A platelet-based immune activation system and its preparation method and application

Abstract

The present invention discloses a platelet-based immune activation system, its preparation method, and its application, belonging to the field of biomedicine. Using platelets as carriers, the system engineered antibodies and cytokines onto the platelet surface. Leveraging the platelets' natural targeting properties to postoperative wounds and tumor adhesion, the antibodies achieve universal and stable antibody labeling of postoperative tumors. Furthermore, the precisely released cytokines fully mobilize immune cells in the tumor microenvironment, promoting their recognition and killing of residual tumors, thereby inhibiting postoperative tumor recurrence and metastasis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a platelet-based immune activation system and a preparation method and application thereof. Background Art

[0002] Surgery is the primary clinical treatment for most solid tumors. However, due to the complex physiological environment of the tumor site and the irregular shape of the tumor itself, complete lesion removal is difficult. Furthermore, there is a two-week postoperative immunosuppression period, which causes immune cell dysfunction, leading to high rates of postoperative tumor recurrence and metastasis, and poor patient prognosis. Therefore, there is an urgent need to develop adjuvant therapies that can inhibit postoperative tumor progression by activating the immune system.

[0003] The successful clinical application of a series of therapeutic antibodies, such as rituximab, trastuzumab, and alemtuzumab, has demonstrated that antibody-dependent cell-mediated cytotoxicity (ADCC) is a promising anti-tumor immunotherapy strategy. In particular, the US FDA has granted Fast Track designation to CYNK-101, a NK cell therapy based on ADCC. The initiation of ADCC requires the specific binding of IgG antibodies to antigenic determinants on the surface of target cells. The Fc domain of the IgG antibody then binds to immune cells such as natural killer (NK) cells, macrophages, eosinophils, and basophils, inducing the activated immune cells to release cytotoxic substances such as perforins and granzymes to kill the target cells. During antibody-mediated ADCC, antibodies specifically bind to corresponding antigenic epitopes on target cells, while effector cells such as NK cells can kill any antibody-bound target cells.

[0004] Endogenous platelets offer the advantages of abundant sources and low immunogenicity as drug carriers. Furthermore, platelets have been reported to target surgical cavities and naturally adhere to tumor cells. Therefore, modifying functional antibodies onto platelets and leveraging their high tumor cell affinity to achieve antibody coating of tumors is a promising approach to overcome the bottleneck of the ADCC effect in clinical applications. Summary of the Invention

[0005] The purpose of the present invention is to provide a platelet-based immune activation system, which uses platelets as carriers to engineer antibodies and cytokines onto the platelet surface. The antibodies leverage the platelets' natural targeting to postoperative wound sites and tumor adhesion properties to achieve postoperative tumor antibody labeling in a universal and stable manner. The precisely released cytokines fully mobilize immune cells in the tumor microenvironment, promoting their recognition and killing of residual tumors, thereby inhibiting postoperative tumor recurrence and metastasis.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] An immune activation system includes platelets with antibodies and / or cytokines attached to the platelet surface.

[0008] Furthermore, the antibody is directly connected to the platelet surface through a chemical bond, or is encapsulated in a nanocarrier and then connected to the platelet.

[0009] In one embodiment of the present invention, the antibody is linked to the sulfhydryl groups on the platelet surface via maleimide.

[0010] Furthermore, the antibody is selected from one or more of human IgG1, human IgG3, mouse IgG2b or mouse IgG2a.

[0011] Furthermore, the cytokine is directly connected to the platelet surface through chemical bonds, or is encapsulated in a nanocarrier and then connected to the platelet.

[0012] In one embodiment of the present invention, the cytokine is first encapsulated in liposomes with maleimide and then linked to the sulfhydryl groups on the platelet surface.

[0013] Furthermore, the cytokine is selected from one or more of interleukin-2, interleukin-3, interleukin-12, interleukin-15, interleukin-18, granulocyte-macrophage colony stimulating factor, interferon-γ, and tumor necrosis factor-α.

[0014] The preparation method of the above immune activation system comprises the following steps:

[0015] Step 1, extracting platelets and modifying thiol groups on their surfaces;

[0016] Step 2, preparing liposomes encapsulating cytokines;

[0017] Step 3, modifying the antibody with a maleimide group;

[0018] In step 4, the platelets from step 1 are linked to the liposomes from step 2 and / or the antibodies from step 3 to obtain a platelet preparation.

[0019] In one embodiment of the present invention, platelets are separated from autologous plasma and temporarily stored in a phosphate buffered saline solution containing prostaglandin E1.

[0020] Application of the above immune activation system in the preparation of postoperative tumor prevention and treatment drugs.

[0021] The present invention constructs a platelet-based immune activation system, which uses a mild and simple method to engineer antibodies and cytokine-encapsulated nanoformulations on the platelet surface. The combination of antibodies and cytokines is flexible and variable and can be applied to the activation of NK cells and even other immune cells.

[0022] Beneficial effects:

[0023] (1) The present invention uses endogenous platelets as carriers, which are abundant in source and help reduce preparation costs. They have low immunogenicity and help solve the problem of rapid clearance faced by conventional drug delivery systems when entering the body, and have clinical translation prospects.

[0024] (2) The immune activation system constructed by the present invention can stably label antibodies on tumor cells in a non-specific binding manner, breaking through the tumor heterogeneity barrier and greatly broadening the application scope of ADCC in the field of tumor treatment.

[0025] (3) The immune activation system constructed by the present invention can achieve local release of cytokines, overcoming the systemic toxicity and weakened efficacy that may be caused by their widespread distribution in the body.

[0026] (4) The co-modification method of antibodies and cytokines proposed in the present invention is universal. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the preparation route of the immune activation system in Example 1.

[0028] Figure 2 This is a scanning electron micrograph of the immune activation system in Example 1.

[0029] Figure 3 This is the result of characterizing the construction of the immune activation system using flow cytometry in Example 2.

[0030] Figure 4 This is the result of flow cytometry characterization of the expression of characteristic proteins of platelets in the immune activation system in Example 3.

[0031] Figure 5 This is the anchoring effect of the immune activation system on NK cells in Example 4 (arrows indicate NK cells).

[0032] Figure 6 The results of the immune activation system on NK cell activation in Example 4, A is CD69+ NK cell characteristic flow cytometry, B is CD69 + NK cell ratio statistics.

[0033] Figure 7 These are the results of the immune activation system stimulating NK cells to secrete cytokines in Example 4, where A is the secretion level of granzyme B and B is the secretion level of perforin.

[0034] Figure 8 The immune activation system in Example 4 is used to inhibit postoperative tumor recurrence. A is the tumor volume growth curve of mice treated with different medications after surgery, and B is the survival period of mice in each group.

[0035] Figure 9 The immune activation effect of the immune activation system in Example 5 is shown in Figure 5. A is the CD69 infiltrating in the tumor tissue of each group of mice. + NK cell ratio statistics, B is IFNγ + NK cell ratio statistics.

[0036] Figure 10 These are the safety assessment results of the immune activation system in Example 6. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the inventive method, steps or conditions are intended to fall within the scope of the present invention. Experimental methods and reagents for which specific conditions are not specified in the examples are based on conventional conditions in the art.

[0038] Example 1

[0039] The preparation of platelet-based immune activation system mainly includes platelet extraction and thiolation, preparation of liposomes encapsulating cytokines, and co-modification of antibodies and cytokines. The basic process is as follows: Figure 1 shown.

[0040] The specific operations are as follows:

[0041] (1) Collect whole blood from the orbit of C57BL / 6 mice, place it in a platelet-rich plasma (PRP) separation tube, and centrifuge it at 3500 rpm for 15 minutes at room temperature. Centrifuge the obtained PRP at 800 × g for 10 minutes to obtain platelets (PLT), which are then resuspended in phosphate buffered saline (PBS) containing 1 μM prostaglandin E1 (PGE1). Take the PLT suspension (approximately 1 × 10 8Thiolated platelets were obtained by reacting the platelets with 0.1 mg / mL 2-iminothiolane hydrochloride (Traut's reagent) for 30 minutes and then centrifuging at 800 × g for 10 minutes.

[0042] (2) Maleimidized liposomes loaded with interleukin-15 (IL-15) were prepared by thin film dispersion. 27 mg of soybean lecithin, 3 mg of DSPE-PEG-Mal, and 6 mg of cholesterol were dissolved in 5 mL of chloroform. The organic solvent was removed under vacuum at 37°C. 3 mL of IL-15 (0.05 mg / mL) in PBS was added and hydrated for 1 hour. The mixture was sonicated for 30 minutes. Free IL-15 was removed by filtration through an ultrafiltration tube (molecular weight cutoff, 30 kD).

[0043] (3) Antibody IgG2b was reacted with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (Sulfo-SMCC) at a molar ratio of 1:1.2 at 4°C for 2 hours, and the excess Sulfo-SMCC was removed by filtration through an ultrafiltration tube (molecular weight cutoff of 3 kD).

[0044] (4) 50 μL of maleimidized liposomes loaded with IL-15 (0.05 mg / mL), 50 μL of maleimidized antibody (0.4 mg / mL) and 100 μL of thiolated platelets (approximately 1 × 10 8 The platelets were reacted in PBS containing PGE1 for 2 h, centrifuged at 800 × g for 10 min, and washed twice with PBS to prepare platelet preparation (PLT-IgG-IL15).

[0045] The structure was observed by scanning electron microscopy. Figure 2 As shown, modification with antibodies and cytokines had no significant effect on platelet morphology.

[0046] Example 2

[0047] Maleimidized liposomes Lip-RhB encapsulating the model drug Rhodamine B (RhB) were prepared by thin film dispersion method. 27 mg soybean lecithin, 3 mg DSPE-PEG-Mal and 6 mg cholesterol were dissolved in 5 mL chloroform, the organic solvent was removed under vacuum at 37°C, 3 mL RhB (0.03 mg / mL) in PBS was added and hydrated for 1 hour, and ultrasonicated for 30 minutes. Free RhB was removed by filtration through an ultrafiltration tube (molecular weight cutoff of 3 kD). To prepare FITC-labeled IgG antibody, 100 mg IgG was dissolved in 10 mL Na2CO3 / NaHCO3 buffer solution at pH = 9. Separately, 10 mg FITC was weighed and dissolved in 100 μL DMSO. After mixing, the mixture was reacted at room temperature in the dark for 12 hours, dialyzed and freeze-dried. Maleimidized FITC-IgG was then further prepared according to the method under specific operation (3) in Example 1. Lip-RhB (0.05 mg / mL) and FITC-IgG antibody (0.4 mg / mL) were co-modified onto the thiolated platelets (approximately 1 × 10 8 The co-loading efficiency of RhB and IgG was determined by flow cytometry.

[0048] like Figure 3 As shown, RhB and antibodies were successfully co-loaded on the platelet surface, and the co-modification ratio was 99.4%.

[0049] Example 3

[0050] The expression of characteristic proteins of modified platelets in the immune activation system was evaluated by flow cytometry. The immune activation system was prepared according to the method of Example 1, and the expression of platelet surface markers (CD61, CD41 and CD62P) was detected and compared with that of natural platelets. Figure 4 As shown, the expression of key platelet markers in both is similar, and this immune activation system retains the inherent characteristics of platelets.

[0051] Example 4

[0052] The activation effect of the prepared immune activation system on NK cells was further analyzed. According to the method in Example 1, the thiolated platelets obtained in the specific operation (1) and the maleimidized liposomes obtained in (2) were reacted in PBS containing PGE1 for 2 hours, centrifuged at 800 × g for 10 minutes, and washed twice with PBS to prepare PLT-IL15. The thiolated platelets obtained in the specific operation (1) and the maleimidized IgG obtained in (3) were reacted in PBS containing PGE1 for 2 hours, centrifuged at 800 × g for 10 minutes, and washed twice with PBS to prepare PLT-IgG. Mouse spleen cells were extracted, and NK cells with a purity greater than 90% were obtained by magnetic bead separation. B16F10 cells were treated with different methods (PBS, IgG, PLT, PLT-IL15, PLT-IgG, and PLT-IgG-IL15), and then the above NK cells were co-incubated with B16F10 cells at a ratio of 5:1 for 1 hour. After washing twice with PBS, they were fixed with 4% paraformaldehyde for 30 minutes and stained with DAPI (1 μg / mL) for 20 minutes. Finally, the binding between NK cells and B16F10 cells was observed under a laser confocal microscope. Figure 5 As shown in the figure, free IgG and free platelets can hardly anchor NK cells around tumor cells. After IgG is modified onto PLT, platelets adhere to tumor cells, forming an antibody coating on the surface of tumor cells, thereby promoting NK cells to recognize tumors and initiate ADCC effects. NK cells co-incubated with B16F10 cells were collected, and the expression of surface immune activation marker CD69 was analyzed by flow cytometry, and the release of cytokines was detected by ELISA kit. Figure 6 As shown, CD69 in PBS, IgG, PLT, PLT-IgG, PLT-IL15 and PLT-IgG-IL15 groups + The NK ratios were 13.5%, 15.1%, 23.4%, 26.6% and 33.8% respectively. Figure 7 As shown in the figure, the secretion of granzyme B and perforin by NK cells in the PLT-IgG-IL15 group were 2.9 times and 2.1 times that of the PBS group, respectively, indicating that the immune activation system of co-modified antibodies and cytokines can significantly activate NK cells and effectively mediate the ADCC effect of NK cells.

[0053] Example 5

[0054] PLT-IL15 and PLT-IgG were prepared according to the method in Example 4. Construction of mouse B16F10 melanoma recurrence model: C57BL / 6 mice were subcutaneously implanted with tumors (1×10 6 B16-Luc cells). When the tumor volume reached approximately 120 mm 3At 4 hr, the tumor tissue was surgically removed, which was recorded as day 0. The surgical site was then administrated with normal saline, PLT, PLT-IL15, PLT-IgG, PLT-IgG-IL15 (1×10 8 PLT, 1 μg IL-15, 10 μg IgG). The tumor volume of the mice was monitored every other day until day 14. The survival period of the mice was continuously monitored until day 60. On day 16, tumor tissues were collected, lymphocytes were isolated, and activated NK cells (CD69 + NK cells, IFNγ + NK cell) ratio. Figure 8 As shown in A, the tumor volume in the saline group and the PLT group increased rapidly, indicating severe tumor recurrence. The tumor growth in the PLT-IL15 and PLT-IgG groups was inhibited to a certain extent. The tumor volume in the PLT-IgG-IL15 group was the smallest 14 days after surgery, and some mice had no recurrence at all. Figure 8 As shown in B, the 60-day survival rate of mice in the PLT-IgG-IL15 group exceeded 83%, which was significantly longer than that in the other groups. Figure 9 As shown in A and 9B, CD69 in the PLT-IgG-IL15 group + NK cells and IFNγ + The proportion of NK cells was the highest, which was 5.6 times and 8.3 times that of the normal saline group, respectively, indicating that the in situ NK cells were effectively activated after surgery.

[0055] Example 6

[0056] After healthy mice were treated with PLT-IgG-IL15, tissue sections of the heart, liver, spleen, lung, and kidney were taken for H&E staining. Figure 10 No lesions were found in any tissue, indicating that the immune activation system has good biosafety.

Claims

1. An immune activation system, characterized in that: The invention comprises platelets, wherein antibodies and cytokines are connected to the surface of the platelets, wherein the antibodies are connected to the sulfhydryl groups on the surface of the platelets via maleimide, and the cytokines are encapsulated in liposomes with maleimide and then connected to the sulfhydryl groups on the surface of the platelets; The antibody is murine IgG2b; The cytokine is interleukin-15.

2. The method for preparing the immune activation system according to claim 1, characterized in that: The following steps are involved: Step 1, extracting platelets and modifying thiol groups on their surfaces; Step 2, preparing liposomes encapsulating cytokines; Step 3, modifying the antibody with a maleimide group; In step 4, the platelets from step 1 are linked to the liposomes from step 2 and the antibodies from step 3 to obtain a platelet preparation.

3. Use of the immune activation system according to claim 1 in the preparation of drugs for the prevention and treatment of tumors after surgery.

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