5, Cy5.5, and Cy7, and can penetrate deeper living tissues; compared with the IRDye800 series, it has better biocompatibility, a longer history, and wider clinical recognition.

[0089] In some embodiments, the magnetic nanoparticles are carboxyl-modified superparamagnetic nanoparticles. As used herein, the term "superparamagnetic nanoparticles" refers to such nanoparticles that will be immediately magnetized when an external magnetic field is applied and will immediately lose their magnetization when the magnetic field is removed. This property of it plays an important role in biological applications.

[0090] The superparamagnetic nanoparticles of the present application have characteristics such as high saturation magnetization intensity, contrast-enhanced imaging effect, and uniform particle size.

[0091] In some embodiments, the saturation magnetization intensity of the magnetic nanoparticles is 65 emu / g or more, and can be, for example, 65 emu / g or more, 70 emu / g or more, 75 emu / g or more, 80 emu / g or more, 85 emu / g or more, 90 emu / g or more, etc.

[0092] In some embodiments, the magnetic nanoparticles can be Perimag, Synomag, Vivotrax, or MagBeads superparamagnetic nanoparticles, preferably MagBeads superparamagnetic nanoparticles. The magnetic nanoparticles of the present application have excellent imaging effects, high saturation magnetization intensity, and magnetothermal effects. Under the same conditions, MagBeads as a tracer for imaging has higher resolution and sensitivity and can achieve linear quantification; as a hyperthermia reagent, MagBeads has high saturation magnetization intensity and good thermal effects.

[0093] In some embodiments, the diameter of the magnetic nanoparticles is 1 to 100 nm, more preferably 10 to 40 nm, and may be, for example, 1 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, etc. The diameter of the magnetic nanoparticles of the present application refers to the D50 particle size. The D50 particle size refers to the particle size corresponding to when the cumulative particle size distribution percentage reaches 50% in a sample, and it is also called the median diameter or median particle size. For example, it can be measured by methods such as transmission electron microscopy and dynamic light scattering. The diameter of superparamagnetic nanoparticles has a great influence on their biodistribution in vivo. Particles with a diameter of 10 to 40 nm can stay in the blood circulation for a longer time and can be phagocytosed by macrophages in lymph nodes and bone marrow through the capillary wall. Solid tumor tissues have the enhanced permeability and retention (EPR) effect, that is, the epithelial cells of the capillary plexus near tumor cells are not tightly arranged, and the pores are distributed between 100 and 800 nm in diameter according to the size of the tumor mass, which is similar to the diameter of nanodrugs. The blood vessel epithelial cells of normal tissues have tight junctions and strong lymphatic clearance functions, making drugs more likely to accumulate between tumor cells than in normal cells. Nanoparticles with too small a size have strong permeability in the blood vessel epithelial cells of normal tissues, thus losing their specificity for tumor tissues and having too short a half-life; nanoparticles with too large a size are very likely to have a first-pass effect or be immediately cleared by hepatic enzymes after entering the blood circulation, and thus are not stable enough.

[0094] In some embodiments, the magnetic nanoparticles are MagBeads superparamagnetic nanoparticles with a saturation magnetization intensity of 65 emu / g or more and a diameter of 10 to 40 nm. In some embodiments, the magnetic nanoparticles may be magnetite magnetic nanoparticles or iron oxide magnetic nanoparticles.

[0095] Since both the CLDN18.2 monoclonal antibody and the near-infrared fluorescent molecule provide amino termini, it is preferred to use DSPE-PEG2000-modified Fe3O4 magnetic nanoparticles (carboxyl terminus) in MagBeads products, that is, Mag3200 as the magnetic nanoparticles of the dual-modal magnetic particle fluorescence probe.

[0096] In some embodiments, the CLDN18.2 monoclonal antibody is a full-length antibody.

[0097] A typical full-length antibody consists of two identical light chains (L) and two identical heavy chains (H). The light chains can be divided into two types, namely kappa (κ) chains and lambda (λ) chains; the heavy chains can be classified into five types, namely μ, δ, γ, α, and ε chains, and the antibodies are defined as IgM, IgD, IgG, IgA, and IgE respectively. The amino acid sequences near the N-terminus of the heavy and light chains vary greatly, while the amino acid sequences of other parts are relatively constant. The regions with relatively large amino acid sequence variations near the N-terminus in the light and heavy chains are called variable regions (V), and the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C). The variable region of the heavy chain (VH) and the variable region of the light chain (VL) are usually the most variable parts of the antibody and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (HVR) and framework regions (FR). The hypervariable regions are also called complementarity-determining regions (CDR), which are loop structures. The CDRs of the heavy chain and the CDRs of the light chain are closely juxtaposed and cooperate with each other through the framework regions, jointly forming a surface that is complementary to the three-dimensional structure of the target antigen or epitope, determining the specificity of the antibody, and being the site where the antibody recognizes and binds to the antigen. The framework regions are the more conserved parts of VH and VL. They generally adopt a β-sheet conformation and are connected by three CDRs that form linker loops. Each VH and VL usually consists of three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0098] The CDRs can be identified according to the Kabat definition, the Chothia definition, the cumulative of the Kabat definition and the Chothia definition, the AbM definition, the contact definition, the IMGT unique numbering definition, and / or the conformational definition, or any CDR determination method well-known in the art. In this application, the CDRs are defined by Kabat.

[0099] The constant region of the light chain (CL) and the constant region of the heavy chain (CH) do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cell cytotoxicity. The lengths of the CLs of different Ig types (κ or λ) are basically the same, but the lengths of the CHs of different Ig classes are different. For example, IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CH1, CH2, CH3, and CH4. The amino acid sequences of the constant regions of the heavy and light chains of antibodies are well-known in the art.

[0100] Full-length antibodies are the most complete antibody molecular structures, with a typical Y-shaped molecular structure. Therefore, in the context of this application, "full-length antibody" and "complete antibody" have the same meaning and can be used interchangeably.

[0101] According to the length of the hinge region, the number of inter-chain disulfide bonds, and the different biological functions caused by the Fc segment, IgG can be further divided into 4 subclasses, namely IgG1, IgG2, IgG3, and IgG4. Among them, the Fc segment of IgG1 has the strongest binding ability to FcγR, while the binding activities of IgG2 and IgG4 are the weakest, which endows the former with the ability of Fc segment-mediated ADCC, while the latter two have very weak such ability. For IgG3, due to its relatively long hinge and high polymorphism, it increases the risks of instability and immunogenicity, and is rarely considered as an anti-tumor antibody drug. In some embodiments, the full-length antibody is IgG1.

[0102] The full-length antibodies of this application can be from a single species, such as human or mouse, or can be humanized antibodies to reduce the body's rejection reaction while maintaining the required specificity and affinity.

[0103] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to increase its homology with the sequence of human antibodies. Generally speaking, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (such as variable region FR and / or constant region) are derived from a human immunoglobulin (receptor antibody). Humanized antibodies usually retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, the ability to enhance immune response, etc. The donor antibody can be an antibody of a mouse, rat, rabbit, or non-human primate (such as cynomolgus monkey) with expected properties (such as antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and / or the ability to enhance immune response).

[0104] As used herein, the term "chimeric antibody" refers to an antibody that combines antibody fragments from different species. Specifically, for example, a monoclonal antibody from one species (such as a mouse), whose Fc constant region is replaced by the Fc constant region from another species (such as a human) via DNA recombination technology. See, for example, patent application PCT / US86 / 02269; EP / 173,494.

[0105] In some embodiments, the CLDN18.2 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1 (VQLQESGAELVRPGTSVKVSCKASRYAFTNYLIEWVKQRPGQGLEWIGMINPGSGGTHYNEKFKVKATLTADKSSSTAYMQLNSLTSDDSAVYFCARGGFGNSFAYWGQGTLVTVSAVGLGGSGAGLVAPGTSVLVSCLASATAPTATLIGTVLGAPGGGLGTIGMIAPGSGGTHTAGLPLVLATLTAALSSSTATMGLASLTSAASAVTPCAAGGPGASPATTGGGTLVTVSA), and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2 (DIMMTQTPLSLTVTAGEKVTMSCKSSQSLLNGGNLKNYLTWYQQKPGQPPKLLTYWASTRESGVPARFTGSGSGTDFTLTISSVQAEDLAVYYCQNGYFYPYTFGGGTKLEIK).

[0106] In some embodiments, the mass ratio of the magnetic nanoparticles, CLDN18.2 monoclonal antibody, and near-infrared fluorescent molecule is 1:0.02 to 1:0.001 to 0.05. For example, it can be 1:0.02:0.001, 1:0.1:0.001, 1:0.2:0.001, 1:0.3:0.001, 1:0.4:0.001, 1:0.5:0.001, 1:0.6:0.001, 1:0.7:0.001, 1:0.8:0.001, 1:0.9:0.001, 1:1:0.001, 1:0.02:0.005, 1:0.02:0.01, 1:0.02:0.02, 1:0.02:0.03, 1:0.02:0.04, 1:0.02:0.05, 1:0.1:0.05, 1:0.2:0.05, 1:0.3:0.05, 1:0.4:0.05, 1:0.5:0.05, 1:0.6:0.05, 1:0.7:0.05, 1:0.8:0.05, 1:0.9:0.05, 1:1:0.05, etc.

[0107] On the other hand, the present application provides a method for constructing any of the bimodal magnetic particle fluorescence probes as described above, comprising the following steps:

[0108] Co-incubating magnetic nanoparticles having a carboxyl terminus with the CLDN18.2 monoclonal antibody; and

[0109] In a dimethylformamide solvent, magnetic nanoparticles with a carboxyl terminus are co-incubated with a near-infrared fluorescent molecule having an N-hydroxysuccinimide group.

[0110] In some embodiments, the pH during the co-incubation of the magnetic nanoparticles and the near-infrared fluorescent molecule is 4 to 6, and can be, for example, 4, 4.5, 5, 5.5, 6, etc.

[0111] In some embodiments, the mass ratio of the magnetic nanoparticles to the CLDN18.2 monoclonal antibody is 1:0.02 to 1, and can be, for example, 1:0.02, 1:0.05, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, etc.

[0112] In some embodiments, the mass ratio of the magnetic nanoparticles to the near-infrared fluorescent molecule is 1:0.001 to 0.05, and can be, for example, 1:0.001, 1:0.003, 1:0.005, 1:0.008, 1:0.01, 1:0.015, 1:0.02, 1:0.025, 1:0.03, 1:0.035, 1:0.04, 1:0.045, 1:0.05, etc.

[0113] In some embodiments, the mass ratio of the magnetic nanoparticles with a carboxyl terminus, the CLDN18.2 monoclonal antibody, and the near-infrared fluorescent molecule having an N-hydroxysuccinimide group is 1:0.02 to 1:0.001 to 0.05, and can be, for example, 1:0.02:0.001, 1:0.1:0.001, 1:0.2:0.001, 1:0.3:0.001, 1:0.4:0.001, 1:0.5:0.001, 1:0.6:0.001, 1:0.7:0.001, 1:0.8:0.001, 1:0.9:0.001, 1:1:0.001, 1:0.02:0.005, 1:0.02:0.01, 1:0.02:0.02, 1:0.02:0.03, 1:0.02:0.04, 1:0.02:0.05, 1:0.1:0.05, 1:0.2:0.05, 1:0.3:0.05, 1:0.4:0.05, 1:0.5:0.05, 1:0.6:0.05, 1:0.7:0.05, 1:0.8:0.05, 1:0.9:0.05, 1:1:0.05, etc.

[0114] This application also provides the use of any of the aforementioned dual-modal magnetic particle fluorescence probes for preparing a drug for treating tumors or a reagent for detecting tumors.

[0115] The tumors targeted by the dual-modal magnetic particle fluorescence probe of the present application are mainly gastric cancers, and it also has diagnostic and therapeutic effects on esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, and colon cancer with high expression levels of CLDN18.2.

[0116] In some embodiments, the dual-modal magnetic particle fluorescence probe is administered to a patient in need by intravenous injection or intratumoral injection.

[0117] As used herein, the term "subject in need" refers to an individual at risk of or suffering from a disease, disorder, or condition.

[0118] In some embodiments, magnetic hyperthermia is used to treat tumors by applying an alternating magnetic field to the tumor site enriched with the dual-modal magnetic particle fluorescence probe to generate a thermal effect.

[0119] Magnetic hyperthermia (MTT) is a novel physical antitumor therapy. It utilizes the heat generation effect of magnetic nanoparticles under the action of an alternating magnetic field and the characteristic that tumor cells have poorer heat tolerance than normal cells. The magnetic nanoparticles are injected into the tumor site, and then an alternating magnetic field is applied to selectively warm the tumor cells. The magnetic heat of the intracellular heat source can regulate tumor cells to trigger immunogenic cell death (ICD). ICD can release antigens and danger-associated molecular patterns (DAMPs), including calreticulin (CRT) on the surface of tumor cells, as well as other factors such as high-mobility group box 1 (HMGB1), adenosine-5'-triphosphate (ATP), and heat shock protein (HSP). Antigen-presenting dendritic cells (DCs) are recruited to recognize, phagocytose the antigens of dead cells, and present them to T cells, activating the adaptive immune response and clearing tumor antigens, thereby producing a long-term antitumor immune effect.

[0120] The present application uses multi-color flow cytometry and multiplex immunofluorescence techniques to analyze the tumor immune microenvironment after magnetic hyperthermia combined with immunotherapy in tumor-bearing mice. Subsequently, transcriptome sequencing analysis is performed on the cell lines before and after magnetic hyperthermia, and flow cytometry, ELISA, and confocal microscopy are used to analyze the spatial localization of key molecules. Finally, mouse bone marrow DC cells are extracted and co-cultured in vitro with the cell debris after magnetic hyperthermia to study the key molecular mechanisms of magnetic hyperthermia-activated immunity, providing a new treatment method for patients with advanced gastric cancer positive for CLDN18.2.

[0121] In some embodiments, the magnetic hyperthermia is administered simultaneously with an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0122] In some embodiments, fluorescence imaging technology and magnetic particle imaging technology are used to detect tumors. Herein, fluorescence imaging technology utilizes the characteristics of fluorescent substances to obtain information about the structure of biological tissues and biomolecules. Its core principle is the absorption of excitation energy and the emission of fluorescent substances. Molecular information in the sample can be obtained by detecting the fluorescence intensity and position. Magnetic Particle Imaging (MPI) is a brand-new quantitative functional imaging technology that uses magnetic substances of nanoparticle tracers injected into the bloodstream to generate real-time three-dimensional images of arterial blood flow and movement. MPI has the advantages of three-dimensional imaging, high temporal resolution, high spatial resolution, high sensitivity, and no harm of ionizing radiation. The tracer used does not contain nephrotoxicity and will not cause serious adverse reactions. MPI does not display anatomical structures, has no background signal interference, and the signal intensity is proportional to the tracer concentration. It is an examination method that can obtain quantitative data. The adoption of the above technologies can ensure the rapid, real-time, and dynamic monitoring of the progression process of gastric cancer peritoneal metastasis while ensuring the accuracy of quantifying the key molecular functions during the progression process.

[0123] In this application, the CLDN18.2 monoclonal antibody and the near-infrared fluorescent molecule are respectively conjugated with magnetic nanoparticles. Finally, the antibody conjugation rate of the obtained product mAb-MNP is 25%-30%, and the ICG conjugation rate is 35%-40%; the antibody conjugation rate of the obtained product IgG-MNP is 30%-35%, and the ICG conjugation rate is 45%-50%.

[0124] Examples

[0125] Example 1 Expression, characteristics, prognosis of CLDN18.2 in clinical samples and its relationship with immunity

[0126] Immunohistochemical specimens (cancer and adjacent tissues) of 563 patients with advanced gastric cancer who underwent radical gastrectomy and were admitted to Peking University Cancer Hospital from January 2008 to December 2012 were selected. All were clearly diagnosed by histopathology and had complete follow-up data, including the patient's gender, age, maximum tumor diameter, tumor differentiation grade, tumor location, tumor invasion depth, lymph node metastasis status, postoperative TNM stage, vascular invasion, etc. Immunohistochemical staining was used to verify the expression of CLDN18.2 in the tumor tissues of gastric cancer patients, and it was found that the expression level of CLDN18.2 in the tumor tissues was significantly higher than that in the adjacent tissues ( Figure 1A)。The gastric cancer tissue microarray was grouped according to the different expression intensities and positive areas of CLDN18.2. Among them, the CLDN18.2 positive (++) group and the CLDN18.2 strongly positive group (+++) were used as the CLDN18.2 high-expression group, and the others were the CLDN18.2 low-expression group. Among the 563 cases of gastric cancer tissues detected and analyzed, CLDN18.2 expression was detected in 306 cases (54.4%) of gastric cancer tissues, and high expression of CLDN18.2 was detected in 90 cases (16.0%) of samples ( Figure 1B and Figure 1C ). The 563 patients were followed up for 1 - 133 months, and the median follow-up time was 35.5 months. The Kaplan-Meier survival analysis results in the PKUCH immunohistochemical data showed that the overall survival time of gastric cancer with high expression of CLDN18.2 in TNM stages I-II was shortened (P = 0.044, Figure 1E ). Survival analysis of the TCGA database found that the overall survival time of gastric cancer with high mRNA expression of CLDN18.2 in TNM stages I-IIIa was shortened (P = 0.003, Figure 1D ). The expression level of CLDN18.2 was correlated with multiple molecules, and there was a negative correlation with the expression of PD-L1 and CD3 molecules (p = 0.002) ( Figure 1F ). TCGA database analysis found that high expression of CLDN18 had lower TIDE (tumor immune dysfunction and exclusion) scores and a more active immune microenvironment status ( Figure 1G ) and ( Figure 1H ), suggesting that the treatment method targeting CLDN18.2 combined with immunotherapy has a better synergistic effect.

[0127] Example 2 Construction of a bimodal magnetic particle fluorescence probe targeting CLDN18.2

[0128] In this application, through preliminary pre-experiments, the biological distribution, half-life, drug-related toxicity and other characteristics of MagBeads with sizes of 10 nm, 20 nm, 30 nm, 40 nm, and 50 nm injected intravenously in 615 tumor-bearing mice were compared, and 20 nm MagBeads were selected as the experimental object; among them, the size of MagBeads refers to the D50 particle size measured by transmission electron microscopy (TEM). Experiments found that 20 nm magnetic nanoparticles have the optimal MPI signal, good magnetic thermal properties, and good biological safety.

[0129] Using the previously prepared and humanized CLDN18.2-specific monoclonal antibody 1D5 (full-length antibody) of the Biochemistry and Molecular Biology Laboratory of Peking University Cancer Hospital (the amino acid sequences of the light and heavy chains of the monoclonal antibody 1D5 can be referred to US20200207857A1) as the main component targeting tumors, it was co-incubated with PEGylated carboxyl-terminal iron oxide magnetic nanoparticles Mag3200 (NANOEAST, Mag3200) activated by EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide). The activated carboxyl groups were coupled to the amino groups on the antibody to form stable amide bonds, and SPIO@1D5 was prepared. Subsequently, in a dimethylformamide solvent, an ICG-NHS fluorescent group (ruixibio, R-ICG-001) was added, the pH value was adjusted, and the incubated ICG was covalently coupled to other carboxyl groups of the magnetic particles to prepare a Mag3200@1D5-ICG bimodal targeting probe. The control probe replaced 1D5 with murine IgG (Solarbio, SP031)( Figure 2A ). The particle size of the probe was detected by dynamic light scattering (DLS) to be approximately 100 nm( Figure 2B ). The surface potential of the probe was detected by electrophoretic light scattering (Zeta) to be approximately -20 mV( Figure 2C ). The ultraviolet-visible absorption spectrum (UV) detected a peak of ICG at 750 - 800 nm( Figure 2D ). Transmission electron microscopy (TEM) detected that the particle size of the probe was stable before and after coupling, and there was no obvious aggregation phenomenon( Figure 2E and Figure 2F ). The saturation magnetization intensity of the probe was detected by a vibrating sample magnetometer (VSM) to be 14.88 emu / g, and the probe had coercivity and superparamagnetism( Figure 2G ).

[0130] The imaging properties of the probe were verified using optical imaging and magnetic particle imaging techniques. It was found that in fluorescence imaging, the signal tended to saturate with the increase in particle concentration, showing a non-linear relationship; in magnetic particle imaging, the signal increased linearly with the increase in ion mass( Figure 2H and Figure 2I ), which confirmed that magnetic particle imaging technology could achieve the quantification of magnetic particles through images. Compared with the previous fluorescent probes, the bimodal probe had magnetic heating properties and, combined with MPI (magnetic particle imaging equipment), could quantitatively visualize the particle distribution in vivo.

[0131] Example 3 Construction of a gastric cancer cell line with high expression of CLDN18.2 to verify the targeting and toxicity of the probe target

[0132] The expression of CLDN18.2 was detected in normal gastric tissue GES-1, human gastric cancer cell lines AGS, SGC7901, MGC803, BGC823, KATOIII, MKN28, MKN45, N87, HGC27, and murine gastric cancer cell line MFC ( Figure 3A ), plasmids stably expressing CLDN18.2 were constructed using AGS and MFC gastric cancer cell lines, and the overexpression efficiency was detected by Western blot and flow cytometry ( Figure 3B - Figure 3F ), and was used to detect the cytotoxic effect of the bimodal targeting probe on gastric cancer cells after incubating at 0 - 50 μg / ml for 48 hours ( Figure 3G - Figure 3H ). When the targeting probe and the control probe were co-incubated with AGS and MFC cells overexpressing CLDN18.2 at 50 μg / ml for 2 hours respectively, it was observed that compared with the control group probe, the targeting group probe was specifically taken up by the cells ( Figure 3I - Figure 3L ). Compared with the radionuclide probes of CLDN18.2 disclosed in, for example, [177Lu]Lu-labeled anti-claudin-18.2 antibody by Zeng Z et al. demonstrated radioimmunotherapy potential in gastric cancer mouse xenograft models. Eur J Nucl Med Mol Imaging. 2023 and Wang S et al. Eur J Nucl Med Mol Imaging. 2023; 50(9):2802 - 17, this nanosensor has the characteristics of nanomaterial cell uptake, and its targeting and therapeutic effects are further improved; compared with radionuclide materials, magnetic materials and fluorescence have no toxic side effects on the human body.

[0133] Example 4 Construction of CDX and PDX optical imaging models with high expression of CLDN18.2

[0134] Subcutaneous tumor models of 615 mice were made with MFC cells with or without overexpression of CLDN18.2, and the bimodal probe or control probe constructed in Example 2 was injected via the tail vein at 100 μg (3 mice in each group). Fluorescence images of mice at different time points within 72 hours after injection were collected, and it was found that the enrichment effect and intratumoral retention effect of the targeting group probe were significantly better than those of the non-specific targeting group ( Figure 4A 、 Figure 4C ). Ex vivo observation of the heart, liver, spleen, lungs, kidneys, and tumors found that the liver enrichment effect of the targeting group probe was significantly weaker than that of the non-specific targeting group ( Figure 4B ), and the tumor signal in the experimental group was significantly stronger than that in the control group ( Figure 4D 、 Figure 4E)。The PDX model with low expression of CLDN18.2 on the left and high expression of CLDN18.2 on the right was used to verify the probe efficacy again (4 in each group). Figure 4F ) It was also found that the experimental group of probes immediately accumulated in the tumor area with high expression of CLDN18.2 after injection. Four hours after injection, the probe signal of the tumor with high CLDN18.2 was significantly higher than that of the tumor with low expression of CLDN18.2. However, the control probe did not immediately accumulate in the tumor area after injection. Although the EPR effect caused a relatively high signal in the tumor area over time, there was no difference in the optical signals of the tumors with high and low expression of CLDN18.2. Figure 4G - Figure 4I ) After 4 hours of injection, when observing the excised heart, liver, spleen, lungs, kidneys and tumors, it was found that the accumulation effects of the probes in the liver and kidneys of the targeted group were significantly weaker than those of the non-specific targeting group. Figure 4J - Figure 4K ) and the tumor signal of the experimental group was significantly stronger than that of the control group. Figure 4L - Figure 4M ) When treating the PDX model, compared with the two single-modal antibody-conjugated fluorescence probes disclosed in, for example, CD24-targeted fluorescence imaging in patient-derived xenograft models of high-grade serous ovarian carcinoma. EBioMedicine. 2020;56:102782 by Kleinmanns K et al. and CDH17 nanobodies facilitate rapid imaging of gastric cancer and efficient delivery of immunotoxin. Biomater Res. 2022;26(1):64 by Ma J et al., the probe of the present application is a dual-modal probe, and the magnetic nanoparticles can better achieve quantitative image-guided magnetic hyperthermia treatment.

[0135] Example 5 Determination of the magnetic hyperthermia treatment time window in mice by magnetic particle imaging

[0136] Using a magnetic particle imaging device (MPI), 100 μg of the probe was injected into the tumors of the mice overexpressing the experimental group probe, the mice overexpressing the control group probe, and the control mice of the experimental group probe respectively. The MPI technique was used to monitor the diffusion and metabolism of the probe in the tumor (3 in each group). The results showed that the diffusion signal value of the probe increased after injection. The MPI signal value in the tumor of the experimental group reached the maximum at 12 hours and was evenly distributed. The signal decreased by half at 72 hours, and the drug reached the half-life. In the two control groups, no obvious diffusion of the probe in the tumor was observed, and it was metabolized faster with the blood and had a shorter half-life. Figure 5A 、 5B ) Also, by observing the optical 3D-CT imaging and nuclear magnetic resonance imaging at the 12th hour Figure 5C 、5D ) confirmed the diffusion of the experimental group's probe in the tumor. After 72 h of injection, the excised heart, liver, spleen, lung, kidney, and tumor were observed, and it was found that the tumor signal in the experimental group was significantly stronger than that in the control group ( Figure 5E 、 5F ). At the 72nd hour, HE, immunohistochemistry, Prussian blue staining, and Odyssey four-modal imaging of the tumor pathological sections ( Figure 5G ) were performed to confirm the diffusion of the control group's probe in the tumor at the microscopic pathological level. Compared with the radionuclide probe navigation of PMC7674096, this paper selected magnetic particle imaging, which is non-radioactive, safer, and more sensitive, as the core technology for image-guided therapy. Compared with the nuclear magnetic imaging navigation disclosed by, for example, Zhang Y et al. in Genetically engineered magnetic nanocages for cancer magneto-catalytic theranostics. Nat Commun. 2020;11(1):5421, magnetic particle imaging has the advantages of higher sensitivity and linear quantification.

[0137] Example 6 Magnetic hyperthermia combined with immunotherapy for CDX and PDX gastric cancer models

[0138] Magnetic nanoprobes were injected into the tumors of MFC tumor-bearing mice overexpressing CLDN18.2. After 12 h under the guidance of MPI technology, the mice were placed in a magnetic field of 20 A, 353 KHz, and 1.6 KW for 10 min of magnetic hyperthermia treatment. Anti-PD1 treatment of 5 mg was given every other day, and the above treatment was repeated every three days for a total of 4 times (5 mice in each group) ( Figure 6A ). Fiber optic temperature sensing was used to measure the temperature of magnetic nanoprobes, mouse tumors, and axillae. The magnetic nanoparticles could be heated to 53 - 55 °C in the magnetic field, and the mouse tumors could be heated to 41 - 43 °C after injecting the probes, while the skin surface temperature was stable below 38 °C ( Figure 6B ). Twenty-one days after modeling, the tumors were dissected, and the longest and shortest diameters and weights of the tumors were measured. It was found that the treatment method of magnetic hyperthermia combined with immunotherapy had significantly better curative effects than simply using magnetic hyperthermia or PD1 antibody treatment ( Figure 6D - Figure 6J ). For the bilateral tumors of PDX mice with high expression of CLDN18.2, magnetic nanoprobes were injected into one side of the tumor and PBS was injected into the other side. After 12 h, a magnetic field was applied or not ( Figure 6K ), and the treatment was repeated every three days for a total of four times (4 mice in each group). Twenty-one days after modeling, tumor dissection and analysis found that the magnetic field could effectively inhibit the tumor growth of the whole body, while the magnetic field combined with magnetic particles could not inhibit tumor growth by itself ( Figure 6L - Figure 6R)。 Different from all other articles on magnetothermal therapy-activated immunity (such as Magnetism-mediated targeting hyperthermia-immunotherapy in "cold" tumor with CSF1R inhibitor by Fang Y et al. Theranostics. 2021;11(14):6860-72 and Combined Magnetic Hyperthermia and Immune Therapy for Primary and Metastatic Tumor Treatments by Pan J et al. ACS Nano. 2020;14(1):1033-44), in this application, based on the image quantification guidance of MPI, it is determined that the best precise heating effect can be achieved by performing magnetothermal therapy 12 hours after the administration of magnetic particles. The drug reaches its half-life 72 hours after administration, and re-administration is required. The optimal time window for drug administration and the application of an external alternating magnetic field is clarified. This application proposes a novel magnetic nanoparticle-magnetic particle imaging-magnetothermal therapy platform to achieve precisely controllable magnetothermal therapy of magnetic nanoparticles in the time and space dimensions. When combined with anti-PD-1 therapy, this treatment can reverse the inhibitory state of the immune microenvironment in patients who are ineffective in anti-PD-1 therapy, convert "cold tumors" into "hot tumors", and thus enhance the effect of immunotherapy.

[0139] As described above, it is only the preferred embodiment of this application and not a limitation in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of this application without departing from the technical solution content of this application still fall within the protection scope of the technical solution of this application.

Claims

1. A dual-modal magnetic particle fluorescence probe for detecting gastric cancer, which comprises a CLDN18.2 monoclonal antibody, a near-infrared fluorescent molecule, and magnetic nanoparticles; The near-infrared fluorescent molecule and the CLDN18.2 monoclonal antibody are respectively connected to the magnetic nanoparticles through amide bonds; The near-infrared fluorescent molecule is indocyanine green ICG; The diameter of the magnetic nanoparticles is 20 nm, and the magnetic nanoparticles are Mag3200; The CLDN18.2 monoclonal antibody is a full-length antibody IgG1; The CLDN18.2 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2; The mass ratio of the magnetic nanoparticles, the CLDN18.2 monoclonal antibody, and the near-infrared fluorescent molecule is 1: 0.02 to 1: 0.001 to 0.

05.

2. The dual-modal magnetic particle fluorescence probe for detecting gastric cancer according to claim 1, wherein the saturation magnetization intensity of the magnetic nanoparticles is 65 emu / g or more.

3. The dual-modal magnetic particle fluorescence probe for detecting gastric cancer according to claim 1 or 2, wherein, The CLDN18.2 monoclonal antibody is a humanized antibody, a murine antibody, or a chimeric antibody.

4. The dual-modal magnetic particle fluorescence probe for detecting gastric cancer according to claim 1 or 2, wherein, The CLDN18.2 is human CLDN18.2 or murine CLDN18.

2.

5. A method for constructing the dual-modal magnetic particle fluorescence probe for detecting gastric cancer according to any one of claims 1 to 4, comprising the following steps: Co-incubating magnetic nanoparticles with a carboxyl terminus with the CLDN18.2 monoclonal antibody; and In a dimethylformamide solvent, co-incubating magnetic nanoparticles with a carboxyl terminus with a near-infrared fluorescent molecule having an N-hydroxysuccinimide group; Wherein the near-infrared fluorescent molecule having an N-hydroxysuccinimide group is indocyanine green ICG; The diameter of the magnetic nanoparticles with a carboxyl terminus is 20 nm, and the magnetic nanoparticles with a carboxyl terminus are Mag3200.

6. The method according to claim 5, wherein the pH during the co-incubation of the magnetic nanoparticles and the near-infrared fluorescent molecule is 4 to 6.

7. Use of the dual-modal magnetic particle fluorescence probe for detecting gastric cancer according to any one of claims 1 to 4 for preparing a drug for treating tumors or a reagent for detecting tumors.

8. The use according to claim 7, wherein the tumor is gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, breast cancer, or lung cancer.

9. The use according to claim 7, wherein, The dual-modal magnetic particle fluorescence probe for detecting gastric cancer is administered to a subject in need by intravenous injection or intratumoral injection.

10. The use according to claim 7, wherein by means of magnetic hyperthermia, heat effect is generated by applying an alternating magnetic field to the tumor site enriched with the dual-modal magnetic particle fluorescence probe for detecting gastric cancer to treat tumors.

11. The use according to claim 10, wherein the magnetic hyperthermia is administered simultaneously with an anti-PD-1 antibody or an anti-PD-L1 antibody.

12. The use according to claim 7, wherein the detection uses fluorescence imaging technology and magnetic particle imaging technology.

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