Nanovaccines with targeted conjugation of immune-activated interleukins and their receptor complexes, their preparation and application

Abstract

This invention relates to a nanovaccine specifically designed to link immune-activated interleukins and their receptor complexes, its preparation, and its application, belonging to the field of biomedical technology. The invention employs recombinant protein expression technology, introducing a cysteine ​​residue at the C-terminus of the IL-15 / IL-15Rα protein. The thiol group of the cysteine ​​is then covalently coupled to the maleimide group on the surface of iron(III) oxide nanoparticles, achieving the targeted coupling of IL-15 / IL-15Rα protein to the nanoparticle surface. This nanovaccine specifically linking IL-15 / IL-15Rα mimics the natural trans-presentation of IL-15 in vivo, acting on CD8. + It promotes the proliferation and activation of T cells and NK cells. Compared with free IL-15 / IL-15Rα recombinant protein, nanovaccines have shown significant advantages in immune activation, tumor-targeted enrichment, reduction of systemic immunotoxicity, and enhancement of anti-tumor immunotherapeutic efficacy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nanovaccine that is directionally linked to an immune-activated interleukin and its receptor complex, its preparation method, and its application in the preparation of antitumor agents. Background Technology

[0002] Tumor immunotherapy aims to modulate the immune system to enhance the body's ability to recognize and kill tumor cells. Cytokines, as small-molecule messengers of the immune system, play a central role in regulating immune cell activation, proliferation, and effector function. Interleukin-15 (IL-15) belongs to the short-chain tetraalpha-helical bundle cytokine family and primarily promotes the activity of cytotoxic T cells (CD8+). + It promotes the proliferation and survival of T cells and natural killer (NK) cells, while having a weaker activation effect on regulatory T (Treg) cells. This characteristic gives it a unique advantage in maintaining durable anti-tumor immunity.

[0003] The biological function of IL-15 depends on its unique receptor system and trans-presentation mechanism. The IL-15 receptor consists of IL-15 receptor α (IL-15Rα or CD215), IL-15 receptor β (IL-15Rβ, also known as IL-2Rβ or CD122), and γc (CD132). Under physiological conditions, IL-15 binds to IL-15Rα with high affinity within the producing cell, forming a stable IL-15 / IL-15Rα complex. This complex is transported to the cell surface and presented to IL-15Rβ and γc on adjacent effector cells via membrane binding. This initiates downstream JAK / STAT, PI3K / Akt, and MAPK signaling pathways, thereby promoting the survival, proliferation, and cytotoxic function of effector cells.

[0004] Despite the significant anti-tumor potential of IL-15, its clinical application in its natural form faces serious challenges. First, IL-15 has an extremely short half-life in vivo; second, systemic administration is prone to inducing dose-limiting immunotoxicities, including cytokine release syndrome (CRS) and capillary leakage syndrome (CLS), as well as the potential risk of autoimmune reactions, making its therapeutic window relatively narrow.

[0005] To overcome these shortcomings, researchers have proposed various improvement strategies. In terms of protein engineering, based on the biological characteristic that IL-15 needs to bind with IL-15Rα to exert its function, a variety of super agonists using the IL-15 / IL-15Rα complex as a template have been developed, aiming to improve the activity and half-life of IL-15. However, it is difficult to solve the problem of tissue-selective delivery, and high-dose systemic administration may still induce systemic immune activation and toxic reactions.

[0006] The rise of nanomedicine delivery systems has provided new insights into the targeted delivery of cytokines, improving the pharmacokinetic characteristics of free IL-15 (such as prolonging in vivo circulation time and accumulation at tumor sites). Currently, IL-15 nanomaterialization research mainly falls into two categories: encapsulated nanoparticles and surface-linked nanocarriers. Encapsulated nanoparticles encapsulate IL-15 within the carrier, protecting it from degradation and prolonging circulation time; however, these carriers face the critical challenge of incomplete IL-15 release. Surface-linked nanocarriers aim to mimic the natural trans-presentation mechanism of IL-15; however, traditional chemical conjugation methods result in IL-15 being randomly oriented onto the nanoparticle surface, potentially masking its receptor-binding domain or causing conformational restriction, leading to a significant reduction in biological activity. Therefore, new approaches are still needed to develop novel IL-15 nanovaccines. Summary of the Invention

[0007] In view of this, the object of the present invention is to provide a nanovaccine with directed linkage to an immune-activated interleukin and its receptor complex, a method for preparing the same, and its application in the preparation of antitumor agents. The nanovaccine with directed linkage to IL-15 / IL-15Rα provided by the present invention can effectively stimulate CD8+. + The expansion of T cells and NK cells, with tumor targeting capabilities, addresses the shortcomings of existing IL-15 / IL-15Rα and related delivery systems, enabling targeted delivery, enhanced immune response, reduced immunotoxicity, and improved anti-tumor efficacy.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: a nanovaccine with directed connection of immune-activated interleukin and its receptor complex, comprising a hydrophilic nanocarrier and an immune-activated interleukin and its receptor complex chemically connected to the hydrophilic nanocarrier.

[0009] The hydrophilic nanocarrier is a magnetic iron oxide nanoparticle with a surface modified with maleimide groups;

[0010] The immune-activated interleukin and its receptor complex are directionally covalently linked to the maleimide group of the hydrophilic nanocarrier via a C-terminal cysteine ​​residue.

[0011] Furthermore, the immune-activated interleukin and its receptor complex are recombinant IL-15 / IL-15Rα proteins.

[0012] Furthermore, the IL-15 / IL-15Rα recombinant protein contains the same amino acid sequence as SEQ ID No. 1;

[0013] Or it may contain at least 90-99% of the same amino acid sequence as SEQ ID No. 1.

[0014] Furthermore, the IL-15 / IL-15Rα recombinant protein contains the same amino acid sequence as SEQ ID No. 2;

[0015] Or it may contain at least 90-99% of the same amino acid sequence as SEQ ID No. 2.

[0016] Furthermore, the number of IL-15 / IL-15Rα recombinant proteins directionally covalently linked to a single hydrophilic nanocarrier is ≥3;

[0017] The hydrophilic nanocarrier has a particle size of 10~100 nm;

[0018] The nanovaccine has a particle size of 20~100 nm.

[0019] Furthermore, the preparation process of magnetic iron oxide nanoparticles with surface-modified maleimide groups is as follows: methoxy-PEG2000-maleimide, benzyl ether, and ferric acetylacetone are mixed and heated to 200-300°C. o C, reacts to form nanoparticles, which are then cooled, washed, and separated by magnetic column separation to obtain the final product.

[0020] The preparation method of the nano-vaccine includes the following steps:

[0021] A nanovaccine was obtained by directionally coupling the nanocarrier and the IL-15 / IL-15Rα recombinant protein.

[0022] The application of the aforementioned nano-vaccines in the preparation of anti-tumor agents.

[0023] The aforementioned nanovaccine is used for in vitro expansion and activation of immune cells.

[0024] The application of the nanovaccine in delivering immune-activated interleukins and their receptor complexes, wherein the immune-activated interleukins include at least one of IL-1α / β, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-23.

[0025] The present invention provides a nanovaccine with directed IL-15 / IL-15Rα linkage, comprising a hydrophilic nanocarrier and a recombinant IL-15 / IL-15Rα protein chemically linked to the hydrophilic nanocarrier;

[0026] The hydrophilic nanocarrier is a magnetic iron oxide nanoparticle with a surface modified with maleimide groups;

[0027] The IL-15 / IL-15Rα recombinant protein is directionally covalently linked to the maleimide group of the hydrophilic nanocarrier via a C-terminal cysteine ​​residue.

[0028] Preferably, the method for preparing the hydrophilic nanocarrier includes the following steps:

[0029] A mixture of acetylacetone iron, benzyl ether, and methoxy-PEG2000-maleimide was subjected to a high-temperature thermal decomposition reaction. After the reaction was completed, hydrophilic nanocarriers were obtained by separation and purification using an LS column.

[0030] Preferably, the immune-activated interleukin and its receptor complex is a recombinant IL-15 / IL-15Rα protein, wherein the recombinant IL-15 / IL-15Rα protein contains the same amino acid sequence as SEQ ID No. 1;

[0031] Or it may contain at least 90-99% of the same amino acid sequence as SEQ ID No. 1.

[0032] Preferably, the immune-activated interleukin and its receptor complex is an IL-15 / IL-15Rα recombinant protein, wherein the IL-15 / IL-15Rα recombinant protein contains the same amino acid sequence as SEQ ID No. 2;

[0033] Or it may contain at least 90-99% of the same amino acid sequence as SEQ ID No. 2.

[0034] Preferably, the number of IL-15 / IL-15Rα recombinant proteins covalently linked to a single hydrophilic nanocarrier is ≥3;

[0035] The hydrophilic nanocarrier has a particle size of 10~100 nm;

[0036] The nanovaccine with directed IL-15 / IL-15Rα has a particle size of 20~100 nm.

[0037] This invention provides a method for preparing the above-mentioned nano-vaccine with directed IL-15 / IL-15Rα, characterized by comprising the following steps:

[0038] A hydrophilic nanocarrier and IL-15 / IL-15Rα recombinant protein were directionally coupled to obtain a hydrophilic nanocarrier linked with IL-15 / IL-15Rα recombinant protein.

[0039] This invention provides the application of the above-mentioned nanovaccines with directional IL-15 / IL-15Rα in the preparation of antitumor agents.

[0040] This invention provides a nanovaccine with directed IL-15 / IL-15Rα linkage, comprising a hydrophilic nanocarrier and a recombinant IL-15 / IL-15Rα protein directionally covalently linked to the hydrophilic nanocarrier. In this invention, the hydrophilic nanocarrier is a magnetic magnetite nanoparticle with a surface modified with maleimide groups; the IL-15 / IL-15Rα recombinant protein is directionally covalently linked to the maleimide groups of the hydrophilic nanocarrier via a C-terminal cysteine ​​residue. This invention uses magnetic magnetite nanoparticles with a surface modified with maleimide groups as the hydrophilic nanocarrier. This carrier not only has good biocompatibility but also good magnetic response performance, facilitating magnetic separation and purification. Furthermore, the maleimide groups on the nanoparticle surface covalently bind to the thiol groups in the C-terminal cysteine ​​residue of the IL-15 / IL-15Rα protein; this linkage is stable and the number of linked proteins is controllable.

[0041] The nanovaccine described herein is used for in vitro expansion and activation of immune cells. Compared with the IL-15 / IL-15Rα recombinant protein, the nanovaccine exhibits at least one of the following significant improvements:

[0042] Higher level CD8 + T amplification;

[0043] Higher levels of NK cell expansion;

[0044] Higher level CD44 + CD8 + Activation of T cells.

[0045] The aforementioned nano-vaccine is used for anti-tumor therapy and has at least one of the following effects:

[0046] Enhance the accumulation of nano-vaccines in tumor tissue;

[0047] Enhance CD8 in tumor tissue + Infiltration of at least one type of cell, including T cells and NK cells;

[0048] Enhance tumor immune response;

[0049] It can mitigate the systemic immunotoxicity caused by recombinant IL-15 / IL-15Rα protein.

[0050] Furthermore, the immune-activating interleukins and their receptor complexes described in this invention are not limited to IL-15 / IL-15Rα recombinant proteins, but may also cover all interleukins and their receptor complexes that have immune-activating activity and can enhance the body's anti-tumor immune function.

[0051] The beneficial effects of this invention are as follows: This invention employs recombinant protein expression technology to introduce a cysteine ​​residue at the C-terminus of the IL-15 / IL-15Rα protein. The thiol group of the cysteine ​​residue is then coupled with the maleimide group on the surface of the iron oxide nanoparticles, achieving directional covalent coupling of the IL-15 / IL-15Rα protein to the nanoparticle surface. This mimics the natural trans-presentation of IL-15 in vivo, thereby specifically activating CD8. + This invention promotes the proliferation, survival, and cytotoxic function of T cells and NK cells, demonstrating excellent immune activation effects in both cell and animal experiments. Compared with free IL-15 / IL-15Rα recombinant protein, the nanovaccine provided by this invention exhibits significant advantages in immune activation, tumor-targeted enrichment, reduction of systemic immunotoxicity, and enhancement of anti-tumor immunotherapeutic efficacy.

[0052] The nanovaccine with targeted IL-15 / IL-15Rα provided by this invention exhibited excellent anti-tumor effects in a subcutaneous melanoma model. The results showed that the nanovaccine significantly inhibited tumor growth, with an anti-tumor effect significantly superior to that of free IL-15 / IL-15Rα recombinant protein, while also enhancing CD8+ in tumor tissue. + It reduces the infiltration of T cells and NK cells, decreases systemic immunotoxicity caused by IL-15 / IL-15Rα, and has good biocompatibility.

[0053] Furthermore, the method for preparing nano-vaccines with directional linkage of IL-15 / IL-15Rα provided by this invention is simple and easy to achieve industrial-scale mass production. Attached Figure Description

[0054] Figure 1 For Fe 3+ Standard curve of concentration.

[0055] Figure 2 This is an electron microscopy characterization image of NPM nanoparticles.

[0056] Figure 3 This is a characterization diagram of the hydrated particle size of NPM nanoparticles.

[0057] Figure 4 Infrared characterization of NPM nanoparticles.

[0058] Figure 5 SDS-PAGE gel electrophoresis image of recombinant IL-15 / IL-15Rα protein.

[0059] Figure 6 This is a graph showing the immunogenicity of the IL-15 / IL-15Rα recombinant protein.

[0060] Figure 7 Image of hIL-15 / IL-15Rα-NPM nanovaccine gel electrophoresis;

[0061] Among them, (a) shows the SDS-PAGE and Native-PAGE images of the hIL-15 / IL-15Rα-NPM nanovaccine;

[0062] (b) is a standard curve of SDS-PAGE grayscale values ​​of hIL-15 / IL-15Rα protein.

[0063] Figure 8 Characterization diagram of hIL-15 / IL-15Rα-NPM nanovaccine;

[0064] Among them, (a) is the hydration particle size diagram of hIL-15 / IL-15Rα-NPM nanovaccine;

[0065] (b) is a graph showing the stability test results of the hIL-15 / IL-15Rα-NPM nanovaccine.

[0066] Figure 9 In vitro activation of memory CD8 for hIL-15 / IL-15Rα-NPM nanovaccine + T cell results;

[0067] Among them, (a) is the sorting memory CD8 + T cell results diagram;

[0068] (b) For memorizing CD8 + CD69 in T cells + Percentage chart;

[0069] (c) is for memorizing CD8 + CD25 in T cells + Percentage chart.

[0070] Figure 10 Biodistribution map of hIL-15 / IL-15Rα-NPM nanovaccine in tumor-bearing mice;

[0071] Among them, (a) is a fluorescence image of 4T1 tumor-bearing mice within 12 h after drug administration;

[0072] (b) In vitro fluorescence images of major organs and tumors 12 h after drug administration.

[0073] Figure 11 A graph evaluating the efficacy of hIL-15 / IL-15Rα-NPM nanovaccine in treating subcutaneous melanoma;

[0074] (a) is a tumor photograph after treatment;

[0075] (b) Tumor weight after treatment.

[0076] Figure 12This is a graph showing the percentage of immune cells in tumor tissue after anti-tumor treatment.

[0077] Among them, (a) represents CD8 in the tumor. + T cell flow cytometry and bar chart;

[0078] (b) shows the flow cytometry and bar chart of NK cells in the tumor.

[0079] Figure 13 This is a graph showing the in vivo safety evaluation of the hIL-15 / IL-15Rα-NPM nanovaccine.

[0080] (a) represents the serum IL-6 level within 96 hours after the first dose;

[0081] (b) Serum IFN-γ levels within 96 h after the first dose;

[0082] (c) Serum ALT activity within 96 h after the first dose;

[0083] (d) Serum AST activity within 96 h after the first dose. Detailed Implementation

[0084] The present invention provides a nanovaccine with directed IL-15 / IL-15Rα linkage, comprising a hydrophilic nanocarrier and a recombinant IL-15 / IL-15Rα protein chemically linked to the hydrophilic nanocarrier;

[0085] The hydrophilic nanocarrier is a magnetic iron oxide nanoparticle with a surface modified with maleimide groups;

[0086] The IL-15 / IL-15Rα recombinant protein is directionally covalently linked to the maleimide group of the hydrophilic nanocarrier via a C-terminal cysteine ​​residue.

[0087] In this invention, the hydrophilic nanocarrier is magnetic iron oxide nanoparticles with surface-modified maleimide groups. Preferably, the maleimide groups are introduced onto the surface of the magnetic iron oxide nanoparticles via methoxy-PEG2000-maleimide (CH3O-PEG2000-Ma1).

[0088] In this invention, the method for preparing the hydrophilic nanocarrier preferably includes the following steps: mixing acetylacetone iron, benzyl ether and methoxy-PEG2000-maleimide, carrying out a high-temperature thermal decomposition reaction, and separating and purifying the mixture using an LS column to obtain the hydrophilic nanocarrier, denoted as NPM.

[0089] In this invention, the preferred mass ratio of iron acetylacetone to methoxy-PEG2000-maleimide is 1~2:4~6. In this invention, the preferred mass ratio of iron acetylacetone to benzyl ether is 0.18~0.35g:2~4mL.

[0090] In this invention, the preferred temperature for the high-temperature thermal decomposition reaction is 200-300°C. o C, the time is preferably 2-3 hours. In this invention, the heating rate is preferably 5-10. o C / min.

[0091] After the high-temperature thermal decomposition reaction was completed, hydrophilic nanocarriers were obtained by LS column chromatography and purified. The nanocarriers were then dispersed in water and placed at 4°C. o Save as C.

[0092] In this invention, the particle size of the hydrophilic nanocarrier is preferably 10~100 nm, more preferably 10~50 nm, and even more preferably 10~20 nm.

[0093] In this invention, the IL-15 / IL-15Rα recombinant protein is of mouse or human origin. In this invention, the IL-15 / IL-15Rα recombinant protein contains the same amino acid sequence as SEQ ID No. 1; or contains at least 90-99% of the same amino acid sequence as SEQ ID No. 1, specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In this invention, from the N-terminus to the C-terminus, SEQ ID No. 1 specifically represents: MGSLQPLATLYLLGMLVASSLGNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSGGSGSGGGGSGGGGSGGGSGASGGSGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTVVTPKVTSQPESPSPSAKEPEAGSGSGSGSHHHHHHGGSAWSHPQFEKGGGSGGGSGGSAWSHPQFEKC. In this invention, the amino acid sequence shown in SEQ ID No. 1 is the amino acid sequence of the murine IL-15 / IL-15Rα recombinant protein.

[0094] Alternatively, in this invention, the IL-15 / IL-15Rα recombinant protein contains the same amino acid sequence as SEQ ID No. 2; or contains at least 90-99% of the same amino acid sequence as SEQ ID No. 2, specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In this invention, from the N-terminus to the C-terminus, SEQ ID No. 2 specifically represents: MGSLQPLATLYLLGMLVASSLGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGSGGGGSGGGGSGGGGSGASGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAGSGSGSGSHHHHHHGGSAWSHPQFEKGGGSGGGSGGSAWSHPQFEKC. In this invention, the amino acid sequence shown in SEQ ID No. 2 is the amino acid sequence of the human IL-15 / IL-15Rα recombinant protein.

[0095] In this invention, the method for preparing the IL-15 / IL-15Rα recombinant protein preferably includes the following steps:

[0096] The amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2 were converted into DNA sequences and cloned into lentiviral plasmid vectors to obtain human or mouse IL-15 / IL-15Rα lentiviral plasmid vectors.

[0097] The human or mouse IL-15 / IL-15Rα lentiviral plasmid vector, VSV-G, Rev, and pMDLg lentiviral packaging plasmids were added to 293T cells and co-transfected with transfection reagents to obtain human or mouse IL-15 / IL-15Rα recombinant protein lentivirus.

[0098] The recombinant protein lentivirus was added to CHO-S cell suspension for cell transfection to obtain stable cell lines expressing human or mouse IL-15 / IL-15Rα;

[0099] The stable cell lines expressing human or mouse IL-15 / IL-15Rα were cultured, and the supernatant of the culture medium was collected and purified to obtain recombinant human or mouse IL-15 / IL-15Rα proteins, which were named hIL-15 / IL-15Rα and mIL-15 / IL-15Rα, respectively.

[0100] In this invention, the number of hIL-15 / IL-15Rα recombinant proteins linked to a single hydrophilic nanocarrier is preferably ≥3, specifically 3 to 50. In this invention, the particle size of the nanovaccine with directed hIL-15 / IL-15Rα linkage is preferably 20 to 100 nm, more preferably 30 to 60 nm.

[0101] The present invention provides a method for preparing the above-mentioned nanovaccine with directional linkage of hIL-15 / IL-15Rα, comprising the following steps: mixing a hydrophilic nanocarrier and hIL-15 / IL-15Rα recombinant protein, performing a directional coupling reaction to obtain hydrophilic nanoparticles linked with hIL-15 / IL-15Rα recombinant protein;

[0102] This invention involves a coupling reaction between a hydrophilic nanocarrier and the hIL-15 / IL-15Rα recombinant protein to obtain a hydrophilic nanocarrier linked to the hIL-15 / IL-15Rα recombinant protein, denoted as hIL-15 / IL-15Rα-NPM. In this invention, the mass ratio of the hydrophilic nanocarrier to the hIL-15 / IL-15Rα recombinant protein is preferably 1:0.5 to 1.5, more preferably 1:0.5. In this invention, the coupling reaction is preferably carried out on a shaker at room temperature for a time of 12 to 24 hours, more preferably 18 to 20 hours.

[0103] After the coupling reaction, the present invention preferably uses an LS column to remove unbound hIL-15 / IL-15Rα free protein, and collects the product hIL-15 / IL-15Rα-NPM and resuspends it in PBS buffer.

[0104] This invention provides the application of the above-described nanovaccine with directed linkage to hIL-15 / IL-15Rα in the preparation of antitumor immunomodulators. In this invention, the tumor preferably includes one or more of the following: lung cancer, liver cancer, melanoma, liver metastases of colon cancer, liver metastases of melanoma, lung metastases of melanoma, pancreatic cancer, rectal cancer, and breast cancer.

[0105] The following detailed description, in conjunction with embodiments, illustrates the nanovaccines linked to IL-15 / IL-15Rα provided by the present invention, their preparation methods, and their application in the preparation of antitumor immunotherapeutic agents. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0106] In the following examples, methoxy-PEG2000-maleimide (CH3-PEG2000-Mal) was purchased from Shanghai Pengshuo Biotechnology Co., Ltd.; and LS magnetic columns were purchased from Miltenyi Biotechnology Co., Ltd., Germany.

[0107] Example 1: Preparation of hydrophilic nanocarriers

[0108] (1) Mix 1 g CH3-PEG2000-Mal, 2.5 mL benzyl ether and 0.24 g ferric acetylacetone, and heat to 250°C. o At C, the reaction proceeded for 2.5 h to form nanoparticles. After cooling, the nanoparticles were washed with n-hexane to obtain a black product. The product was then dispersed in deionized water, and insoluble matter was removed by centrifugation. The solution was separated by an LS magnetic column and dispersed in water to obtain 10 mL of hydrophilic iron oxide nanocarriers. o Stored in a C refrigerator. The prepared iron oxide nanoparticles were named NPM.

[0109] Structural characterization of NPM nanoparticles

[0110] (1) Detection of iron ion concentration by Prussian blue colorimetric method: The nanoparticle solution was treated with 3 mol / L hydrochloric acid and heated to allow it to fully dissociate and release Fe. 3+ Subsequently, it reacted with potassium ferrocyanide to form a Prussian blue complex. After dilution, the absorbance was measured at a wavelength of approximately 710 nm, and based on Fe... 3+ The concentration is calculated using a standard curve. A quantitative standard curve for iron ion concentration is shown below. Figure 1 As shown, R 2 =0.999. Calculations show that the Fe content in the NPM nanoparticle solution is... 3+ The concentration was 1.86 mg / mL.

[0111] (2) Transmission electron microscopy image of NPM nanoparticles as shown Figure 2 As shown, by Figure 2 It can be seen that the NPM particle size is 14.1 ± 2.02 nm.

[0112] (3) Hydrated particle size diagram of NPM nanoparticles as shown in Figure Figure 3 As shown, by Figure 3 It can be seen that the hydrated particle size of NPM nanoparticles is 25.4±2.1 nm and the PDI is 0.22±0.03, indicating that the nanoparticles have good dispersibility in water.

[0113] (4) Infrared characterization of NPM nanoparticles as shown in the figure Figure 4 As shown, by Figure 4 It can be seen that CH 3- PEG2000-Mal at approximately 690cm -1The presence of a characteristic absorption peak at the point of origin, which is attributed to the maleimide group, is also observed in the infrared spectrum of the NPM nanoparticles, indicating that the maleimide group has been successfully introduced into the nanoparticles.

[0114] Example 2 Preparation of IL-15 / IL-15Rα recombinant protein

[0115] The amino acid sequence of the constructed mouse IL-15 / IL-15Rα recombinant protein is shown in SEQ ID No. 1 above, and the amino acid sequence of the human IL-15 / IL-15Rα recombinant protein is shown in SEQ ID No. 2 above.

[0116] (1) Construction of IL-15 / IL-15Rα recombinant protein lentiviral plasmid vector: The amino acid sequences of SEQ ID No.1 and SEQ ID No.2 above were converted into DNA sequences and cloned into lentiviral plasmid vectors containing green fluorescent protein elements, and named hIL-15 / IL-15Rα lentiviral plasmid vector and mIL-15 / IL-15Rα lentiviral plasmid vector, respectively.

[0117] (2) Packaging of IL-15 / IL-15Rα recombinant protein lentivirus: 1 μg of hIL-15 / IL-15Rα lentivirus plasmid vector and mIL-15 / IL-15Rα lentivirus plasmid vector were mixed with 0.3 μg VSV-G, 0.2 μg Rev, and 0.5 μg pMDLg lentivirus packaging plasmid, respectively, and jetPRIME transfection reagent was added. The mixture was then slowly added to 293T cells. After 6 h, the medium was replaced with fresh medium. On the third day, the supernatant of the medium was treated according to the instructions of the JetPRIME lentivirus concentrate to obtain lentivirus. After aliquoting, the lentivirus was stored at -80°C. o Save as C.

[0118] (3) Construction of stable CHO-S cell lines expressing IL-15 / IL-15Rα recombinant protein: The above-mentioned hIL-15 / IL-15Rα recombinant protein lentivirus and mIL-15 / IL-15Rα recombinant protein lentivirus were added to CHO-S cell suspension, and 2 μg / mL Polybrene was added. After transfection and expansion to a certain number of cells, cells with green fluorescent positive signals were separated by flow cytometry for further expansion. Finally, the construction of stable cell lines expressing hIL-15 / IL-15Rα and mIL-15 / IL-15Rα recombinant proteins was completed.

[0119] (4) Purification of IL-15 / IL-15Rα recombinant protein: The stable cell lines expressing hIL-15 / IL-15Rα and mIL-15 / IL-15Rα recombinant proteins were expanded to six 1 L culture flasks. Cell viability was monitored, and when cell viability was below 90%, the culture supernatant was collected by centrifugation at 3000 rpm for 30 min. The protein was concentrated using a 10 kDa Millipore tangential flow ultrafiltration membrane, and the culture medium was replaced with His binding buffer (pH=8.0). Two-step purification was performed using a Cytiva protein purifier with HisTrap HP column and Strep-Tactin affinity chromatography column. The collected protein solution was replaced with PBS buffer using a PD-10 column, and the protein solution was filtered through a 0.22 μm sterile filter membrane to obtain purified IL-15 / IL-15Rα recombinant protein. The protein concentration was determined using a Bradford protein quantification kit and then stored at -80°C. o Store in a refrigerator (C).

[0120] The purity of the purified hIL-15 / IL-15Rα and mIL-15 / IL-15Rα recombinant proteins was determined by SDS-PAGE. The resulting SDS-PAGE gel electrophoresis images are shown below. Figure 5 As shown, by Figure 5 As can be seen, the protein bands are clear and there are no obvious impurities, indicating that the prepared hIL-15 / IL-15Rα and mIL-15 / IL-15Rα recombinant proteins have high purity.

[0121] Test Example 1: In vitro activity verification of human and mouse IL-15 / IL-15Rα recombinant protein

[0122] (1) Whole spleen cells labeled with CFSE fluorescent dye

[0123] CFSE is a dye that can penetrate the cell membrane and form a stable fluorescent label inside the cell. Its fluorescence intensity halves with each cell division, and cell proliferation can be analyzed by detecting fluorescence decay. Mouse spleens were harvested, ground on a ground glass slide, and passed through a 300-mesh filter to obtain a single-cell suspension. The suspension was centrifuged at 1800 rpm for 5 min, the supernatant was discarded, and 5 mL of erythrocyte lysis buffer was added for lysis at room temperature for 5 min. Lysis was terminated by adding 5 mL of PBS, and the suspension was centrifuged again at 1800 rpm for 5 min. The supernatant was discarded, and the cell pellet at the bottom of the centrifuge tube was collected. The cells were resuspended in 1640 complete culture medium (containing sodium pyruvate, MEM non-essential amino acids, Glutamax, HEPES, and β-mercaptoethanol) until the cell density reached 1 × 10⁻⁶ cells / mL. 7Add CFSE solution to a concentration of 5 μM, centrifuge tubes at 2 × 10⁶ cells / mL, and incubate for 10 min, shaking occasionally every 3-5 min. Add 2 mL of PBS, centrifuge at 1800 rpm for 5 min to remove unbound CFSE, discard the supernatant, add 1 mL of culture medium, mix well, and count the cells. Then, centrifuge at 2 × 10⁶ cells / mL. 6 Cells were seeded per well in 48-well plates and divided into Control group, mIL-15 / IL-15Rα group and hIL-15 / IL-15Rα group. Culture medium, 100 ng / mL mIL-15 / IL-15Rα and 100 ng / mL hIL-15 / IL-15Rα were added respectively.

[0124] (2) In vitro activity verification of IL-15 / IL-15Rα

[0125] After co-incubation for 72 h, cells were collected in 2 mL centrifuge tubes, centrifuged at 1800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 20 μL of FACS buffer. Then, the cells were incubated with FACS buffer containing anti-CD3 PE-Cy7, anti-CD4 BV510, anti-CD8 BV421, and anti-NK1.1 Percp-Cy5.5 at room temperature in the dark for 1 h. The cells were then resuspended in 800 μL of PFA buffer. o Incubate at C in the dark for 1 h, centrifuge and discard the supernatant, then resuspend in 300 μL of FACS buffer and detect CD8+ using flow cytometry. + CFSE signal intensity in T cells and NK cells.

[0126] CFSE signal intensity graph for in vitro activity verification of IL-15 / IL-15Rα is shown below. Figure 6 As shown, by Figure 6 It can be seen that CD8 + The CFSE signal intensity peaks in both T cells and NK cells showed a shift back, and the CFSE fluorescence shift rate in the recombinant protein experimental group was significantly increased. This indicates that the prepared recombinant proteins mIL-15 / IL-15Rα and hIL-15 / IL-15Rα possess good immunomodulatory activity and can effectively stimulate CD8+. + T cell and NK cell proliferation. Given that both the prepared human and mouse recombinant proteins are active, the human recombinant protein was selected for the preparation of nanovaccines and subsequent anti-tumor research in order to achieve clinical translation studies.

[0127] Example 3: Preparation of a nanovaccine with targeted linkage of hIL-15 / IL-15Rα

[0128] (1) 3 mL of PBS buffer containing 1 mg of NPM nanoparticles and 0.5 mg of the hIL-15 / IL-15Rα recombinant protein obtained in Example 2 were placed on a shaker and reacted for 12 h. Unbound free hIL-15 / IL-15Rα recombinant protein was removed using an LS column to obtain 3 mL of nanoparticle solution conjugated with hIL-15 / IL-15Rα recombinant protein. The nanoparticles were named hIL-15 / IL-15Rα-NPM.

[0129] Take 30 μL of the above nanoparticle solution, add 5× protein loading buffer, and incubate in a metal bath at 95°C. o Heating at C for 10 min followed by electrophoretic analysis, the number of protein linkages was calculated using the gray values ​​of the protein bands.

[0130] (2) The obtained hIL-15 / IL-15Rα-NPM nanoparticle gel electrophoresis image is shown below. Figure 7 As shown. Figure 7 Image (a) shows SDS-PAGE and Native-PAGE gel electrophoresis results; Figure 7 (b) is the SDS-PAGE grayscale value quantitative standard curve of hIL-15 / IL-15Rα protein.

[0131] ImageJ software was used to analyze the gray values ​​of protein bands in the SDS-PAGE gel electrophoresis image in Figure (a), and a standard curve was plotted to compare gray values ​​with protein mass. This curve was used to calculate the number of hIL-15 / IL-15Rα protein linkages. The resulting standard curve is shown below. Figure 7 As shown in (b), the results show the R-value of the standard curve. 2 =0.9974, indicating that this method has high accuracy for protein quantification; combined with the quantification of Fe in Example 1 3+ Method for calculating Fe in solution 3+ The content was calculated to be 12, which is derived from the nanoparticle-linked recombinant protein content. Figure 7 As shown in the Native-PAGE gel electrophoresis image (a), there was no free hIL-15 / IL-15Rα protein in the gel lanes of the hIL-15 / IL-15Rα-NPM nanovaccine group. This is because the hIL-15 / IL-15Rα protein coupled to the NPM nanoparticles could not enter the lanes, indicating that hIL-15 / IL-15Rα and NPM nanoparticles are covalently bound.

[0132] Structural characterization of hIL-15 / IL-15Rα-NPM nanovaccine

[0133] The hydrated particle size of the hIL-15 / IL-15Rα-NPM nanovaccine prepared in Example 3 was determined using dynamic light scattering (DLS), and its stability was tested after 1, 3, 6, and 12 weeks.

[0134] The hydration particle size (a) and stability test results (b) of the hIL-15 / IL-15Rα-NPM nanovaccine are shown in the figure. Figure 8 As shown. By Figure 8 As can be seen in (a), the average hydrated particle size of the hIL-15 / IL-15Rα-NPM nanovaccine is 43.1 ± 1.2 nm, and the PDI is 0.21 ± 0.017. Figure 8 As can be seen from (b) in the figure, the particle size of the nano-vaccine only fluctuated slightly during the test, showing good stability.

[0135] Test Example 2: hIL-15 / IL-15Rα-NPM in vitro activation of memory CD8 + T-cell validation

[0136] (1) Mouse memory CD44 high CD8 + T-cell isolation and extraction

[0137] Mouse spleens were harvested, ground using a ground glass slide, and passed through a 300-mesh filter to obtain a single-cell suspension. The suspension was centrifuged at 800 g for 8 min, the supernatant was discarded, and 5 mL of erythrocyte lysis buffer was added for lysis at room temperature for 5 min. Lysis was terminated with 5 mL of PBS, and the suspension was centrifuged again at 800 g for 8 min. The supernatant was discarded, and the cell pellet was resuspended in FACS buffer. The suspension was then incubated with FACS buffer containing anti-B220 FITC and anti-CD4 FITC at room temperature in the dark for 30 min. Unbound antibodies were washed with 2 mL of separation buffer, and the suspension was resuspended in 4 mL of FACS buffer. 100 μL of Anti-FITC MagneticBeads was added, and the centrifuge tubes were incubated on ice for 20 min. After incubation, the centrifuge tubes were magnetically separated on a magnetic rack for 2 min. After magnetic separation, the supernatant was transferred to a new centrifuge tube, centrifuged to remove unbound beads, and the cell pellet was resuspended in FACS buffer. The suspension was then incubated with anti-CD3 PE-Cy7, anti-CD8 APC, and anti-CD44. Incubate APC-Cy7 in FACS buffer solution at room temperature in the dark for 30 min, then wash unbound antibodies with 2 mL of separation buffer, resuspend in culture medium, and sort using a flow cytometer to obtain high-purity memory CD44. high CD8+ T cells.

[0138] like Figure 9 As shown in (a), the vast majority of cells obtained after sorting are CD8. + T cells, and memory cells CD44 high CD8 + T cells account for a significant portion of CD8. + The presence of 88.1% of T cells indicates the acquisition of high-purity memory CD8. + T cells will be used for subsequent experimental research.

[0139] (2) Mouse memory CD8 + T-cell activation assay

[0140] Memory CD44 high CD8 + T cells at 2×10 4 Cells / well density were added to 96-well U-shaped plates and co-incubated with different concentrations (0, 10, 20, 50, 100, 500 ng / mL) of hIL-15 / IL-15Rα and hIL-15 / IL-15Rα-NPM at 37°C. o Cells were cultured at 5% CO2 for 24 h and 48 h, and then collected. They were stained at room temperature for 1 h with anti-CD8 APC, anti-CD44 APC-Cy7, anti-CD25BV421, and anti-CD69 PE, and then further stained at 4°C. o Mouse CD8+ was fixed for 40 min, centrifuged, and resuspended in 300 μL of FACS buffer. Flow cytometry was used to detect mouse memory CD8+. + Indicators of T cell activation.

[0141] hIL-15 / IL-15Rα-NPM in vitro activation of memory CD8 + T cells such as Figure 9 As shown in (b) and (c), after co-incubation for 24 h, the nanovaccine significantly upregulated memory CD8 compared with free hIL-15 / IL-15Rα protein. + T cell CD69 and CD25 expression indicate that the nanovaccine is more effective than free hIL-15 / IL-15Rα protein in activating memory CD8. + T cells.

[0142] Test Case 3: In vivo tumor targeting validation of hIL-15 / IL-15Rα-NPM

[0143] (1) hIL-15 / IL-15Rα and hIL-15 / IL-15Rα-NPM labeled Alexa Fluor 647 fluorescent small molecules

[0144] Take the protein solution and add 1 / 10 volume of 1 M NaHCO3 buffer to adjust the pH of the reaction system. Add AF647 solution at a dye:protein molar ratio of 4:1, mix gently, and react at room temperature in the dark. Remove unbound free dye using an ultrafiltration centrifuge tube, centrifuging multiple times until the filtrate is colorless. o Store in a cool, dark place in a refrigerator for later use.

[0145] The prepared hIL-15 / IL-15Rα / AF647 solution was used to obtain the product hIL-15 / IL-15Rα-NPM / AF647 NPs according to the method described in Example 3. o Store in a cool, dark place in a refrigerator for later use.

[0146] (2) Small animal live imaging

[0147] BALB / c mouse 4T1 breast cancer was constructed in situ. On day 7, the 4T1 tumor-bearing mice were divided into two groups: hIL-15 / IL-15Rα / AF647 and hIL-15 / IL-15Rα-NPM / AF647. hIL-15 / IL-15Rα / AF647 and hIL-15 / IL-15Rα-NPM / AF647 (hIL-15 / IL-15Rα dose: 2 μg) were injected into the tumor-bearing mice via the tail vein. Small animal in vivo imaging was performed at 2 h, 4 h, 6 h, 8 h and 12 h after injection. The mice were sacrificed after 12 h of in vivo imaging, and their heart, liver, spleen, lung, kidney, brain, lymph nodes and tumors were collected for fluorescence imaging.

[0148] like Figure 10 As shown, (a) is an in vivo fluorescence imaging image of mice within 12 h after drug administration, and (b) is an in vitro fluorescence imaging image of major organs and tumor tissues 12 h after drug administration. It can be seen that the nanovaccine hIL-15 / IL-15Rα-NPM / AF647 accumulates more effectively in the tumor site than the free protein hIL-15 / IL-15Rα / AF647, indicating that the nanovaccine has stronger tumor targeting.

[0149] Test Example 4: In vivo antitumor efficacy and safety study of hIL-15 / IL-15Rα-NPM nanovaccine

[0150] A B16 subcutaneous tumor model was established using female C57BL / 6 mice aged 7-9 weeks. On day 7 post-inoculation, tumors with a volume of approximately 30-40 mm were selected. 3Mice were then randomly divided into three groups of five mice each: a control group, an hIL-15 / IL-15Rα free protein group, and an hIL-15 / IL-15Rα-NPM nanovaccine group. Treatment was administered intravenously; the control group received PBS, and the doses of hIL-15 / IL-15Rα and hIL-15 / IL-15Rα-NPM (IL-15 / IL-15Rα protein) were 2 μg and 2 μg respectively. Five injections were administered, with two days between each injection. Tumor tissue was collected two days after the last injection to evaluate the treatment efficacy.

[0151] Take tumor tissue and cut it into 1 mm pieces 3 Place small pieces in a 5 mL centrifuge tube, add 3 mL of digestion solution containing 0.02 mg / mL DNase and 1 mg / mL type IV collagenase, and incubate at 37°C. o Incubate at C for 30 min. Transfer tissue blocks to 50 mL centrifuge tubes containing 70 μm filter membranes and grind to prepare single-cell suspension. After centrifugation, remove supernatant, add 5 mL of erythrocyte lysis buffer, lyse at room temperature for 5 min, add an equal volume of PBS for neutralization, centrifuge at 1800 rpm for 5 min, and discard supernatant. Add 5 mL of Percoll separation buffer (37.5%), centrifuge at 2000 rpm for 20 min (drop-off rate set to 0), discard supernatant and collect the lower cell pellet. Resuspend each tumor tissue in 300 μL of FACS buffer. Add 20 μL of cell suspension to 2 mL EP tubes, followed by 20 μL of FACS buffer containing anti-CD45APC-Cy7, anti-CD3 BV510, anti-CD8 BV421, and anti-NK1.1 Percp-Cy5.5. Incubate at room temperature in the dark for 1 h. Wash unbound antibodies with FACS buffer by centrifugation, then resuspend in 800 μL of PFA buffer. o Incubate at C (protected from light) for 1 h, centrifuge and discard the supernatant, then resuspend in 300 μL of FACS buffer. Detect CD8+ in tumor tissue using flow cytometry. + T cell and NK cell infiltration status.

[0152] Venous blood was collected from mice at 0 h, 4 h, 24 h, 76 h, and 96 h after the first administration. After standing at room temperature for 2 h, the blood was then... o Serum was collected by centrifugation at 4000 rpm under C conditions, and the levels of interferon-γ (IFN-γ) and interleukin-6 (IL-6), as well as the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were detected.

[0153] Evaluation of nano-vaccine therapy for B16 subcutaneous tumors Figure 11As shown, (a) is an image of the tumor; (b) is a statistical chart of tumor weight analysis. Figure 11 As shown in (a), the nanovaccine group significantly inhibited the progression of B16 subcutaneous tumors, and the tumor volume was significantly reduced, as shown in (a). Figure 11 As shown in (b), the tumor weight in the hIL-15 / IL-15Rα-NPM nanovaccine group was significantly lower than that in the Control group and the hIL-15 / IL-15Rα group. These results indicate that the hIL-15 / IL-15Rα-NPM nanovaccine has superior therapeutic efficacy against B16 subcutaneous tumors compared to the free hIL-15 / IL-15Rα protein.

[0154] CD8 in tumor tissue + T cell and NK cell infiltration status, such as Figure 12 As shown, (a) represents CD8⁺ in the tumor. (a) T cell flow cytometry and bar chart, (b) NK cell flow cytometry and bar chart in tumor, CD8⁺ in tumor of nanovaccine group The proportions of T cells and NK cells were significantly increased, indicating that the hIL-15 / IL-15Rα-NPM nanovaccine significantly increased CD8⁺ levels in tumor tissue compared to free hIL-15 / IL-15Rα protein. Infiltration of T cells and NK cells.

[0155] With the first administration time defined as 0 h and the second administration time as 72 h, the safety evaluation of the nano-vaccine is as follows: Figure 13 As shown, (a) and (b) represent the changes in the levels of IL-6 and IFN-γ in the peripheral blood serum of mice within 96 hours after the first administration; Figure 13 As shown in Figure (a), after the first dose, serum IL-6 levels in the hIL-15 / IL-15Rα group showed a burst of increase, reaching a peak of 55 pg / mL at 24 h, which was 11 times the baseline level and far exceeded the CRS warning threshold (3 times the upper limit of normal, i.e., 15 pg / mL). After a second dose at 72 h, IL-6 levels remained above the safety threshold. In contrast, IL-6 levels in the hIL-15 / IL-15Rα-NPM group remained at an extremely low level of less than 15 pg / mL throughout the entire treatment, with no abnormal increase after either dose. Figure 13 As shown in (b), IFN-γ in the hIL-15 / IL-15Rα group peaked at 24 h, reaching 6.75 times the baseline, and increased again after a second dose at 72 h. In contrast, IFN-γ in the nanovaccine group only showed a slight increase and quickly returned to baseline. These results indicate that hIL-15 / IL-15Rα can induce mild cytokine release syndrome (CRS), while hIL-15 / IL-15Rα-NPM effectively avoids the risk of CRS.

[0156] Figure 13(c) and (d) show the activities of AST and ALT in peripheral blood serum of mice within 96 hours after the first administration. The gray area in the figure represents the normal safety range indicated by the kit. In the hIL-15 / IL-15R group, liver enzymes significantly increased and far exceeded the normal range after the first administration, and remained abnormal after the second administration, showing typical acute liver injury. In contrast, liver enzymes in the hIL-15 / IL-15Rα-NPM nanovaccine group showed only small physiological fluctuations without persistent damage, and the levels at all time points were significantly lower than those in the free group. The results confirm that the nanovaccine can effectively alleviate the acute hepatotoxicity of free protein hIL-15 / IL-15Rα and significantly improve its safety in vivo.

[0157] In summary, the nanovaccine with targeted IL-15 / IL-15Rα provided by this invention can stimulate higher levels of CD8 compared to the IL-15 / IL-15Rα recombinant protein. + The expansion of T cells and NK cells, and activation of CD44 + CD8 + T cells; simultaneously, this nanovaccine exhibits tumor-targeting properties, significantly enhancing CD8 levels in tumor tissue in a melanoma subcutaneous tumor model. + The infiltration of T cells and NK cells effectively avoids key issues such as rapid metabolism of free proteins and reduced systemic immunotoxicity, resulting in superior anti-tumor effects.

[0158] 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanovaccine that targets the complex of interleukin and its receptor activated by immune activation, characterized by, It includes a hydrophilic nanocarrier and an immune-activated interleukin and its receptor complex chemically linked to the hydrophilic nanocarrier. The hydrophilic nanocarrier is a magnetic iron oxide nanoparticle with a surface modified with maleimide groups; The immune-activated interleukin and its receptor complex are directionally covalently linked to the maleimide group of the hydrophilic nanocarrier via a C-terminal cysteine ​​residue.

2. The nanovaccine of claim 1, wherein, The immune-activated interleukin and its receptor complex are recombinant IL-15 / IL-15Rα proteins.

3. The nanovaccine of claim 2, wherein, The IL-15 / IL-15Rα recombinant protein contains the same amino acid sequence as SEQ ID No. 1; Or it may contain at least 90-99% of the same amino acid sequence as SEQ ID No.

1.

4. The nanovaccine according to claim 2, characterized in that, The IL-15 / IL-15Rα recombinant protein contains the same amino acid sequence as SEQ ID No. 2; Or it may contain at least 90-99% of the same amino acid sequence as SEQ ID No.

2.

5. The nano-vaccine according to claim 2, characterized in that, The number of IL-15 / IL-15Rα recombinant proteins directionally covalently linked to a single hydrophilic nanocarrier is ≥3; The hydrophilic nanocarrier has a particle size of 10~100 nm; The nanovaccine has a particle size of 20~100 nm.

6. The nanovaccine according to claim 1, characterized in that, The preparation process of the magnetic ferroferric oxide nanoparticles with surface modified maleimide group is as follows: mixing methoxy-PEG2000-maleimide, benzyl ether and acetylacetone iron, heating to 200-300 o C, reacting to form nanoparticles, cooling, washing, and separating by a magnetic column to obtain the nanoparticles.

7. The method for preparing the nano-vaccine according to any one of claims 1 to 6, characterized in that, Includes the following steps: A nanocarrier and IL-15 / IL-15Rα recombinant protein were directionally coupled to obtain a nanovaccine with IL-15 / IL-15Rα recombinant protein.

8. The use of the nanovaccine according to any one of claims 1-6 in the preparation of antitumor agents.

9. The use of the nanovaccine according to any one of claims 1-6 in in vitro expansion and activation of immune cells.

10. The use of the nanovaccine according to any one of claims 1-6 in the delivery of immune-activated interleukins and their receptor complexes, wherein the immune-activated interleukins include at least one of IL-1α / β, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-23.

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