Artificial antigen presenting cell based on polymer nanosheet as well as preparation method and application of artificial antigen presenting cell

Abstract

The invention relates to the technical field of nano materials and immunity, and provides an artificial antigen presenting cell based on a polymer nanosheet as well as a preparation method and application of the artificial antigen presenting cell. According to the preparation method of the artificial antigen presenting cell, uniform distribution of an N3 group on a two-dimensional polymer nanosheet is realized through a crystallization-driven self-assembly process, then an antibody is subjected to DBCO group modification, and finally the two-dimensional polymer nanosheet is modified with the antibody through a click chemical reaction to form the artificial antigen presenting cell. The interaction with the T cells is realized, and the T cells are activated and amplified. According to the invention, the two-dimensional polymer nanosheet is used as an artificial antigen presenting cell for activating the T cell, and the nanosheet structure effectively increases the contact area between the antibody and the T cell, so that a strong specific immunoreaction can be generated, and an anti-tumor effect is achieved; and the material has the advantages of controllable size, uniform morphology, stable structure, high biocompatibility, biodegradability and the like.

Description

Technical Field

[0001] The present invention relates to the fields of polymer materials and biomedical materials, and in particular to an artificial antigen-presenting cell based on crystallization-driven self-assembled anisotropic organic polymer nanosheets, and a preparation method and application thereof. Background Art

[0002] In terms of clinical treatment, although conventional treatments such as surgery, chemotherapy, and radiotherapy have made some progress, they still face clinical challenges such as limited efficacy, significant adverse reactions, and high recurrence rates when treating complex conditions and advanced cancers. Notably, the emergence of revolutionary tumor immunotherapy has brought new hope to cancer treatment. It modulates the body's immune response, enhancing the immune system's ability to recognize and eliminate tumor cells. Compared with conventional treatments, this therapy exhibits unique advantages such as precise targeting, long-lasting immune memory, and mild adverse reactions, providing a new breakthrough in the treatment of malignant tumors.

[0003] However, in anti-tumor therapy, the immune response relies on the activation and proliferation of T cells. Naive T cells within the host are activated through interaction with natural antigen-presenting cells (APCs), particularly dendritic cells. They then undergo proliferation and differentiation, forming a population of T cells with effector functions and memory immunity. These cells then take on the task of killing target cells. In recent years, T cell-based adoptive immunotherapy, such as chimeric antigen receptor (CAR) T cell therapy and T cell receptor-engineered T cell therapy, has achieved remarkable results in the treatment of malignant tumors. These treatments involve extracting T cells from the patient, activating, expanding, or modifying them in vitro to enhance their immune activity, and then returning them to the patient to fight cancer. The effectiveness of these therapies relies heavily on the activation and expansion of T cells by APCs. Although T cells can be activated using autologous CARs, isolating CARs from the body is difficult, inefficient, costly, and prone to T cell exhaustion. These issues limit their clinical application in T cell therapy.

[0004] Artificial antigen-presenting cells (aAPCs) have become a system that is easier to produce and more economically viable than natural ones. A large number of cell-based and cell-free systems have been designed as APC substitutes. Current aAPCs include carbon nanotubes, liposomes, and gels. These contain inorganic components or polystyrene coatings, which can lead to certain biotoxicity or indegradability in the body.

[0005] Therefore, it is necessary to develop a safer and more effective artificial antigen-presenting cell to achieve strong T cell activation and proliferation efficiency and obtain good immunotherapy effects. Summary of the Invention

[0006] In response to the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an artificial antigen-presenting cell based on crystallization-driven self-assembled two-dimensional polymer nanosheets, as well as a preparation method and application thereof. The present invention forms an anisotropic organic polymer nanosheet through crystallization-driven self-assembly. After connecting it to an antibody through a click chemistry reaction, it is used as an artificial antigen-presenting cell, which can activate and amplify T cells, stimulate specific immune responses and tumor immunotherapy, enhance the body's immune response, and has biocompatibility and biodegradability.

[0007] The present invention proposes a method for preparing artificial antigen-presenting cells based on crystallization-driven self-assembled two-dimensional polymer nanosheets that can activate and amplify T cells. The working mechanism is as follows Figure 1 As shown:

[0008] The present invention uses two-dimensional polymer nanosheets formed by epitaxial growth of high molecular weight polymers through crystallization-driven self-assembly as artificial antigen-presenting cells to activate T cells. First, the two-dimensional polymer nanosheets and antibodies are modified with click groups, so that the antibodies are connected to the two-dimensional polymer nanosheets through a click chemistry reaction. When the two-dimensional polymer nanosheets are combined with the antibodies, artificial antigen-presenting cells with a two-dimensional nanosheet structure are formed. After interacting with T cells, they can cause T cell activation and amplification. The present invention uses the two-dimensional polymer nanosheets as artificial antigen-presenting cells to activate T cells. Its nanosheet structure effectively increases the contact area between antibodies and T cells, thereby generating a strong specific immune response and achieving an anti-tumor effect. The material has the advantages of controllable size, uniform morphology, stable structure, high biocompatibility and biodegradability.

[0009] According to the above principles, the purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention provides a method for preparing artificial antigen-presenting cells, comprising the following steps:

[0011] S1. Providing a polymer, wherein the polymer comprises an antibody-targeting polymer, wherein the antibody-targeting polymer is modified with an azide (N3) group, and the polymer forms azide-modified two-dimensional polymer nanosheets through a crystallization-driven self-assembly method;

[0012] S2. providing an antibody, and modifying the antibody with a dibenzocyclooctyne (DBCO) group to obtain a DBCO-modified antibody;

[0013] S3. A click chemistry reaction is performed between the two-dimensional polymer nanosheets modified with azide groups and the antibodies modified with DBCO groups to obtain artificial antigen-presenting cells.

[0014] Since the antibody-targeting polymer carries an N3 group, the two-dimensional polymer nanosheet formed by the antibody-targeting polymer also carries an N3 group. The N3 group and the DBCO group are both click groups. The N3 group and the DBCO group can undergo a click chemistry reaction. In the present invention, the antibody is connected to the two-dimensional polymer nanosheet through a click chemistry reaction to obtain an artificial antigen-presenting cell.

[0015] Specifically, in step S1, the thickness of the formed azide group-modified two-dimensional polymer nanosheet is preferably less than 15 nm, and the length of the azide group-modified two-dimensional polymer nanosheet is 500 nm to 4000 nm, and the width is 100 nm to 1000 nm.

[0016] Specifically, in step S2, the antibody is selected from one or more of CD3 antibody and CD28 antibody.

[0017] Specifically, step S2 is to mix DBCO-PEG4-NHS with the antibody and incubate at 4 degrees Celsius for 4 to 8 hours to obtain a DBCO group-modified antibody.

[0018] Specifically, step S3 is to incubate the azide group-modified two-dimensional polymer nanosheets and the DBCO group-modified antibody at 4 degrees Celsius for 8 to 24 hours to obtain artificial antigen-presenting cells.

[0019] Preferably, the high molecular polymer further comprises PCL-b-PDMA, and the antibody targeting high molecular weight is PCL-N3; and the step S1 specifically comprises the following steps:

[0020] S11, dissolving PCL-b-PDMA in anhydrous ethanol, heating it, and then treating it with liquid nitrogen to obtain a seed solution;

[0021] S12. Dissolve PCL-b-PDMA and PCL-N3 in a good solvent to obtain a polymer mixture, add the polymer mixture to the seed solution, and shake thoroughly to obtain azide group-modified two-dimensional polymer nanosheets.

[0022] Specifically, in step S11, PCL-b-PDMA is placed in anhydrous ethanol, fully dissolved, and then placed in an oil bath at 70 degrees Celsius for 4 hours, and then placed in liquid nitrogen for 1 to 2 minutes to obtain a seed solution.

[0023] Specifically, in step S12, the good solvent includes tetrahydrofuran.

[0024] Specifically, in step S1, the antibody distribution conditions such as the size of the two-dimensional polymer nanosheets, the spacing and number of surface antibodies, etc. can be adjusted by adjusting parameters such as the content of the antibody-targeting polymer and the distribution ratio of each group.

[0025] In a specific embodiment, the mass ratio of PCL-b-PDMA to PCL-N3 in the polymer mixture is 1:1, and the mass ratio of the total polymer in the polymer mixture to the total polymer in the seed solution is 160:1.

[0026] In one specific embodiment, step S2 comprises mixing DBCO-PEG4-NHS with antibodies against CD3 and CD28, respectively, and incubating at 4 degrees Celsius for 4 to 8 hours to obtain CD3-DBCO and CD28-DBCO. Step S3 comprises adding CD3-DBCO and CD28-DBCO to a two-dimensional polymer nanosheet modified with an azide group to obtain an antibody-linked two-dimensional nanosheet as an artificial antigen-presenting cell. The equivalent ratio of CD3 or CD28 to DBCO-PEG4-NHS is 1:3, and the equivalent ratio of the azide-modified two-dimensional polymer nanosheet to CD3-DBCO and CD28-DBCO is 2:1:1.

[0027] Optionally, in step S1, the epitaxial polymer may further include PCL homopolymer, a fluorescent polymer containing a fluorescent group, etc., and the fluorescent polymer is preferably PCL-FITC.

[0028] Specifically, the preparation method of PCL-N3 is as follows: PCL and 3-azidopropionic acid are dissolved in dry DCM in a certain ratio, stirred and reacted in DCM, and catalysts dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) are added during the reaction. After 48 to 60 hours, the product is placed in ether for precipitation, and finally the precipitate is ventilated and dried, and then vacuum dried for 12 to 24 hours to obtain the product PCL-N3.

[0029] The present invention also provides artificial antigen-presenting cells prepared according to the above-mentioned method for preparing artificial antigen-presenting cells.

[0030] The present invention also provides the use of the artificial antigen-presenting cells in the preparation of agents for T cell activation and expansion, such as the preparation of tumor immunotherapy drugs.

[0031] The present invention has the following beneficial effects:

[0032] The present invention provides a method for preparing artificial antigen-presenting cells based on crystallization-driven self-assembled two-dimensional polymer nanosheets. First, a uniform distribution of N3 groups on the two-dimensional polymer nanosheets is achieved through a crystallization-driven self-assembly process. The material has the advantages of controllable size, uniform morphology, stable structure, high biocompatibility, and biodegradability. Then, the antibody is modified with a DBCO group, and then the antibody is modified on the two-dimensional polymer nanosheet by a click chemistry reaction to form an artificial antigen-presenting cell with a two-dimensional nanosheet structure, which interacts with T cells and can cause T cell activation and amplification. The present invention uses two-dimensional polymer nanosheets as artificial antigen-presenting cells for activating T cells. The surface of the anisotropic organic polymer nanosheet is easy to modify, which is not only conducive to adjusting the spacing and number of the surface-modified antibody distribution, but also its nanosheet structure is conducive to increasing the contact area between the antibody and the T cell, which can cause a strong specific immune response, thereby showing great application prospects in the field of tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the working principle of the crystallization-driven self-assembled polymer two-dimensional nanosheets of the present invention as artificial antigen-presenting cells to activate T cells;

[0034] Figure 2 This is a low-magnification transmission electron microscopy image of the seed solution prepared in Preparation Example 2;

[0035] Figure 3 This is a high-magnification transmission electron microscopy characterization image of the two-dimensional polymer nanosheet prepared in Preparation Example 5;

[0036] Figure 4 This is a high-magnification confocal microscopy characterization image of the two-dimensional polymer nanosheet prepared in Preparation Example 6;

[0037] Figure 5 This is a gel permeation chromatography characterization chart of the antibody targeting molecule PCL-N3 prepared in Example 1;

[0038] Figure 6 This is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis characterization image of CD3 / DBCO / ATTO532 and CD28 / DBCO / ATTO532 prepared in Preparation Example 4;

[0039] Figure 7 This is a high-magnification confocal microscopy characterization of a two-dimensional polymer nanosheet connected to a dye with a DBCO group;

[0040] Figure 8 This is a graph showing the safety evaluation results of the material concentration after the two-dimensional polymer nanosheets prepared in Preparation Example 5 were co-incubated with Jurkat T cells;

[0041] Figure 9Evaluation results of T cell activation titer after co-incubation of Jurkat T cells with different concentrations of CD3-DBCO and CD28-DBCO prepared in Preparation Example 3;

[0042] Figure 10 These are the evaluation results of the activation titer of T cells after co-incubation of CD3-DBCO and CD28-DBCO prepared in Preparation Example 3 and the artificial antigen-presenting cells prepared in Example 1 with Jurkat T cells. DETAILED DESCRIPTION

[0043] The present invention will be described in detail in conjunction with specific embodiments to provide a clear and comprehensive explanation of the technical solutions. If specific techniques or conditions are not clearly stated in the examples, reference is made to those described in the literature or in accordance with the product specifications. The reagents or instruments used are commercially available conventional products unless the manufacturer is indicated.

[0044] The following are examples of the present invention, which are intended to further illustrate the embodiments of the present invention. It should be noted that the following examples are only specific cases within the scope of application, and the process parameters involved are for reference only and are not limited to the specific values ​​described below. Those skilled in the art can adjust the parameters within a reasonable range based on the description herein and in combination with actual needs.

[0045] Crystallization-driven self-assembly (CDSA) is an advanced polymer assembly technique. Its core lies in the introduction of crystalline segments into polymer blocks, using crystallization as the driving force to form complex assembly structures of zero-, one-, two-, and multidimensional dimensions. Through the combined regulation of phase separation induced by a hydrophilic solvent and crystallization driven by the crystalline blocks, the precise construction of anisotropic polymer micelles can be achieved. The introduction of a crystallization driving force guides the orderly stacking of polymers along specific directions, making it easier to construct stable high-dimensional, multi-layered heterogeneous micelle structures. During the active crystallization-driven self-assembly process, the ends of the assembly retain crystallization activity, which can induce the epitaxial growth of subsequently added crystalline polymers on the micelle surface, forming nested co-micelle structures. This mechanism enables the spatially anisotropic distribution and nanoscale patterning of multiple molecules. Through the crystallization-driven self-assembly method, two-dimensional, size-tunable, water-dispersible particle micelles with spatially defined chemical properties can be obtained.

[0046] Based on this, the present invention provides artificial antigen-presenting cells based on crystallization-driven self-assembled polymer two-dimensional nanosheets, which are used to activate T cells. When the two-dimensional polymer nanosheets are combined with the antibody CD3 / CD28, they can serve as artificial antigen-presenting cells. After interacting with T cells, they can cause T cell activation and amplification, generating a powerful specific immune response, thereby achieving an anti-tumor effect.

[0047] Preparation Example 1 Synthesis of Antibody Targeting Molecule PCL-N3

[0048] PCL and 3-azidopropionic acid were dissolved in dry DCM in a 1:1 mass ratio and stirred in DCM. Catalysts DCC and DMAP were added during the reaction. After 48 to 72 hours, the product was precipitated in ether. The precipitate was finally dried by ventilation and then vacuum dried for 24 hours to obtain the product PCL-N3.

[0049] Preparation Example 2 Preparation of Quasi-Seed Solution

[0050] 10 mg of PCL-b-PDMA was weighed and dissolved in 2 mL of anhydrous ethanol, placed in a 70 °C oil bath for 4 hours, and then immediately placed in liquid nitrogen for 1 to 2 minutes to obtain a quasi-seed solution with a concentration of 5 mg / mL.

[0051] Preparation Example 3 DBCO Group Modification of Antibody

[0052] CD3 and CD28 were respectively mixed with DBCO-PEG4-NHS at an equivalent ratio of 1:3 and incubated at 4 degrees Celsius for 4 to 8 hours to obtain CD3-DBCO and CD28-DBCO.

[0053] Preparation Example 4: DBCO Group and Fluorescent Modification of Antibodies

[0054] CD3 and CD28 were respectively mixed with DBCO-PEG4-NHS and ATTO532-NHS in an equivalent ratio of 2:3:3, and incubated at 4 degrees Celsius for 4 to 8 hours to obtain CD3 / DBCO / ATTO532 and CD28 / DBCO / ATTO532.

[0055] Preparation Example 5 Preparation of N3 Group-Modified Two-Dimensional Polymer Nanosheets

[0056] The quasi-seed solution prepared in Preparation Example 2 was diluted with anhydrous ethanol to 0.01 mg / mL to obtain a seed solution. 4 mg PCL-N3 was weighed and dissolved in 100 μL of tetrahydrofuran with 4 mg PCL-b-PDMA to obtain a polymer solution. 10 μL of the polymer solution was added to 500 μL of the seed solution and thoroughly shaken to obtain uniformly sized two-dimensional polymer nanosheets modified with N3 groups, which were designated as PCL-N3 nanosheets. The nanosheet ethanol system was converted to a water system by solvent replacement for subsequent use.

[0057] Preparation Example 6 Preparation of N3-modified two-dimensional polymer nanosheets with fluorescent properties

[0058] The seed solution prepared in Preparation Example 4 was diluted to 0.01 mg / mL with anhydrous ethanol to obtain a seed solution. 4 mg of PCL-N3 was weighed and dissolved in 100 μL of tetrahydrofuran with 4 mg of PCL-b-PDMA and a trace of fluorescently modified PCL-FL to obtain a polymer solution. 10 μL of the polymer solution was added to 500 μL of the seed solution and thoroughly shaken to obtain uniformly sized fluorescent N3-modified two-dimensional polymer nanosheets.

[0059] Example 1

[0060] The N3-modified two-dimensional polymer nanosheets prepared in Preparation Example 5 were uniformly mixed with the CD3-DBCO and CD28-DBCO prepared in Preparation Example 3 in an equivalent ratio of 2:1:1, and incubated for 8 to 24 hours to obtain artificial antigen-presenting cells.

[0061] <Performance Test>

[0062] (1) Transmission electron microscopy observation of physical morphology

[0063] The quasi-seed solution prepared in Preparation Example 2 was diluted to a concentration of 0.02 mg / mL, 10 μL was dropped onto the carbon film surface of the copper mesh and dispersed, and then dried for TEM characterization. Figure 2 As shown, it can be seen that the prepared seed micelles are evenly distributed and uniform in size.

[0064] 10 μL of the two-dimensional polymer nanosheets with N3 groups prepared in Preparation Example 5 was dropped onto the carbon film surface of the copper mesh and dispersed. After drying, it was used for TEM characterization. The test results are shown in Figure 5. Figure 3 As shown, it can be seen that the prepared two-dimensional polymer nanosheets are evenly distributed and uniform in size, with a size of about 0.8 μm.

[0065] (2) Confocal microscopy analysis and observation of physical morphology

[0066] The two-dimensional polymer nanosheets with fluorescence and N3 groups prepared in Example 6 were diluted 10 times with anhydrous ethanol, 10 μL of the diluted material was dropped on a glass slide, covered with a cover glass, and evaporated at room temperature for half an hour. The sample was sealed and stored for confocal imaging. The imaging results are shown in FIG. Figure 4 As shown, it can be seen that the prepared two-dimensional polymer nanosheets are evenly distributed and uniform in size.

[0067] (3) Gel permeation chromatography verification experiment

[0068] Weigh 2 mg of PCL-N3 prepared in Preparation Example 1 and dissolve it in 1 mL of dimethylformamide solution. After it is completely dissolved, filter it with a 0.22 μm filter membrane. After filtration, test the molecular weight on the machine. The result is as follows: Figure 5 As shown, the results showed the successful synthesis of PCL-N3.

[0069] (4) Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis verification experiment

[0070] Take 200mL of 5× electrophoresis solution and add 800mL of ultrapure water to obtain 1× electrophoresis solution. Take 4mL of the lower gel solution in the gel kit, add 4mL of the lower gel buffer and 80μL of the improved coagulant, mix well and add to the glass plate for making gel, then add 1-2mL of anhydrous ethanol to press the gel, and solidify for about 15-20 minutes. After solidification, pour out the anhydrous ethanol. Take 1mL of the upper gel solution, add 1mL of the upper gel buffer and 20μL of the improved coagulant, mix well and add to the glass plate, gently insert the comb to solidify for 15-20 minutes. Take 1μL of CD3 / DBCO / ATTO532 and CD28 / DBCO / ATTO532 prepared in Example 4, mix well with 99μL of non-reducing loading buffer, incubate in a metal bath at 100 degrees Celsius for 5 minutes, and then take out. Place the gel in the electrophoresis tank and add electrophoresis solution. Remove the comb and add 10 μL of marker to the first well. Then add 20 μL of CD3 / DBCO / ATTO532 and CD28 / DBCO / ATTO532 to the following wells, filling the gel to the scale line. Run at 80V for 30 minutes, then change to 120V and run for 120 minutes. After the run, cut the gel and keep the lower gel for imaging. The results are as follows: Figure 6 shown.

[0071] Result analysis: Since the fluorescent dye ATTO532 and the DBCO group were linked to CD3 through the same reaction, the SDS-PAGE bands showed a relative shift, which confirmed that the above preparation example had successfully linked the DBCO group to the CD3 antibody and the CD8 antibody.

[0072] (5) Verification of the interface between two-dimensional nanosheets and DBCO-containing dyes

[0073] The seed solution prepared in Preparation Example 2 was diluted to 500 μL of 0.01 mg / mL seed solution, 4 mg PCL and 4 mg PCL-b-PDMA were weighed and dissolved in 100 μL of tetrahydrofuran. After complete dissolution, 10 μL was added to 500 μL of 0.01 mg / mL seed solution. After immediate strong oscillation and mixing, it was allowed to stand for 15 minutes. A first epitaxial layer without fluorescence and N3 group was extended and grown on the periphery of the seed micelle to obtain a preliminary nanosheet.

[0074] According to the preparation method of Preparation Example 6, 4 mg of PCL-N3 was weighed and dissolved in 100 μL of tetrahydrofuran with 4 mg of PCL-b-PDMA and a trace amount of fluorescently modified PCL-FL to obtain a polymer solution. 10 μL of the fluorescent polymer solution was added to the first nanosheet solution, and the mixture was immediately vigorously shaken and mixed. After standing for 30 minutes, a second epitaxial layer with green fluorescence and N3 groups was extended and grown outside the first epitaxial layer.

[0075] During the preparation of the second nanosheet having green fluorescence and N3 groups, 5 μL of 2 mg / mL Cy3-DBCO dye was added, mixed evenly, and incubated for 8 to 24 hours to obtain the first nanosheet.

[0076] After the second nanosheet having green fluorescence and N3 groups is prepared according to the above method, 5 μL of 2 mg / mL Cy3-DBCO dye is added, mixed evenly, and incubated for 8 to 24 hours to obtain the second nanosheet.

[0077] The above-prepared preliminary nanosheets without fluorescence and N3 groups, the first nanosheet obtained by adding Cy3-DBCO during the preparation of the second epitaxial layer, and the second nanosheet obtained by adding Cy3-DBCO after the preparation of the second epitaxial layer were diluted 10 times with anhydrous ethanol. 10 μL of the diluted material was dropped on a glass slide, covered with a cover glass, evaporated at room temperature for half an hour, and sealed to preserve the sample for confocal photography. The photography results are shown as follows: Figure 7 shown.

[0078] Result analysis: Cy3-DBCO dye co-localized with the second epitaxial layer containing a green fluorescent group and an N3 group, confirming that the reaction between N3 and DBCO is highly selective and indirectly proving that PCL-N3 was successfully synthesized in the present invention.

[0079] (6) Cell safety evaluation of nanosheets

[0080] Jurkat T cells that had grown to T25 were collected by centrifugation and plated in a 96-well plate. When the cells grew to about 80% in the well plate, PCL-N3 nanosheets prepared in Preparation Example 5 at different concentrations were added to different well plates. Three replicate wells were set for each concentration, and three replicate wells were set for the control group without adding the material. After the material and Jurkat T cells were co-incubated for 24 hours, the culture medium in the replicate wells was aspirated, and the cells were washed three times with PBS. Subsequently, the CCK-8 solution was diluted 10 times with PBS, and 100 μL of the prepared CCK-8 solution was added to each replicate well. The cells were incubated at 37 degrees Celsius for 40 to 50 minutes, and the OD value of each replicate well was measured with a microplate reader. The data analysis results are shown in the figure. Figure 8 shown.

[0081] The results showed that the nanosheet material still had good biosafety at a concentration of 0.8 mg / mL and had no obvious toxic effects on cells.

[0082] (7) Verification of the effect of antibody concentration on T cell activation

[0083] Jurkat T cells that have grown on T25 were collected by centrifugation and 5×10 5 Cells were plated in 96-well plates at 400 μg / ml. CD3-DBCO and CD28-DBCO prepared in Preparation Example 3 were added to the plates at different concentrations. Three replicates were set for each concentration. Three replicates were set for the control group without adding any material. After mixing, the cells were incubated in a cell culture incubator at 37°C for 24 hours. The cells were harvested by centrifugation at 350g for 5 minutes and washed 1-2 times with PBS. After adding Aqua and Fc block, the cells were incubated on ice for 20 minutes, followed by a wash with FACS buffer. The cells were then stained with Anti-CD25 and Anti-CD107a, incubated on ice for 20 minutes, and washed once with FACS buffer. The resulting cell pellet was resuspended in 250 μL FACS buffer.

[0084] Flow cytometry was used to detect the activation of T cells at different antibody concentrations. Figure 9 shown.

[0085] The results showed that at a concentration of 300 ng / mL of CD3-DBCO / CD28-DBCO, the activation effect of Jurkat T cells reached a plateau. Therefore, in the subsequent activation experiments, 300 ng / mL was selected as the concentration of CD3-DBCO / CD28-DBCO for activating Jurkat T cells.

[0086] (8) Evaluation of T cell activation by two-dimensional nanosheets

[0087] Jurkat T cells that have grown on T25 were collected by centrifugation and 5×10 5 Cells were plated in 96-well plates at 100 ng / mL. CD3-DBCO and CD28-DBCO prepared in Preparation Example 3 and artificial antigen-presenting cells from Example 1 were added to the plates at a final concentration of 300 ng / mL. Three replicate wells were set up for each concentration. A control group without any material was also set up in three replicate wells. After mixing thoroughly, the cells were incubated in a cell culture incubator at 37°C for 24 hours. The cells were harvested by centrifugation at 350g for 5 minutes and washed one to two times with PBS. After adding Aqua and Fc block, the cells were incubated on ice for 20 minutes, followed by a single wash with FACS buffer. The cells were then stained with Anti-CD69, Anti-CD25, and Anti-CD107a, incubated on ice for 20 minutes, and washed once with FACS buffer. The resulting cell pellet was resuspended in 250 μL of FACS buffer.

[0088] Flow cytometry was used to detect the activation of T cells by nanosheets compared with free antibodies. Figure 10 shown.

[0089] The results showed that the artificial antigen-presenting cells prepared by the present invention, after modifying antibodies on two-dimensional polymer nanosheets, can significantly activate Jurkat T cells compared with free antibodies, thereby achieving the purpose of activating T cells.

[0090] The manufacturers of the materials used in the above embodiments and performance tests are as follows:

[0091] Anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0092] Dimethylformamide and tetrahydrofuran were purchased from Aladdin;

[0093] DBCO-PEG4-NHS was purchased from J&K;Kingwei;

[0094] Culture medium and PBS were purchased from Gibco;

[0095] The gel kit and maker were purchased from Unionville;

[0096] Flow cytometry antibodies were purchased from Biolegend;

[0097] CD3 and CD28 were purchased from Bio X cell;

[0098] The CCK8 kit was purchased from Liji Company.

[0099] In summary, the artificial antigen-presenting cells based on crystallization-driven self-assembled two-dimensional polymer nanosheets provided by the present invention have the advantages of uniform morphology, stable structure, and high biocompatibility. They interact with T cells, can cause T cell activation and expansion, and can induce a strong specific immune response, and have great application prospects in the field of tumor immunotherapy.

[0100] The above describes the embodiments of the present invention in conjunction with the accompanying drawings. However, the present invention is not limited to the above examples and various modifications can be made based on the inventive purpose of the present invention. Any changes, modifications, substitutions, combinations, or simplifications based on the methods and technical contents disclosed in the present invention shall be considered equivalent replacement methods and shall fall within the scope of the present invention as long as they are within the spirit and principles of the present invention.

Claims

1. A method for preparing artificial antigen-presenting cells, characterized in that: The steps include: S1. Providing a polymer, wherein the polymer comprises an antibody-targeting polymer, wherein the antibody-targeting polymer is modified with an azide group, and forming azide-modified two-dimensional polymer nanosheets by crystallization-driven self-assembly of the polymer; S2. providing an antibody, and performing DBCO group modification on the antibody to obtain a DBCO group-modified antibody; S3. A click chemistry reaction is performed between the two-dimensional polymer nanosheets modified with azide groups and the antibodies modified with DBCO groups to obtain artificial antigen-presenting cells.

2. The method for preparing artificial antigen-presenting cells according to claim 1, wherein: The antibody is selected from one or more of CD3 antibody and CD28 antibody.

3. The method for preparing artificial antigen-presenting cells according to claim 1, wherein: The thickness of the two-dimensional polymer nanosheet modified by the azide group is less than 15 nm, the length of the two-dimensional polymer nanosheet modified by the azide group is 500 nm to 4000 nm, and the width is 100 nm to 1000 nm.

4. The method for preparing artificial antigen-presenting cells according to claim 1, wherein: The high molecular polymer further includes PCL-b-PDMA, and the antibody targeting high molecular weight polymer is PCL-N3.

5. The method for preparing artificial antigen-presenting cells according to claim 4, characterized in that: The step S1 specifically includes the following steps: S11, dissolving PCL-b-PDMA in anhydrous ethanol, heating it, and then treating it with liquid nitrogen to obtain a seed solution; S12. Dissolve PCL-b-PDMA and PCL-N3 in a good solvent to obtain a polymer mixture, add the polymer mixture to the seed solution, and shake thoroughly to obtain azide group-modified two-dimensional polymer nanosheets.

6. The method for preparing artificial antigen-presenting cells according to claim 1, wherein: The antibody-targeting polymer is PCL-N3. The preparation method of PCL-N3 is as follows: PCL and 3-azidopropionic acid are dissolved in dry DCM, stirred and reacted in DCM, during which catalysts dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) are added and reacted for 48 to 60 hours, and then placed in ether for precipitation. Finally, the precipitate is ventilated and dried, and then vacuum dried for 12 to 24 hours to obtain the product PCL-N3.

7. The method for preparing artificial antigen-presenting cells according to claim 1, wherein: Specifically, step S2 comprises mixing DBCO-PEG4-NHS with the antibody and incubating the mixture at 4 degrees Celsius for 4 to 8 hours to obtain a DBCO group-modified antibody.

8. The method for preparing artificial antigen-presenting cells according to claim 1, wherein: In step S3, the two-dimensional polymer nanosheet modified with an azide group and the antibody modified with a DBCO group are incubated at 4 degrees Celsius for 8 to 24 hours to obtain artificial antigen-presenting cells.

9. An artificial antigen-presenting cell, characterized in that The artificial antigen-presenting cell is prepared according to the method for preparing the artificial antigen-presenting cell according to any one of claims 1 to 8.

10. Use of the artificial antigen-presenting cell according to claim 9 in the preparation of a medicament for T cell activation and expansion.

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