A NK cell engager and preparation method thereof
By using NK cell adapters made of Bi2S3 nanomodifications to combine with cell membrane fragments of cancer cells and activate NK cells, it solves the problems of limited effectiveness of existing immunotherapy methods and immune-related adverse events, and achieves effective identification of tumor cells. and killing.
Patent Information
- Application Number
- CN202411337195.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-25
- Publication Date
- 2025-01-21
- Estimated Expiration
- 2044-09-25
AI Technical Summary
Existing immunotherapy methods, such as ICIs, have limited effects and immune-related adverse events, making it difficult to fully control tumor progression, especially for the treatment of single ICIs.
Bi2S3 nanomodification is used as the core component of the NK cell engager. By combining with the cell membrane fragments of cancer cells, it forms a targeting envelope layer, activates NK cells, and enhances its ability to recognize and kill tumor cells.
Significantly activates NK cells and improves their ability to recognize and kill cancer cells, providing a new tumor immunotherapy solution, especially suitable for the treatment of digestive tract and colon cancers.
Smart Images
Figure CN118845712B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a NK cell engager and a preparation method thereof. Background Art
[0002] Cancer is a highly complex disease characterized not only by the overgrowth of malignant cells but also by altered immune responses. In the occurrence and development of tumors, suppression and reprogramming of the immune system play a key role. The goal of immunotherapy is to reactivate antitumor immune cells and overcome the immune escape mechanisms of tumors. Immunotherapy methods such as immune checkpoint inhibitor therapy (ICIs), monoclonal antibody therapy, adoptive cell therapy, and tumor vaccine therapy are often used to treat cancer. However, only a small number of patients with immunogenic tumors can benefit from single treatment methods such as ICIs. The occurrence of immune-related adverse events (irAEs) cannot be ignored. They can affect almost all organ systems, limit the benefits of drugs, and seriously endanger the prognosis of patients. In essence, ICIs only inhibit the immune escape of tumor cells. Considering the multiple aspects of the immune system and the complex tumor immune microenvironment, as well as the slow onset (usually 3 months), the therapeutic effect of ICIs is greatly hindered. In addition, due to the continuous discovery of irAEs, a single immunotherapy method is no longer sufficient to fully control tumor progression, especially for the treatment of a single ICI. It often needs to be used in combination with other immunotherapies or targeted therapies to improve efficacy and reduce the occurrence of irAEs.
[0003] NK cell immunotherapy is another anti-tumor method with great clinical prospects after T lymphocyte immunotherapy. First, NK cells can release killing activity mediators as unique cytotoxicity; second, NK cells can also secrete some cytokines and chemokines, such as IFN-γ, to evoke other immunological responses; third, NK cells can also bind to antibodies on the surface of tumor cells to establish an adaptive immune system. Therefore, the mechanism of NK cell activation includes natural cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). However, the cost of NK cell therapy often stops patients. On the other hand, the use of nanobiomaterials as a NK cell engager (NKCEs) is expected to enhance the process of NK cell tropism, exudation, and active enrichment, promote the intratumoral recruitment of NK cells, and the use of nanobiomaterials to improve the recognition accuracy and efficiency of NK cells may be another potential strategy for NK cell immunotherapy. Summary of the invention
[0004] In order to solve the problems existing in the prior art, the present invention provides a nano material use, a NK cell engager and a preparation method thereof, and provides an activation effect on NK cells by discovering new nano materials.
[0005] The technical solution adopted by the present invention is:
[0006] In a first aspect, the present invention provides a use of a nanomaterial, including a nano-modified substance of Bi2S3, which is used to activate NK cells.
[0007] In combination with the first aspect, the present invention provides a first implementation mode of the first aspect, wherein nanoparticles containing Bi2S3 are modified by PVP to obtain a nano-modified product.
[0008] In a second aspect, the present invention further provides a NK cell engager, which uses the above-mentioned nano-modifier containing Bi2S3 to form a targeted coating layer formed by cell membrane fragments extracted from cancer cells on the surface of the nano-material.
[0009] In combination with the second aspect, the present invention provides a first embodiment of the second aspect, which is applied in the treatment of gastrointestinal cancer by activating NK cells.
[0010] In combination with the second aspect, the present invention provides a second embodiment of the second aspect, which is applied in the treatment of colon cancer by activating NK cells.
[0011] In a third aspect, the present invention further provides a preparation method for preparing the above-mentioned NK cell engager, which is as follows:
[0012] After the BiC3 aqueous solution and the modifier are mixed and stirred, the Na2S aqueous solution is added and mixed and heated to obtain the Bi2S3 nano-modifier, which is then mixed with the obtained cancer cell membrane fragments and extruded through a micro liposome extruder to obtain the Bi2S3 NK cell engager.
[0013] In combination with the third aspect, the present invention provides a first implementation manner of the third aspect, wherein the modifier is an aqueous solution of PVP.
[0014] In combination with the third aspect, the present invention provides a second implementation of the second aspect, wherein after the cell membrane fragments of cancer cells are mixed with the nano-modified Bi2S3, they are first incubated at 3-6°C for 2-3 hours.
[0015] In combination with the third aspect, the present invention provides a third implementation of the second aspect, and the specific steps are as follows:
[0016] Firstly, a BiCl3 aqueous solution and a PVP aqueous solution are mixed and stirred in water at a mass ratio of 0.5-0.8 at room temperature to form a mixed solution, and then an excess of Na2S aqueous solution is added to the mixed solution, stirred and mixed, and then transferred to a 90°C water bath environment and heated for 30-45 minutes to react to form PVP-modified, after the reaction is completed, cooled to room temperature and centrifuged to remove free PVP, and finally the PVP-modified Bi2S3 nanomaterial is dispersed in methanol for later use;
[0017] Then, cell membrane fragments were extracted from the tumor cells using a membrane protein extraction kit, and then the reserved Bi2S3 nanomaterials were mixed with the cell membrane fragments at a mass ratio of 0.4-0.7, incubated at 4-6°C for 2 hours, and then extruded through a 400nm porous polycarbonate film using a micro-liposome extruder for at least 10 times. Finally, the excess cell membrane fragments were removed by centrifugation at 4-6°C to obtain the NK cell engager of the Bi2S3 nanomaterial.
[0018] The beneficial effects of the present invention are:
[0019] The present invention synthesizes a new nanomaterial and finds that the material is associated with NK cells and can activate NK cells, thereby providing a new solution for tumor immunotherapy; at the same time, the NK cell engager made based on the nanomaterial has a significant activation effect and can be used in clinical immunotherapy for certain types of cancer cells;
[0020] The present invention can also efficiently synthesize the NK cell engager of the modified Bi2S3 nanomaterial through a defined preparation method. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 is a scanning electron microscope image of the Bi2S3 nanomaterial in an embodiment of the present invention;
[0022] Figure 2 Bi2S3@M in the embodiment of the present invention CT26 Transmission electron microscope image of
[0023] Figure 3 are solid images of mouse tumors after different treatments in the embodiments of the present invention;
[0024] Figure 4 Schematic diagram of NK cell surface receptors under different treatment methods in the embodiments of the present invention;
[0025] Figure 5 Schematic diagram of the expression of NK cell ligand proteins under different treatment methods in the embodiments of the present invention;
[0026] Figure 6are images of receptors and ligands associated with the surface of NK cells in different treatment methods in the embodiments of the present invention;
[0027] Figure 7 It is a schematic diagram of the expression of receptors and ligands associated with NK cells under different treatment methods in the embodiments of the present invention;
[0028] Figure 8 This is a graph showing the ratio of CD49b membrane surface glycoproteins in detecting the ratio of mouse spleen reactive NK cells by flow cytometry in an embodiment of the present invention;
[0029] Fig. 9 is a schematic diagram of the proportion of CD4+T cells in the spleen tissue of a fluorescently labeled mouse obtained by flow cytometry in an embodiment of the present invention;
[0030] Fig.10 is a schematic diagram of the proportion of fluorescently labeled CD8 cells in mouse spleen tissue obtained by flow cytometry in an embodiment of the present invention;
[0031] Fig.11 It is a schematic diagram of the proportion of the reactant CD206 used to reflect the proportion of macrophages after flow cytometry analysis in an embodiment of the present invention. DETAILED DESCRIPTION
[0032] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments.
[0033] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments.
[0034] Therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the present application for which protection is sought, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.
[0035] Embodiment 1:
[0036] This embodiment discloses a novel use of nanomaterials, which is a nano-modified product made of Bi sulfide as the main material, specifically a modified product of Bi2S3, which is used to activate NK cells, thereby achieving the effect of clearing cancer cells in the living body through the immune system.
[0037] This embodiment also discloses a NK cell engager made of Bi2S3 nano-modifier, that is, a targeting thin film layer formed by cell membrane fragments of extracted cancer cells is covered on the surface of the obtained Bi2S3 nano-modifier.
[0038] The NK cell engager is mainly used in immunotherapy for digestive tract cancer in vivo, especially in immunotherapy for colon cancer.
[0039] This embodiment provides a method for manufacturing the Bi2S3 nano-modified product and the NK cell engager, which is as follows:
[0040] The preparation method of Bi2S3 nano-modification is as follows: first, a BiC3 aqueous solution and a PVP aqueous solution are mixed in water at a mass ratio of 0.5-0.8 at room temperature to form a mixed solution, and then an excess of Na2S aqueous solution is added to the mixed solution, stirred and mixed, and then transferred to a 90°C water bath environment and heated for 30-45 minutes to react to form PVP-modified. After the reaction is completed, it is cooled to room temperature and centrifuged to remove free PVP. Finally, the PVP-modified Bi2S3 nanomaterial is dispersed in methanol for later use.
[0041] Reference Figure 1 , Figure 1 The figure shows the structure of Bi2S3. At two different magnifications, it can be seen that Bi2S3 is a porous spherical structure. Figure 1 This shows that the nano-modified Bi2S3 can be successfully synthesized through this preparation method.
[0042] Then, the method for preparing the NK cell engager is as follows: cell membrane fragments are extracted from tumor cells using a membrane protein extraction kit, and then the reserved Bi2S3 nanomaterials are mixed with the cell membrane fragments at a mass ratio of 0.4-0.7, incubated at 4-6°C for 2h, and then extruded through a 400nm porous polycarbonate film using a micro liposome extruder for at least 10 times, and finally centrifuged at 4-6°C to remove excess cell membrane fragments to obtain the NK cell engager of the Bi2S3 nanomaterial.
[0043] Reference Figure 2 , the figure shows the morphology of Bi2S3 nanomaterials coated by targeted thin film layers, and Figure 1 The difference is that they all exist in the solution in a separate state.
[0044] In order to verify the effect of the NK cell engager, experiments were conducted on mice in this example.
[0045] Experimental procedures
[0046] 1. Preparation of NK cell nanoengagers
[0047] First, BiCl3 aqueous solution (28.8 mg / mL, 10 mL) and PVP aqueous solution (40 mg / mL, 10 mL) were added to 20 mL of water, and the mixture was stirred at room temperature for 45 min. Next, Na2S aqueous solution (1.3 g / mL, 320 μL) was added to the above mixture. The resulting solution was stirred for another 5 min and then transferred to a 90 °C water bath for 30 min. After the reaction was completed, the PVP-modified Bi2S3 nanosphere solution was cooled to room temperature and centrifuged to remove excess free PVP. Finally, the PVP-modified Bi2S3 was dispersed in methanol.
[0048] In order to construct Bi2S3@MemCT26 (Bi2S3@M CT26 ), cell membrane fragments were extracted from CT26 cells using a membrane protein extraction kit. Subsequently, Bi2S3 (1 mg / mL, 0.5 mL) was mixed with CT26 cell membrane fragments (2 mg / mL, 0.5 mL) and incubated at 4°C for 2 hours. The membrane was physically extruded 11 times through a 400 nm porous polycarbonate film using an Avantiminer extruder. Excess membrane fragments were removed by centrifugation (7000 rpm, 10 min, 4°C) to obtain Bi2S3@M CT26 .
[0049] Figure 1 The Bi2S3 nanosphere structure obtained in this experiment is shown. Figure 2 The Bi2S3@M CT26 Structure diagram of .
[0050] 2. Group test on the activation effect of NK cells
[0051] First, the experimental groups were divided into:
[0052] 1) Normal saline group
[0053] 2) Isotype control group
[0054] 3) aPD-1 group
[0055] 4) Bi2S3@M CT26 Group
[0056] The so-called isotype control group refers to other existing activation methods, while the aPD-1 group refers to the group that enhances tumor immunotherapy by binding to PD-1 antibody (aPD-1).
[0057] 2.1 Immunohistochemistry: Tumor tissue sections were routinely dewaxed and hydrated, followed by antigen repair and blocking, incubation with primary antibody overnight at 4°C, incubation with secondary antibody for 60 min, color development, nuclear counterstaining with hematoxylin, dehydration and sealing, observation under an optical microscope, and photography. IPP6.0 software was used for analysis.
[0058] 2.2. Western blot method: 40 mg of total protein from tumor tissue of each group of mice was extracted, separated by electrophoresis after preparation of gel, and the protein was transferred to PVDF membrane, blocked with 5% skim milk powder for 2 h, and primary antibody (NKG2D, RAE-1) was added. The membrane was incubated at 4°C overnight, washed 3 times with TBST, 5 min / time, and secondary antibody was added. The membrane was incubated at room temperature for 2 h, washed 3 times with TBST, 5 min / time, and developed with ECL luminescence kit. The relative expression of proteins was statistically analyzed by ImageJ software.
[0059] 2.3. Flow cytometry: After the treatment, the spleen of the mouse was removed, ground and filtered into a single cell suspension, centrifuged, the supernatant was discarded, red blood cell lysis buffer was added for reaction, the cells were washed with PBS, and the immune cells were stained with fluorescently labeled antibodies (anti-CD4, anti-CD8, anti-CD86, anti-CD206, anti-CD11b) and analyzed by flow cytometry.
[0060] 3. Experimental results
[0061] Reference Figure 3 The figure shows the size of tumor tissues of mice after four treatments. The isotype control group and aPD-1 group can delay tumor growth, and the tumors of mice in the Bi2S3@MCT26 group were significantly reduced and significantly inhibited, with a more obvious effect.
[0062] As an indicator of NK cell activity, NKG2D is an activating receptor on the surface of NK cells, and RAE-1 is one of its main ligands. During the process of NK cell activation, tumor cells can escape NK cell killing by downregulating the expression of NKG2D. Figure 4 and Figure 5 In the mouse tumor tissues treated with Bi2S3@MCT26, the expression of NKG2D and RAE-1 proteins in the mouse tumor tissues was significantly increased.
[0063] Reference Figure 6 and Figure 7 The immunohistochemistry method also confirmed that the expression of NKG2D and RAE-1 proteins in mouse tumor tissues was significantly increased after Bi2S3@MCT26 treatment. The above results confirmed that Bi2S3@MCT26 can activate NK cells through NKG2D and RAE-1 to exert anti-cancer immune effects, suggesting that promoting NK cell activation may provide a new solution for tumor immunotherapy.
[0064] Flow cytometry was used to detect the proportions of NK cells, NKG2D and RAE-1 on the surface of NK cells in the spleen of each group of mice to evaluate the relevant immune response.
[0065] Reference Figure 6-Figure 11 ,The results showed that the proportion of NK cells in the spleen tissue of mice treated with Bi2S3@MCT26 was 3.67%, while the proportions of NK cells in the saline group, isotype control group and aPD-1 group were 0.51%, 0.68% and 1.26%, respectively.
[0066] Reference Figure 8 Compared with the saline group, the proportion of CD4+T cells in the spleen tissue of mice treated with Bi2S3@MCT26 increased, while there was no significant difference among the other groups.
[0067] Reference Figure 9-11 In addition, the results of mouse spleen CD8+T cells, M1, and M2 macrophages were not significant. The flow cytometry results showed that Bi2S3@MCT26 could strongly and significantly target NK cells, thereby activating NK cell-related immune responses, further proving that after Bi2S3@MCT26 treatment, NK cells may play a more important role in anti-tumor immunity.
[0068] The above experiments have verified that the NK cell engager based on the Bi2S3 nano-modifier prepared in this example can be efficiently prepared through this preparation method and can effectively activate NK cells.
[0069] The present invention is not limited to the above optional implementations, and anyone can derive other various forms of products under the enlightenment of the present invention. The above specific implementations should not be understood as limiting the scope of protection of the present invention. The scope of protection of the present invention should be based on the definition in the claims, and the description can be used to interpret the claims.
Claims
1. A NK cell engager, characterized in that: The invention comprises a Bi2S3 nano-modifier modified by PVP, and a targeted coating layer formed by CT26 cell membrane fragments extracted from CT26 cells is formed on the surface of the nano-modifier.
2. Use of the NK cell engager according to claim 1 in the preparation of a drug for treating colon cancer.
3. A method for preparing the NK cell engager according to claim 1, characterized in that: The specific steps are as follows: After the BiCl3 aqueous solution and the modifier PVP are mixed and stirred, the Na2S aqueous solution is added and mixed and heated to obtain a Bi2S3 nano-modifier; then the Bi2S3 nano-modifier is mixed with CT26 cell membrane fragments and extruded through a micro liposome extruder to obtain a NK cell engager.
4. The preparation method according to claim 3, characterized in that: CT26 cell membrane fragments were mixed with Bi2S3 nano-modifiers and incubated at 3-6°C for 2-3 hours.
5. The preparation method according to claim 4, characterized in that: The specific steps are as follows: Firstly, a BiCl3 aqueous solution and a PVP aqueous solution are mixed and stirred in water at a mass ratio of 0.5-0.8 at room temperature to form a mixed solution, and then an excess Na2S aqueous solution is added to the mixed solution, and after stirring and mixing, the mixture is transferred to a 90° C. water bath environment and heated for 30-45 minutes to react to form a PVP-modified Bi2S3 nano-modification product. After the reaction is completed, the mixture is cooled to room temperature and centrifuged to remove free PVP, and finally the PVP-modified Bi2S3 nano-modification product is dispersed in methanol for later use; Then, cell membrane fragments were extracted from CT26 cells using a membrane protein extraction kit, and the reserved Bi2S3 nano-modifiers were mixed with the cell membrane fragments at a mass ratio of 0.4-0.7, incubated at 4-6°C for 2 hours, and then extruded through a 400nm porous polycarbonate film using a micro-liposome extruder for at least 10 times. Finally, the excess cell membrane fragments were removed by centrifugation at 4-6°C to obtain the NK cell engager.