Mature activated dendritic cells and methods and uses thereof

Abstract

The application discloses mature activated dendritic cells and an inducing method and application thereof, and the inducing method comprises the following steps: obtaining PBMCs by separating from peripheral blood, and obtaining mononuclear cells by adherent purification; inducing and culturing the mononuclear cells by using complete culture medium containing IL-4 and GM-CSF; collecting cells after 6-7 days of induction and culture, and co-incubating the collected cells with FPC-NPs@TAAs to obtain the mature activated dendritic cells; the inducing method can efficiently induce maturation of DCs from PBMCs, and further, the mature DCs can induce PBMCs to differentiate into tumor-specific CTLs under the condition that no exogenous IL-2 is added; in addition, the DC-CTL prepared by the application can specifically lyse tumor cells in vitro, and has excellent anti-tumor effect.

Description

Technical Field

[0001] This invention relates to the field of mature activated dendritic cell induction technology, specifically to a mature activated dendritic cell, its induction method, and its application. Background Technology

[0002] Tumor immunotherapy is an emerging approach to cancer treatment; among it, cell therapy, which utilizes the body's own cells to eliminate cancer, represents a significant breakthrough in the field. Compared to broad-spectrum cancer treatments, cell therapy typically offers precision and personalization, providing targeted cancer treatment. Currently, it can be categorized into autologous cell therapy, allogeneic cell therapy, genetically modified cells, or simply expanding or activating the original functions of cells.

[0003] IL-2 is a classic T-cell growth factor widely used to maintain the proliferation and activity of CTLs during in vitro induction. However, high doses of IL-2 can lead to severe systemic toxicity, such as vascular leakage syndrome, limiting its safety. Furthermore, while IL-2 promotes the proliferation of effector T cells, it also preferentially expands regulatory T cells with immunosuppressive functions, weakening the anti-tumor effect of CTLs. Therefore, finding new strategies that can replace the function of IL-2 while maintaining both safety and high efficiency is crucial for optimizing CTL cell preparation processes.

[0004] Dendritic cells (DCs) take up, process, and present tumor antigens to T cells, inducing naive T cells to differentiate into tumor-specific cell-mediated cytokines (CTLs). This is a crucial step in initiating the tumor immune response. DC-CTL combined immunotherapy involves inducing the differentiation of the patient's autologous monocytes into immature DCs in vitro. After loading these DCs with tumor-associated antigens, they are matured under adjuvant stimulation. The mature DCs are then co-cultured with autologous differentiated T cells to obtain DC-CTLs capable of specifically recognizing and killing tumor cells, which are then reinfused into the patient to exert a therapeutic effect. Meanwhile, the maturation state of DCs is a core factor determining their ability to effectively stimulate a CTL response. Therefore, developing more efficient DC maturation induction protocols that can effectively activate CTLs even without exogenous IL-2 supplementation has significant clinical translational value. Summary of the Invention

[0005] The purpose of this invention is to provide a mature activated dendritic cell, its induction method, and its application. This invention constructs F-PEI and CpG dual adjuvant nanoparticles (FPC-NPs) using carrier-free self-assembly technology and loads them with TAAs to form a personalized tumor vaccine FPC-NPs@TAAs. This efficiently induces the maturation of DCs derived from PBMCs. Furthermore, these mature DCs induce PBMCs to differentiate into tumor-specific CTLs without the addition of exogenous IL-2. In addition, the DC-CTLs prepared by this invention can specifically lyse tumor cells in vitro and have excellent anti-tumor effects.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: The first aspect of this invention provides a method for inducing mature and activated dendritic cells, the induction method comprising the following steps: (a) PBMCs were isolated from peripheral blood and then purified by adherence to a wall to obtain mononuclear cells; (b) Monocytes were induced to grow using a complete culture medium containing IL-4 and GM-CSF; (c) After 6-7 days of induction culture, cells are collected and co-incubated with FPC-NPs@TAAs to obtain the mature activated dendritic cells; The FPC-NPs@TAAs are formed by the self-assembly of F-PEI, CpG and TAAs.

[0007] Preferably, the concentration of IL-4 in the complete culture medium containing IL-4 and GM-CSF is 8~12 ng / mL, and the concentration of GM-CSF is 15~25 ng / mL.

[0008] Preferably, the culture medium is changed every 2-3 days during the induction culture process; the incubation time is 20-30 hours.

[0009] Preferably, the FPC-NPs@TAAs are prepared by the following method: FPC-NPs are self-assembled by electrostatic adsorption of fluorinated PEI (F-PEI) and CpG in a mass ratio of 1:(0.8~1.2). Then, FPC-NPs are mixed with TAAs in a mass ratio of 1:(0.2~0.4) and shaken for 30 min to obtain FPC-NPs@TAAs.

[0010] A second aspect of the present invention provides mature activated dendritic cells obtained by the above-described induction method.

[0011] A third aspect of the present invention provides the application of mature and activated dendritic cells obtained by the above-described induction method in the preparation of DC-CTL cells.

[0012] A fourth aspect of the present invention provides a method for preparing DC-CTL cells, the method comprising the following steps: (1) PBMCs were isolated from peripheral blood; (2) PBMCs and the mature activated dendritic cells obtained by the induction method were seeded into cell culture plates coated with CD3 and CD28 antibodies and cultured in RPMI 1640 complete medium containing 10% FBS, 1% penicillin-streptomycin mixture (1% penicillin-streptomycin mixture), and 50 μM β-mercaptoethanol to obtain the DC-CTL cells.

[0013] Preferably, the ratio of PBMCs to mature and activated dendritic cells is (1.5~2.5):1; the culture time is 4~5 days; and the culture medium is changed every 2~3 days during the culture process.

[0014] The fifth aspect of the present invention provides a DC-CTL cell prepared by the above-described preparation method.

[0015] The sixth aspect of this invention provides the application of DC-CTL cells prepared by the above-described method in the preparation of antitumor drugs.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least the following: This invention utilizes self-assembly technology to simultaneously load two synergistic immune adjuvants, the TLR4 agonist F-PEI and the TLR9 agonist CpG, and loads tumor-associated antigens (TAAs) through positive and negative charge adsorption to form a personalized tumor vaccine, FPC-NPs@TAAs. This allows for the efficient induction of DCs derived from PBMCs to mature without the intervention of exogenous carriers. Furthermore, these mature DCs induce PBMCs to differentiate into tumor-specific CTLs without the addition of exogenous IL-2, thereby avoiding IL-2-related adverse effects and optimizing relevant process control parameters. This strategy not only reduces the cost of DC-CTL cell preparation but also fundamentally improves the efficacy and safety of immunotherapy by optimizing the functional state of DCs, providing a new technical pathway for the clinical translation of adoptive cell immunotherapy for tumors. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0018] Figure 1The expression of CD86, CD80 and CD40 on the surface of PBMC-induced DCs in Experiment Example 1 of this invention is shown in the figures after treatment with FPC-NPs (mDC-1) or FPC-NPs@TAAs (mDC-2). Figure 2 The table shows the expression of MHC I and MHC II on the surface of PBMC-induced DCs without treatment (iDC), after treatment with FPC-NPs (mDC-1) or FPC-NPs@TAAs (mDC-2) in Experimental Example 1 of this invention. Figure 3 The proportions of T cells and CD8+ T cells induced by PBMCs after different treatments (without IL-2, iDC, mDC-1, mDC-2, IL-2) in Experimental Example 2 of this invention; Figure 4 The target cell specific lysis ratios under different effect-to-target ratios in Experiment Example 3 of this invention; Figure 5 The figures show the tumor volume change curve and survival curve of mice after prophylactic inoculation with DC-CTL cells in Experiment Example 4 of this invention. Detailed Implementation

[0019] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0020] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] This invention provides a method for inducing mature and activated dendritic cells, the induction method comprising the following steps: (a) PBMCs were isolated from peripheral blood and then purified by adherence to a wall to obtain mononuclear cells; (b) Monocytes were induced to grow using a complete culture medium containing IL-4 and GM-CSF; (c) After 6-7 days of induction culture, cells are collected and co-incubated with FPC-NPs@TAAs to obtain the mature activated dendritic cells; The FPC-NPs@TAAs are formed by the self-assembly of F-PEI, CpG and TAAs.

[0022] In one embodiment, the concentration of IL-4 in the complete culture medium containing IL-4 and GM-CSF is 8-12 ng / mL, and the concentration of GM-CSF is 15-25 ng / mL.

[0023] In one embodiment, the culture medium is changed every 2 to 3 days during the induction culture process; the incubation time is 20 to 30 hours.

[0024] Preferably, the FPC-NPs@TAAs are prepared by the following method: FPC-NPs are self-assembled by electrostatic adsorption of fluorinated PEI and CpG in a mass ratio of 1:(0.8~1.2). Then, FPC-NPs are mixed with TAAs in a mass ratio of 1:(0.2~0.4) and shaken for 30 min to obtain FPC-NPs@TAAs. The final concentration of FPC-NPs@TAAs added is 15~25 μg / mL based on CpG.

[0025] Another embodiment of the present invention provides mature activated dendritic cells obtained by the above-described induction method.

[0026] Another embodiment of the present invention provides the application of mature activated dendritic cells obtained by the above-described induction method in the preparation of DC-CTL cells.

[0027] Another embodiment of the present invention provides a method for preparing DC-CTL cells, the method comprising the following steps: (1) PBMCs were isolated from peripheral blood; (2) PBMCs and mature activated dendritic cells obtained by the induction method were seeded into cell culture plates coated with CD3 and CD28 antibodies and cultured in RPMI 1640 complete medium containing 10% FBS, 1% penicillin-streptomycin mixture and 50 μM β-mercaptoethanol to obtain the DC-CTL cells.

[0028] In one embodiment, the ratio of PBMCs to mature and activated dendritic cells is (1.5~2.5):1; the culture time is 4~5 days; and the culture medium is changed every 2~3 days during the culture process.

[0029] Another embodiment of the present invention provides a DC-CTL cell prepared by the above preparation method.

[0030] Another embodiment of the present invention provides the application of DC-CTL cells prepared by the above method in the preparation of antitumor drugs.

[0031] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0032] Example 1 This embodiment describes a method for inducing mature and activated dendritic cells, the induction method comprising the following steps: (a) Take fresh blood into an anticoagulant tube and dilute it with an equal volume of diluent; separate PBMCs according to the instructions of the lymphocyte separation kit (750 g, 25 min, 8 / 3 speed); aspirate the white membrane layer with a 1 mL syringe, centrifuge with 10 mL of washing buffer (250 g, 10 min), seed it into a cell culture plate, and culture it in complete medium without factors for 5-6 hours. After some cells adhere to the plate, discard the medium, wash gently once with 1 mL of PBS to obtain monocytes; (b) Continue culturing with complete medium containing IL-4 and GM-CSF factors. The day of extraction is recorded as day 0. On day 3 and day 5, the medium is replaced halfway to replenish new cytokines. (c) On day 6, cells were collected, centrifuged, and re-seeded in 12-well plates. The cells were then co-incubated with FPC-NPs@TAAs (CpG concentration 20 μg / mL) for 24 h to obtain the mature, activated dendritic cells (denoted as mDC-2). The FPC-NPs@TAAs were prepared using the following method: FPC-NPs are self-assembled by electrostatic adsorption of fluorinated PEI and CpG in a mass ratio of 1:1. Then, FPC-NPs are mixed with TAAs in a mass ratio of 1:0.3 and shaken for 30 min to obtain FPC-NPs@TAAs.

[0033] Example 2 This embodiment describes a method for preparing DC-CTL cells, which includes the following steps: (1) Antibody coating one day in advance: The cell culture plate was rinsed twice with sterile PBS and coated overnight at 4 ℃ with CD3 and CD28 antibodies (5 μg / mL, 500 μL / well); (2) Blocking: Remove unbound antibody suspension; add 500 μL of PBS solution containing 1% BSA to each well and block at room temperature for 30 min; wash once with sterile PBS to obtain cell culture plates coated with CD3 and CD28 antibodies; (3) Lymphocyte separation: Take fresh blood and dilute it with an equal volume of diluent. Use lymphocyte separation solution to separate lymphocytes from PBMCs. After centrifuging at 750 g for 25 min, carefully aspirate the white membrane layer with a 1 mL syringe and wash it by centrifuging (250 g, 10 min) with 10 mL washing solution. (4) Cell stimulation culture: Cells were diluted to 5 × 10⁶ 5 / well, cultured in 1640 complete medium containing 10% FBS, 1% penicillin-streptomycin and 50 μM β-mercaptoethanol (37 ℃, 5% CO2), with the addition of mature activated dendritic cells from Example 1. The day of extraction was designated as day 0, the medium was changed halfway on day 2, and the cells were collected on day 4 to obtain the DC-CTL cells (denoted as G5).

[0034] Comparative Example 1 This comparative example illustrates a method for inducing mature activated dendritic cells. This induction method is essentially the same as that in Example 1, except for step (c): On day 6, cells are collected, centrifuged, and re-seeded in 12-well plates. The cells are then co-incubated with FPC-NPs (CpG concentration 20 μg / mL) for 24 h to obtain the mature activated dendritic cells (denoted as mDC-1). The FPC-NPs are prepared using the following method: FPC-NPs were made by blending fluorinated PEI and CpG in a mass ratio of 1:1 and shaking for 30 min.

[0035] Comparative Example 2 This comparative example is a method for inducing immature activated dendritic cells. The induction method is basically the same as that in Example 1, except that step (c) is as follows: On day 6, cells are collected, centrifuged, and then re-coated into 12-well plates and incubated for 24 hours to obtain the immature activated dendritic cells (denoted as iDC).

[0036] Comparative Example 3 This comparative example is the preparation method of DC-CTL cells (denoted as G4). The preparation method is basically the same as that of Example 2, except that the mature activated dendritic cells of Example 1 are replaced with the mature activated dendritic cells of Comparative Example 1.

[0037] Comparative Example 4 This comparative example is the preparation method of DC-CTL cells (denoted as G3). The preparation method is basically the same as that of Example 2, except that the mature activated dendritic cells of Example 1 are replaced with the immature activated dendritic cells of Comparative Example 2.

[0038] Comparative Example 5 This comparative example illustrates the preparation method of DC-CTL cells (denoted as G6), which is basically the same as that in Example 2, except that the mature activated dendritic cells in Example 1 are replaced with IL-2 at a concentration of 20 ng / mL, serving as a positive control.

[0039] Comparative Example 6 This comparative example illustrates the preparation method of DC-CTL cells (denoted as G2), which is basically the same as the preparation method in Example 2, except that the mature activated dendritic cells from Example 1 are removed as a negative control.

[0040] Experimental Example 1 Dendritic cells were obtained according to the induction methods of Example 1 and Comparative Examples 1-2, respectively. After centrifugation and washing, they were stained with anti-CD11c, CD80, CD86, CD40, MHC I, and MHC II fluorescent antibodies at 4 °C for 30 min. After washing and fixation, the expression ratios of co-stimulatory molecules and major histocompatibility complexes on the surface of dendritic cells were detected by flow cytometry. The results are as follows: Figures 1-2 As shown; Figure 1 The expression of CD86, CD80, and CD40 on the surface of PBMC-induced DCs without treatment (iDC), after treatment with FPC-NPs (mDC-1) or FPC-NPs@TAAs (mDC-2); Figure 2 The expression of MHC I and MHC II on the surface of PBMC-induced DCs without treatment (iDC), after treatment with FPC-NPs (mDC-1) or FPC-NPs@TAAs (mDC-2); Depend on Figures 1-2 It can be known that: Stimulation with FPC-NPs@TAAs resulted in significantly higher proportions of CD80, CD86, CD40, MHC I, and MHC II than with FPC-NPs and the control group, indicating that it can effectively induce DC maturation.

[0041] Experimental Example 2 DC-CTL cells were obtained according to the preparation methods of Examples 2 and Comparative Examples 3-6, respectively. The collected cells were incubated with CD3e-FITC and CD8a-PerCP Cy5.5 antibodies at 4 °C for 30 min, washed, fixed with tissue cell fixation solution, and data were collected and analyzed using flow cytometry. The results are as follows: Figure 3 As shown; Figure 3 In the formula, G2: no IL-2, G3: iDC, G4: mDC-1, G5: mDC-2, and G6: IL-2.

[0042] Depend on Figure 3 It can be seen that the DC-induced group treated with FPC-NPs@TAAs induced more T cells than the control group, with a proportion of about 80%, which was also significantly higher than the positive control group with conventional IL-2 addition; at the same time, the proportion of CD8+ T cells was also higher.

[0043] Experimental Example 3 This experimental example studies the ability of DC-CTL cells to specifically lyse tumor cells: 4T1 target cells were labeled with Calcein-AM fluorescent dye, and the viability of DC-CTL effector cells (prepared using the methods described in Example 2 and Comparative Examples 3-6) was detected by Calcein release assay. Target cells (2 × 10⁻⁶) 6 Stain with 5 μM Calcein-AM (2 mM) at 37 ℃ for 20 min. After washing effector cells and target cells twice with PBS, they were seeded in 96-well plates at appropriate ratios (1:20, 1:10, 1:1, 10:1, 20:1) and incubated at 37 ℃ for 6 h. The cell supernatant was collected and transferred to a new plate. The fluorescence intensity at Ex = 485 nm and Em = 530 nm was measured.

[0044] Tumor-specific lysis percentage (%) = (Experimental release group - Spontaneous release group) / (Maximum release group - Spontaneous release group) × 100%. The maximum release group was obtained using 3% Triton X-100, and the spontaneous release group contained only labeled target cells, without effector cells.

[0045] Experimental results are as follows Figure 4 As shown, Figure 4 In the formula, G2: no IL-2, G3: iDC, G4: mDC-1, G5: mDC-2, and G6: IL-2.

[0046] Depend on Figure 4 It can be seen that the percentage of tumor cell-specific lysis increases with the increase of effector-target ratio. T cells generated by FPC-NPs@TAAs-induced mDC stimulation showed superior killing ability to the IL-2-added group at multiple effector-target ratios (e.g., 1:10, 1:1, 10:1, 20:1), and were the strongest among all groups.

[0047] Experiment Example 4 PBMCs were extracted on Day 11 and induced into dendritic cells (DCs). PBMCs were extracted on Day 4 and induced into T cells. DCs from different groups were collected and co-cultured with T cells. Controls were set up with IL-2 or no additional stimulant. On Day 0, DC-CTL cells or T cells (i.e., cells from Example 2 and Comparative Examples 3-6) were collected, washed, and injected into mice via tail vein for immunization. Eight mice were in each group. On Day 7, 4 T1 cells were subcutaneously inoculated into the right hind limb of mice. Tumor volume was observed and recorded. When the tumor volume exceeded 1500 mm, the tumor was classified as a tumor. 3 At that time, the mice were euthanized, and survival curves were plotted; the results are as follows. Figure 5 As shown, Figure 5In the figure, A represents the tumor volume change curves of different groups of mice, and B represents the survival curves of mice; G1: Control group, G2: no IL-2, G3: iDC, G4: mDC-1, G5: mDC-2, G6: T cells treated with IL-2; Depend on Figure 5 It can be seen that, compared with the control group and other groups, DC-CTL cells generated by FPC-NPs@TAAs-induced mDC stimulation have a stronger ability to preventively inhibit tumor growth after reinfusion.

[0048] 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 therein. Such 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, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for inducing mature and activated dendritic cells, characterized in that, The induction method includes the following steps: (a) PBMCs were isolated from peripheral blood and then purified by adherence to a wall to obtain mononuclear cells; (b) Monocytes were induced to grow using a complete culture medium containing IL-4 and GM-CSF; (c) After 6-7 days of induction culture, cells are collected and co-incubated with FPC-NPs@TAAs to obtain the mature activated dendritic cells; The FPC-NPs@TAAs are formed by the self-assembly of F-PEI, CpG and TAAs.

2. The induction method according to claim 1, characterized in that, The complete culture medium containing IL-4 and GM-CSF has a concentration of 8-12 ng / mL for IL-4 and a concentration of 15-25 ng / mL for GM-CSF.

3. The induction method according to claim 1, characterized in that, The culture medium is changed every 2-3 days during the induction culture process; the incubation time is 20-30 h.

4. The induction method according to claim 1, characterized in that, The FPC-NPs@TAAs are prepared by the following method: FPC-NPs are self-assembled by electrostatic adsorption of fluorinated PEI and CpG in a mass ratio of 1:(0.8~1.2). Then, FPC-NPs are mixed with TAAs in a mass ratio of 1:(0.2~0.4) and shaken for 30 min to obtain FPC-NPs@TAAs.

5. Mature activated dendritic cells obtained by the induction method according to any one of claims 1 to 4.

6. The use of mature activated dendritic cells obtained by the induction method according to any one of claims 1 to 4 in the preparation of DC-CTL cells.

7. A method for preparing DC-CTL cells, characterized in that, The preparation method includes the following steps: (1) PBMCs were isolated from peripheral blood; (2) PBMCs and mature activated dendritic cells obtained by the induction method according to any one of claims 1 to 4 are seeded into cell culture plates coated with CD3 and CD28 antibodies and cultured in RPMI 1640 complete medium containing 10% FBS, 1% penicillin-streptomycin mixture and 50 μM β-mercaptoethanol to obtain the DC-CTL cells.

8. The preparation method according to claim 7, characterized in that, The ratio of PBMCs to mature and activated dendritic cells is (1.5~2.5):1; the culture time is 4~5 days; the culture medium is changed every 2~3 days during the culture process.

9. DC-CTL cells prepared by the method of claim 7 or 8.

10. The use of DC-CTL cells prepared by the method of claim 7 or 8 in the preparation of antitumor drugs.

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