A dendritic cell vaccine loaded with a novel antigen peptide and a preparation method and application thereof

Abstract

This invention discloses a dendritic cell vaccine loaded with novel antigenic peptides, its preparation method, and its applications, relating to the biomedical field. The active ingredient of the dendritic cell vaccine loaded with novel antigenic peptides of this invention comprises dendritic cells loaded with peptide segments; wherein, the peptide segments include any one of the following: a first antigenic peptide with an amino acid sequence as shown in SEQ ID NO.2, a second antigenic peptide with an amino acid sequence as shown in SEQ ID NO.4, and a third antigenic peptide with an amino acid sequence as shown in SEQ ID NO.9. The dendritic cells loaded with these peptide segments can effectively promote T cell proliferation (proliferation rate 67.8%) and directionally induce CD4+ T cells to differentiate into Th1-type cells (proportion 62.3%) and CD8+ T cells to become effector CTLs (proportion 58.6%), thereby increasing the secretion level of immune effector molecules and can be used for anti-tumor therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a dendritic cell vaccine loaded with a novel antigenic peptide, its preparation method, and its application. Background Technology

[0002] Malignant tumors are a major disease that seriously threatens human health, ranking among the highest in incidence and mortality worldwide. Tumor immunotherapy, by restarting and maintaining the body's normal anti-tumor immune response, has become a revolutionary treatment strategy following surgery, radiotherapy, and chemotherapy. Among them, dendritic cell (DC)-based tumor vaccines, by utilizing the powerful antigen-presenting capacity of DCs, activate tumor-specific T cells both in vivo and in vitro, demonstrating unique therapeutic advantages and promising application prospects.

[0003] Dendritic cells (DCs), acting as "sentinels" of the body's immune system, can take up and process tumor antigens and present them to T cells, initiating specific anti-tumor immune responses. They serve as a crucial bridge connecting innate and adaptive immunity. In recent years, with a deeper understanding of the biological characteristics of DCs and advancements in in vitro culture technology, DC vaccines have achieved preliminary efficacy in preclinical and clinical studies of various solid tumors, including melanoma, prostate cancer, and renal cell carcinoma. However, the clinical translation of DC vaccines still faces several technical bottlenecks, mainly in the following aspects: First, in terms of antigen peptide screening, the identification of highly immunogenic tumor antigens is the primary step in DC vaccine development. Although bioinformatics prediction technologies based on tumor genomic data have facilitated antigen peptide screening, existing research largely remains at the algorithmic prediction level, lacking systematic in vitro immunological functional verification. This results in limited ability of the selected candidate peptides to actually induce T cell responses, making it difficult to effectively activate specific anti-tumor immunity. Second, regarding DC in vitro culture and antigen loading, current DC differentiation induction largely relies on classic cytokine combination schemes, but this system still has room for optimization in terms of DC maturity, survival rate, and functional stability. Meanwhile, standardized procedures for loading antigenic peptides (such as concentration, timing, and cofactor addition) have not yet been established, leading to significant parameter variations among different studies. This directly impacts the antigen presentation efficiency of dendritic cells (DCs) and the uniformity and reproducibility of vaccines. Thirdly, regarding the molecular mechanisms of immune activation, although signaling pathways such as PI3K / Akt and NF-κB have been confirmed to participate in T cell activation and proliferation, the synergistic regulatory network and key molecular nodes of these pathways in the DC vaccine-mediated specific immune response remain unclear. This lag in mechanistic research limits the precise regulation of the immune effects of DC vaccines and the development of synergistic strategies.

[0004] In summary, current DC vaccine research generally suffers from problems such as insufficient immunogenicity of antigenic peptides, non-standardized culture and loading processes, and unclear T-cell activation mechanisms, leading to unstable clinical efficacy and severely restricting its large-scale application. Therefore, screening and identifying novel tumor antigenic peptides with high immunogenicity, establishing optimized DC in vitro culture and antigen loading systems, and systematically elucidating their molecular regulatory mechanisms for T-cell activation and their in vitro and in vivo anti-tumor effects are of significant clinical importance for promoting the clinical translation of DC vaccines and improving the efficiency of tumor immunotherapy. Summary of the Invention

[0005] The first aspect of the present invention is to provide a dendritic cell vaccine loaded with a novel antigenic peptide.

[0006] A second aspect of the present invention is to provide a method for preparing a dendritic cell vaccine loaded with a novel antigenic peptide.

[0007] The third objective of this invention is to provide a method for constructing a DC-T co-cultivation model.

[0008] The fourth aspect of this invention aims to provide the use of a dendritic cell vaccine loaded with a novel antigenic peptide in the preparation of products that promote T cell proliferation and / or induce T cell differentiation into Th1 types.

[0009] The fifth aspect of this invention aims to provide the application of a dendritic cell vaccine loaded with a novel antigenic peptide in the preparation of an antitumor drug.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a dendritic cell vaccine loaded with a novel antigenic peptide, wherein the active ingredient comprises dendritic cells loaded with the peptide. The peptide segment includes any one of the following: a first antigenic peptide with an amino acid sequence as shown in SEQ ID NO.2; a second antigenic peptide with an amino acid sequence as shown in SEQ ID NO.4; and a third antigenic peptide with an amino acid sequence as shown in SEQ ID NO.9.

[0011] In some embodiments of the present invention, the ratio of dendritic cells to peptides is 1×10⁻⁶. 6 Cells: 0.1~200μg. Preferably 1×10⁻⁶. 6 cells: 2~30μg.

[0012] In some embodiments of the present invention, the dendritic cells are induced to differentiate from peripheral blood mononuclear cells.

[0013] In some embodiments of the present invention, the induced differentiation includes culturing the peripheral blood mononuclear cells in a culture medium containing GM-CSF, IL-4, TNF-α, TLR agonist and IL-12.

[0014] In some embodiments of the present invention, the TLR agonist comprises poly(I:C).

[0015] In some embodiments of the present invention, the induced differentiation includes: first culturing the peripheral blood mononuclear cells in a culture medium containing the GM-CSF, the IL-4, the TLR agonist and the IL-12, and then culturing them in a culture medium containing the TNF-α to obtain the cells.

[0016] In some embodiments of the present invention, the concentration of GM-CSF is 100~200 ng / mL.

[0017] In some embodiments of the present invention, the concentration of IL-4 is 50~100 ng / mL.

[0018] In some embodiments of the present invention, the concentration of TNF-α is 15~35 ng / mL.

[0019] In some embodiments of the present invention, the concentration of the TLR agonist is 15-25 μg / mL. Preferably, the concentration of the TLR agonist is 20 μg / mL. In some embodiments of the present invention, the concentration of IL-12 is 5-15 ng / mL. Preferably, the concentration of IL-12 is 10 ng / mL.

[0020] In some embodiments of the invention, the dendritic cell vaccine further comprises pharmaceutically acceptable excipients.

[0021] A second aspect of the present invention provides a method for preparing a dendritic cell vaccine loaded with a novel antigenic peptide as described in the first aspect, comprising: contacting the peptide with the dendritic cells and obtaining the vaccine after screening.

[0022] In some embodiments of the present invention, the ratio of dendritic cells to peptides is 1×10⁻⁶. 6 Cells: 0.1~200μg. Preferably 1×10⁻⁶. 6 cells: 2~30 μg.

[0023] A third aspect of the present invention provides a method for constructing a DC-T co-culture model, which includes co-culturing dendritic cells loaded with novel antigenic peptides as described in the first aspect with T cells to obtain the model.

[0024] A fourth aspect of the invention provides the use of a dendritic cell vaccine loaded with a novel antigenic peptide as described in the first aspect in the preparation of products that promote T cell proliferation and / or induce T cell differentiation into Th1 types.

[0025] A fourth aspect of the invention provides the use of a dendritic cell vaccine loaded with a novel antigenic peptide as described in the first aspect in the preparation of an antitumor drug.

[0026] The dendritic cell vaccine loaded with novel antigenic peptides according to embodiments of the present invention, its preparation method, and its application have at least the following beneficial effects: (1) This invention successfully screened three novel antigenic peptides with high immunogenicity—peptide 2 (CEACAM5), peptide 4 (CTNNB1p.T41A), and peptide 9 (KRASp.Q61H)—through a dual strategy of "bioinformatics prediction-in vitro functional verification," providing a high-quality antigenic basis for vaccine preparation. Simultaneously, this invention optimized and established a highly efficient DC in vitro culture system. After inducing differentiation for 5 days with GM-CSF (150 ng / mL), IL-4 (100 ng / mL), TLR agonist (20 μg / mL), and IL-12 (10 ng / mL), TNF-α (25 ng / mL) was added for maturation induction. The differentiation rate of monocytes into DCs reached 89.7%, and the DCs highly expressed antigen-presenting molecules. The peptide concentration was 20 μg / mL, the loading time was 12 h, and the DC-to-peptide ratio was 1×10⁻⁶. 6 Cells: 20μg, which increased the positive rate of DC surface antigen peptide-MHC complex to 91.3%, providing technical support for the standardized preparation of vaccines. (2) Through experiments, this invention found that antigen peptide loading can significantly enhance the immunostimulatory function of DCs. After co-culturing with T cells, it effectively promotes T cell proliferation (proliferation rate 67.8%) by activating the PI3K / Akt / mTOR and NF-κB signaling pathways, and directionally induces CD4+ T cells to differentiate into Th1 type (proportion 62.3%) and CD8+ T cells to become effector CTLs (proportion 58.6%), and enhances the secretion level of immune effector molecules. Moreover, the two signaling pathways have been confirmed as core regulatory targets. In vitro functional verification showed that the CTLs induced by this DC vaccine exhibited highly efficient and specific tumor killing activity against different tumor cells, and were effector-target ratio dependent. At an effector-target ratio of 50:1, the killing rate against HCT116 and HepG2 cells was the highest, reaching 79.5% and 74.3%, respectively; it also had a strong killing effect on A549 cells; the killing rate against MCF-7 cells was relatively low, at about 62.8%. These results demonstrate that the CTLs induced by this DC vaccine possess broad-spectrum and highly effective in vitro antitumor activity. The DC cell vaccine of this invention exhibits highly efficient, specific, and stable antitumor potential, providing solid experimental evidence and scientific support for the large-scale production, clinical translation, and development of new targets for tumor immunotherapy using DC vaccines. Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a flow cytometry diagram of the surface antibody CD86 on DC cells in the in vitro culture system of Group A of this invention.

[0028] Figure 2 This is a flow cytometry diagram of the surface antibody CD86 on DC cells in the in vitro culture system of Group B of this invention.

[0029] Figure 3 This is a flow cytometry diagram of the DC surface antibody CD86 in the in vitro culture system of group C of this invention.

[0030] Figure 4 This is a diagram showing the D4 differentiation state of the DC cells in the in vitro culture system of Group A of this invention.

[0031] Figure 5 The positive rate of surface antigen peptide-MHC complex at different maturation stages of DC in the in vitro culture system of Group A of this invention.

[0032] Figure 6 The flow cytometry diagrams show the ability of different peptides in this invention to induce DCs to secrete IFN-γ, where A is the blank control, B is the positive control, C is the CEACAM5 peptide, D is the CTNNB1P.T41A peptide, and E is the KRASp.Q61H peptide.

[0033] Figure 7 The results show the effects of different peptides induced by DC on the proliferation rate of CD8+ T cells.

[0034] Figure 8 The results of the experiment on the secretion of pro-inflammatory cytokines by DCs loaded with antigenic peptides according to the present invention are shown.

[0035] Figure 9 This is a flowchart illustrating the construction process of the DC-T co-cultivation model of this invention.

[0036] Figure 10 The results of the T cell proliferation rate and p65 nuclear translocation rate are presented in this invention.

[0037] Figure 11 This is a diagram showing the activation state of T cells (D4) in the DC+T cell group loaded with antigen peptides according to the present invention.

[0038] Figure 12 The results show the phosphorylation level of T cells in the DC+T cell group loaded with antigen peptides according to this invention.

[0039] Figure 13 The results of T cell phenotype and function detection in this invention are shown, where A represents IL-2 and IFN-γ secretion, B represents perforin and granzyme B expression levels, C represents siRNA interference, and D represents T cell phenotype and function.

[0040] Figure 14 This is the result of the tumor cell killing rate detection in this invention. Detailed Implementation

[0041] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0042] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0043] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0044] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0045] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0046] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0047] Example 1: Screening of DC in vitro culture system This embodiment established a highly efficient DC in vitro culture system to determine the optimal loading scheme for antigenic peptides, laying the foundation for subsequent vaccine preparation. The specific experiments in this study are as follows.

[0048] 1. Experimental Methods This experiment designed three cytokine combinations based on a DC culture system. Each combination contained poly(I:C) (20 μg / mL) and IL-12 (10 ng / mL). The different cytokine combinations are as follows: Group A: Cultured with 150 ng / mL GM-CSF, 100 ng / mL IL-4, 25 ng / mL TNF-α, 20 μg / mL poly(I:C) and 10 ng / mL IL-12; Group B: Induced culture with 150 ng / mL GM-CSF, 100 ng / mL IL-4, 20 μg / mL poly(I:C) and 10 ng / mL IL-12; Group C: Cultured with 150 ng / mL GM-CSF, 100 ng / mL IL-4, 10 ng / mL IL-1β, 20 μg / mL poly(I:C) and 10 ng / mL IL-12.

[0049] Taking group A as an example, the specific cultivation method is as follows: (1) PBMC isolation: Peripheral blood was collected, and the intermediate white membrane layer was collected by Ficoll density gradient centrifugation. The cells were washed twice with D-PBS, and then resuspended in RPMI 1640 to adjust the cell concentration to 2×10⁻⁶. 6 PBMC suspension was obtained by measuring 1 / mL of PBMC.

[0050] (2) Plate culture: Add the above PBMC suspension to a 6-well plate, 2 mL / well, and culture at 37 ℃ and 5% CO2 for 2 h. Discard the supernatant and non-adherent cells, and retain the adherent monocytes (i.e. DC precursors).

[0051] (3) Differentiation induction: Add 2 mL of complete culture medium containing 150 ng / mL GM-CSF, 100 ng / mL IL-4, 20 μg / mL poly(I:C) and 10 ng / mL IL-12 to each well, and culture at 37℃ and 5% CO2. Change half of the medium and add an equal amount of cytokines every 2-3 days to ensure stable factor concentrations. On day 5 of induction, add 25 ng / mL TNF-α for maturation induction.

[0052] (4) Morphological observation and harvest: On the 6th-7th day of culture, cells can be seen to grow in suspension and show typical dendritic protrusions, which are mature DCs; collect the cells by centrifugation and count them for later use.

[0053] The expression of the DC surface antibody CD86 was detected by flow cytometry. The positivity rate of the DC surface antigen peptide-MHC complex was also detected by flow cytometry.

[0054] 2. Experimental Results Flow cytometry plots of CD86 surface antibody from different groups of DCs are shown below. Figures 1-3 As shown, group A (GM-CSF 150 ng / mL + IL-4 100 ng / mL + poly(I:C) 20 μg / mL + IL-12 10 ng / mL induction for 5 days, TNF-α 25 ng / mL maturation induction) showed the best results, and its DC differentiation D4 state diagram is shown below. Figure 4 As shown, dendritic cells obtained using the Group A culture system highly expressed CD86, MHC-I, and MHC-II molecules (positive rate 89.08%-96.3%), specifically as follows. Figure 5 As shown.

[0055] Example 2 Screening of novel antigenic peptides with high immunogenicity In this embodiment, 12 candidate peptides were obtained using bioinformatics methods, and further screening was performed on them based on the DC culture system screened above, resulting in novel antigenic peptides with high immunogenicity. The specific experimental process is as follows.

[0056] 1. Experimental Materials Sample sources for the experiment: peripheral blood from healthy volunteers (ethically approved) and A549 (lung cancer cell line).

[0057] Reagents: 1640 basal medium, GM-CSF, IL-4, IL-2, TNF-α, IL-1β, poly(I:C) and IL-12 and other cytokines, CD3, CD28 antibody; flow cytometry antibodies (CD86, MHC-I, MHC-II, CD3, CD4, CD8, etc.).

[0058] Instruments: Flow cytometer, ELISA reader, CO2 incubator, clean bench.

[0059] 2. Experimental Methods First, this invention uses databases such as NetMHC and IEDB to predict MHC binding affinity, immunogenicity score, and antigen processing and presentation efficiency, and screens 12 candidate peptides, the details of which are shown in Table 1.

[0060] Table 1:

[0061] Furthermore, antigen peptide screening was performed on the DC in vitro culture system constructed above, specifically including the following steps: (1) PBMC segregation and induced differentiation: Peripheral blood was collected, centrifuged using Ficoll density gradient centrifugation, and the intermediate white membrane layer was collected. The cells were washed twice with D-PBS, and then the cell concentration was adjusted to 2 × 10⁻⁶ cells using RPMI 1640 counting. 6 PBMC suspension was obtained by increasing the number of cells per mL. The PBMC suspension was then added to 6-well plates at 2 mL / well and incubated at 37 °C with 5% CO2 for 2 h. The supernatant and non-adherent cells were aspirated, retaining the adherent monocytes (DC precursors). 500 μL of plasma was added to each well, along with 100 ng / mL IL-4, 150 ng / mL GM-CSF, 20 μg / mL poly(I:C), and 10 ng / mL IL-12 for DC cell culture. The medium was changed and an equal amount of cytokines was added every other day to ensure stable factor concentrations.

[0062] Supernatant and non-adherent cells were transferred to pre-coated plates 2 hours prior for T cell culture: Coating medium was prepared by diluting CD3 and CD28 to 2 μg / mL and 4 μg / mL, respectively, with PBS. 2 mL of this medium was added to each well of a 6-well plate and incubated at room temperature for 2 hours. The coating medium was then removed, and the supernatant and non-adherent cells were transferred to the plates. 150 μL of plasma and 100 ng / mL IL-2 were added to each well. The medium was changed and an equal amount of cytokines was added every other day to ensure stable cytokine concentrations, resulting in T cell culture.

[0063] (2) DC-loaded antigen peptides and co-culture: On day 5 of induction using the above method, the 12 candidate peptides or OVA-positive peptides mentioned above were added to the DC culture wells at a final concentration of 20 μg / mL, and incubated for 2 hours. Excess peptides were aspirated, the cells were washed once with PBS, and the DC culture medium was replaced for continued culture. On day 6 of induction, 25 ng / mL TNF-α was added for maturation induction. By days 6-7, cells were observed to grow in suspension and develop typical dendritic protrusions, indicating mature DCs. On day 7, T cells were added at a cell ratio of 1:100 for co-culture (i.e., DC-T co-culture) for 7 days, during which plasma and IL-2 were supplemented to maintain factor stability.

[0064] (3) Secondary addition of peptides to stimulate T cells to secrete IFN-γ: After co-culturing DC-T cells for 7 days, the above 12 candidate peptides or OVA positive peptides were added to the culture wells again at a concentration of 20 μg / mL. After incubation for 6 hours, the cells were collected to detect IFN-γ.

[0065] (4) Flow cytometry detection of IFN-γ: After 6 hours of loading culture, cells from each group were digested and transferred to sterile centrifuge tubes, centrifuged at 1000 r / min and 4 ℃ for 5 min, the supernatant was discarded, and the cells were washed twice with flow cytometry staining buffer. The supernatant was discarded after each centrifugation to remove residual culture medium and unbound peptides. Subsequent flow cytometry analysis was then performed. The specific steps are as follows: S1. Fixation treatment: Add 500 μL of 1× fixation membrane-breaking solution to each group of cells and incubate at 4 ℃ in the dark for 20 min to fix the cells and break the cell membrane, so as to facilitate the binding of intracellular IFN-γ to the antibody. S2. Washing after membrane rupture: Add flow cytometry staining buffer, centrifuge at 1000 r / min and 4 ℃ for 5 min, discard the supernatant, and repeat the washing twice to remove residual fixation membrane rupture solution to avoid affecting antibody binding efficiency.

[0066] S3. Fluorescent antibody staining: Add 1 μL of IFN-γ fluorescently labeled antibody to each group of cells, gently pipette to mix, and incubate at 4°C in the dark for 30 min. Occasionally gently invert the centrifuge tube to ensure uniform antibody binding.

[0067] S4. Washing after staining: Add flow cytometry staining buffer, centrifuge at 1000 r / min and 4 ℃ for 5 min, discard the supernatant, and wash twice to remove unbound fluorescent antibodies and reduce non-specific staining interference.

[0068] S5. Resuspending for instrumentation: Add 200 μL of flow cytometry staining buffer to each group of cells, gently pipette to resuspend the cells, and prepare a single-cell suspension to avoid cell aggregation.

[0069] The proportion of TNF-γ positive cells was analyzed by flow cytometry.

[0070] (5) Detection of the activation ability of T cells by DCs loaded with antigen peptides: The activation capacity of DCs on T cells after DC loading was detected using the CCK-8 assay. The specific method is as follows: S1. Construction of cell co-culture system: Human peripheral blood mononuclear cells (PBMCs) in logarithmic growth phase were collected, and T lymphocytes were obtained by density gradient centrifugation. The cell concentration was adjusted to 1×10⁶ cells / mL. 6 Dendritic cells were cultured at 37 ℃ and 5% CO2 for 2 h at a concentration of 10 cells / mL for later use. Antigen-loaded dendritic cells (DCs) and unloaded dendritic cells (DCs) were prepared, and the concentration was adjusted to 2 × 10⁻⁶ cells / mL. 5 / mL. Then, co-culture was performed using 96-well cell culture plates, with experimental groups, blank control groups, and T-cell control groups set up, with 3 replicates in each group to ensure experimental reproducibility.

[0071] ① Experimental group: Add 100 μL of the T cell suspension prepared above (1×10⁻⁶) to each well. 5 (cells / well), then add 100 μL of loaded DC suspension (2×10) 4 (cells / well), so that the ratio of T cells to loaded DC cells is 100:1, gently pipette to mix, and construct a loaded DC-T cell co-culture system.

[0072] ② Blank control group: Add 100 μL of T cell suspension (1×10⁻⁶) to each well. 5 (cells / well), then add 100 μL of blank control DC suspension (2 × 10⁻⁶ cells / well). 4 A blank DC-T cell co-culture system was constructed with cells / wells, and the cell ratio was also 100:1.

[0073] ③T cell control group: Add 100 μL of T cell suspension (1×10⁻⁶) to each well. 5 (cells / well), then add 100 μL of RPMI-1640 complete culture medium, without adding DC cells, as a control for spontaneous T cell proliferation.

[0074] S2. Co-culture incubation: Place the above 96-well culture plate in a 37℃, 5% CO2 constant temperature incubator and co-culture statically for 72h. Avoid shaking the culture plate during this period to ensure stable cell growth and interaction.

[0075] S3, CCK-8 assay: After co-culture, add 10 μL of CCK-8 reagent to each well, gently pipette to mix, avoiding the formation of air bubbles, and continue to incubate in a 37℃, 5% CO2 incubator in the dark for 2 hours to ensure that the CCK-8 reagent reacts fully with the cells.

[0076] S4. Absorbance detection: Remove the culture plate and use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm. Before measurement, gently shake the culture plate for 10 seconds to ensure that the liquid in the well is uniform. Measure each well 3 times and take the average value as the final OD value of the well.

[0077] S5. Result Calculation and Judgment: Based on the OD value of the T cell control group alone, the T cell proliferation rate of the experimental group and the blank control group was calculated separately, where: Proliferation rate = [(OD value of experimental group - OD value of blank control) - (OD value of DC control - OD value of blank control)] / [(OD value of T cell control - OD value of blank control)] × 100%.

[0078] If the metabolic activity of DC itself is very low (DCs usually do not proliferate and the OD value is close to that of the blank control), the formula can be simplified to: (OD experimental group - blank control) / (OD_T cell control - blank control) × 100%; by comparing the T cell proliferation rate of the experimental group and the blank control group, the activation ability of DCs after loading on T cells can be determined. The higher the proliferation rate, the stronger the activation ability of DCs on T cells after loading on T cells.

[0079] SPSS 22.0 software was used for the above analysis. Quantitative data are expressed as mean ± standard deviation. The t-test or ANOVA was used for comparisons between groups. P5 was considered statistically significant.

[0080] 3. Experimental Results Twelve candidate peptides were screened using bioinformatics. Flow cytometry was used to detect the ability of these peptides to induce dendritic cells (DCs) to secrete IFN-γ. The results are as follows: Figure 6 As shown in Table 2.

[0081] Table 2:

[0082] The results showed that three peptides (peptide 2, peptide 4, and peptide 9) exhibited high IFN-γ expression. Furthermore, the activation capacity of loaded DCs for T cells was assessed using the CCK-8 assay, and the results are as follows: Figure 7 As shown, the T cell proliferation rate increased by 2.3-3.1 times, confirming that these three peptides have high immunogenicity.

[0083] Example 3 Construction and maturation characterization of DCs loaded with antigenic peptides In this embodiment, a DC loaded with antigen peptides was constructed based on the three peptide segments (peptide 2, peptide 4, and peptide 9) selected above, and its performance was characterized. The specific experiments are as follows.

[0084] 1. Experimental Methods (1) Construction of DC loaded with antigenic peptides Two control groups were set up: a DC group loaded with antigen peptide and a control group without loaded DC. The peptide concentration was 20 μg / mL, the loading time was 12 h, and the DC to peptide ratio was 1×10. 6 cells: 20μg.

[0085] Specifically as follows: ① Antigen peptide-loaded DC experimental group: Mature DC cells were collected according to the above method and resuspended in RPMI 1640. (The dosage was 1×10⁻⁶ cells / mL). 6 Cells / mL were seeded into culture plates and stimulated with antigenic peptides (20 μg / mL each of peptide 2, peptide 4, and peptide 9). Each group was divided into 3 replicates, and incubated in an incubator for 6 h, 12 h, and 24 h, respectively. The supernatant was collected: centrifuged at 300 g for 10 min at 4 °C, and the supernatant was used for analysis.

[0086] ② Empty DC control group (DC cells without antigen peptide loading): Collect mature DC cells at a ratio of 1×10 6 Cells / mL were seeded into a culture plate, with a total of 9 wells. The supernatant from 3 wells was collected at 6 h, 12 h, and 24 h for analysis.

[0087] (2) Detection of pro-inflammatory / anti-inflammatory cytokines Based on the constructed DCs loaded with antigenic peptides and empty DCs as controls, ELISA was used to detect IL-12, TNF-α and IL-10 secreted by DCs.

[0088] 2. Experimental Results The results of the experiment on the secretion of pro-inflammatory / anti-inflammatory cytokines by DCs loaded with antigenic peptides are as follows: Figure 8As shown, compared with the unloaded mature DC group, the DCs loaded with antigen peptides in this invention have significantly increased levels of pro-inflammatory cytokines (IL-12: 215.3 pg / mL; TNF-α: 320.5 pg / mL) and decreased levels of anti-inflammatory factor IL-10 (45.2 pg / mL), indicating enhanced maturation function.

[0089] The above results indicate that loading antigenic peptides helps promote the secretion of pro-inflammatory cytokines by dendritic cells and enhances their maturation function.

[0090] Example 4: Effects of DCs loaded with antigenic peptides on T cells This embodiment constructs a DC-T co-culture model based on the aforementioned DCs loaded with antigenic peptides to further investigate the T cell activation signaling pathway and functional response characteristics, aiming to identify regulatory targets. The construction process of the DC-T co-culture model is as follows: Figure 9 As shown, the specific experiment is as follows.

[0091] 1. Experimental Methods First, collect 20 mL of peripheral blood and centrifuge using a Ficoll density gradient for 20 min to collect the intermediate white membrane layer. Wash twice with D-PBS and adjust the cell concentration to 2 × 10⁻⁶ cells using RPMI 1640 counting. 6 PBMC suspension was obtained by increasing the number of cells per mL. The PBMC suspension was then added to 6-well plates at 2 mL / well and incubated at 37°C with 5% CO2 for 2 hours. The supernatant and non-adherent cells were aspirated, retaining the adherent monocytes (DC precursors). 500 μL of plasma was added to each well, along with 100 ng / mL IL-4, 150 ng / mL GM-CSF, 20 μg / mL poly(I:C), and 10 ng / mL IL-12 for DC cell culture. The medium was changed and an equal amount of cytokines was added every other day to ensure stable factor concentrations.

[0092] Supernatant and non-adherent cells were transferred to pre-coated plates 2 hours prior for T cell culture: Coating medium was prepared, and CD3 and CD28 were diluted to 2 μg / mL and 4 μg / mL, respectively, with PBS. 2 mL of this medium was added to each well of a 6-well plate and incubated at room temperature for 2 hours. The coating medium was then removed, and the supernatant and non-adherent cells were transferred to the plates. 150 μL of plasma and 100 ng / mL IL-2 were added to each well. The medium was changed and an equal amount of cytokines was added every other day to ensure stable cytokine concentrations.

[0093] On day 5 of induction, three peptides (peptide 2, peptide 4, and peptide 9) were added sequentially to the DC culture wells, each at a final concentration of 20 μg / mL, and incubated for 2 h. Excess peptides were aspirated, the cells were washed once with PBS, and the DC culture medium was replaced for further culture. On day 6 of induction, 25 ng / mL TNF-α was added for maturation induction. By days 6-7, cells showed suspension growth and the appearance of typical dendritic protrusions, indicating mature DCs. On day 7, T cells were added at a cell ratio of 1:100 and co-cultured for 7 days, supplementing with plasma and 100 ng / mL IL-2 during this period to maintain factor stability, resulting in DC-T cells, which were labeled as the antigen-peptide-loaded DC-T cell group.

[0094] Simultaneously, three control groups were set up: a DC+T cell group without antigen peptide loading, a T cell group alone, and an siRNA interference group (PI3KsiRNA, NF-κBsiRNA). The relevant information for the PI3KsiRNA used is as follows: 5'-GCA UGA AGA UGA AGG UGA UTT-3' (SEQ ID NO. 13); 5'-AUC ACC UUC AUC UUC AUG CTT-3' (SEQ ID NO. 14).

[0095] The relevant information for NF-κBsiRNA is as follows: 5'-GGA UUA CUU CGA GAA GAA UTT-3' (SEQ ID NO. 15); 5'-AUU CUU CUC GAA GUA AUC CTT-3' (SEQ ID NO. 16).

[0096] Cells were collected 48–72 h after siRNA interference transfection, and total RNA was extracted. The RNA was reverse transcribed into cDNA, and PI3K and NF-κB specific primers were designed. Using GAPDH / β-actin as an internal control, the relative expression levels (2...) were calculated. -ΔΔCt Law).

[0097] Western blot was used to detect the expression of phosphorylated Akt, mTOR, and p65 proteins in T cells, and immunofluorescence was used to detect p65 nuclear translocation. Flow cytometry was used to analyze the proportion of CD4+ and CD8+ T cell subsets and IFN-γ expression to detect T cell phenotype and function.

[0098] 2. Experimental Results The results of T cell proliferation rate and p65 nuclear translocation rate detection are as follows: Figure 10 As shown, the activation state of T cells (D4) in the antigen-peptide-loaded DC+ T cell group is illustrated in the figure. Figure 11As shown, the phosphorylation level detection results are as follows: Figure 12 As shown, the proliferation rate of T cells in the DC group loaded with antigen peptides reached 67.8%; the PI3K / Akt / mTOR and NF-κB signaling pathways in T cells were activated, phosphorylated Akt and mTOR increased by 3.2 times and 2.8 times, respectively, and the p65 nuclear translocation rate increased by 4.5 times.

[0099] T cell phenotype and function test results are as follows: Figure 13 As shown, after co-culture, the proportion of CD4+ T cells differentiating into Th1 type reached 62.3%, and the secretion of IFN-γ and IL-2 increased by 2.5-3.0 times; the proportion of CD8+ effector T cells was 58.6%, and the expression of perforin and granzyme B increased by 4.1 times and 3.7 times, respectively; siRNA interference confirmed that the two signaling pathways are key regulatory targets.

[0100] The above results indicate that co-culturing DCs loaded with antigen peptides with T cells in this invention effectively promotes T cell proliferation (proliferation rate 67.8%) by activating the PI3K / Akt / mTOR and NF-κB signaling pathways, and directionally induces CD4+ T cells to differentiate into Th1 type (proportion 62.3%), which helps to enhance the secretion level of immune effector molecules.

[0101] Example 5: Validation of in vitro antitumor effect This embodiment verifies the in vitro antitumor effect of the DC loaded with the above-mentioned antigenic peptide in vitro. The specific experiment is as follows.

[0102] 1. Experimental Materials Effector cells (CTLs): obtained using the DC-T co-culture model construction method described in Example 4 above. CTLs are CD8+ cells. + Cytotoxic T cells are a functional subset of T cells that are responsible for antigen-specific killing of target cells. They are the main effector cells of cell-mediated immunity and the main cell type of T cells obtained in Example 4 above.

[0103] Target cells: A549 (human lung adenocarcinoma cells), HepG2 (human liver cancer cells), MCF-7 (human breast cancer cells), HCT116 (human colon cancer cells).

[0104] Reagents: LDH release assay kit, IFN-γ-PE antibody.

[0105] 2. Experimental Methods (1) Target cell plating Various tumor cells in logarithmic growth phase (A549, HepG2, MCF-7, HCT116) were collected, trypsinized, centrifuged, and resuspended to a concentration of 1×10⁵ / mL; 100 μL of cell suspension (1×10⁵ / mL) was added to each well of a 96-well culture plate. 4(each well), incubate overnight at 37°C in a 5% CO2 incubator to ensure full adhesion to the walls.

[0106] (2) Co-incubation of target cells Collect CTL cells, count them, and adjust them to the appropriate concentration. Set up the following groups (each group has 3 replicates): CTL killing group: CTL + tumor cell group (effect-to-target ratio 10:1, 20:1, 50:1), CTL normal cell group, tumor cell group alone, background control wells: containing only culture medium. For the 50:1 effect-to-target ratio, the CTL density needs to be adjusted to 5 × 10⁶ cells / well. 6 The cells were diluted at a ratio of 1 / mL to obtain effector cell suspensions of 20:1 and 10:1.

[0107] Discard the old culture medium of the target cells in the 96-well plate. According to the experimental design, add 100 μL of effector cell suspension with different effector-to-target ratios to the experimental wells. Add an equal volume of culture medium or effector cell suspension to each control well. Add the suspension slowly to avoid generating air bubbles. Incubate the plate at 37 ℃ in a 5% CO2 incubator for 4–6 h.

[0108] (3) LDH detection One hour before the end of co-culture, 10 μL of LDH lysis buffer was added to each well of the tumor cell group. After the culture was completed, the cells were centrifuged at 400 xg for 5 min, and 100 μL of supernatant was transferred from each well to a new 96-well plate. 100 μL of LDH detection working solution was added, and the plates were incubated at room temperature in the dark for 30 min. Then, 50 μL of stop solution was added, and the OD value of each well was measured at 490 nm using a microplate reader.

[0109] (4) Calculate the kill rate.

[0110] The killing rate of CTLs against tumor cells was detected using the LDH release assay. Among other things: Kill rate (%) = (OD experimental wells - effector cell spontaneous release wells - target cell spontaneous release wells) / (OD maximum release wells - target cell spontaneous release wells).

[0111] All OD values ​​must be corrected by subtracting the OD value of the "background reference hole".

[0112] 3. Experimental Results The tumor cell killing rate of DC-T cells co-cultured with A549, HepG2, MCF-7, and HCT116 cells at different effector-to-target ratios of 10:1, 20:1, and 50:1 is as follows: Figure 14As shown, the results indicated that DC-T cells exhibited significant killing effects against all four types of tumor cells, and the killing efficiency increased significantly in a dose-dependent manner with increasing effector-to-target ratio. Specifically, under effector-to-target ratios of 10:1, 20:1, and 50:1, the killing rates of DC-T cells against A549 cells were 22.5%, 45.5%, and 72.3%, respectively; against HepG2 cells, 38.5%, 59.2%, and 74.3%, respectively; against MCF-7 cells, 26.1%, 45.4%, and 62.8%, respectively; and against HCT116 cells, 41.4%, 59.2%, and 79.5%, respectively.

[0113] In summary, this invention successfully screened three novel antigenic peptides with high immunogenicity—peptide 2, peptide 4, and peptide 9—through a dual strategy of "bioinformatics prediction and in vitro functional verification," providing a high-quality antigenic basis for vaccine preparation. Simultaneously, an efficient in vitro DC culture system was optimized and established. After 5 days of induction with GM-CSF (150 ng / mL) and IL-4 (100 ng / mL), TNF-α (25 ng / mL) was added for maturation induction. The differentiation rate of monocytes into DCs reached 89.7%, and the DCs highly expressed antigen-presenting molecules. The optimal peptide concentration was 20 μg / mL, the loading time was 12 h, and the DC-to-peptide ratio was 1 × 10⁻⁶. 6 Cells: 20μg, which increased the positive rate of DC surface antigen peptide-MHC complex to 91.3%, providing technical support for the standardized preparation of vaccines. Furthermore, this invention, through experiments, discovered that antigen peptide loading can significantly enhance the immunostimulatory function of DCs. When co-cultured with T cells, it effectively promotes T cell proliferation (proliferation rate 67.8%) by activating the PI3K / Akt / mTOR and NF-κB signaling pathways, directionally induces CD4+ T cells to differentiate into Th1 types (proportion 62.3%), and CD8+ T cells to become effector CTLs (proportion 58.6%), and increases the secretion level of immune effector molecules. Both signaling pathways were confirmed as core regulatory targets. In vitro functional validation showed that the CTLs induced by this DC vaccine exhibited highly efficient and specific tumor-killing activity against different tumor cells, in an effector-to-target ratio dependent manner. At an effector-to-target ratio of 50:1, the killing rate was highest against HCT116 and HepG2 cells, reaching 79.5% and 74.3%, respectively; it also showed a strong killing effect on A549 cells; the killing rate against MCF-7 cells was relatively low, around 62.8%. These results demonstrate that the CTLs induced by this DC vaccine possess broad-spectrum and highly effective in vitro antitumor activity. The DC cell vaccine of this invention exhibits highly efficient, specific, and stable antitumor potential, providing solid experimental evidence and scientific support for the large-scale production, clinical translation, and development of new targets for tumor immunotherapy using DC vaccines. The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A dendritic cell vaccine loaded with a novel antigenic peptide, characterized in that, The active ingredient comprises dendritic cells loaded with peptides; The peptide segment includes any one of the following: a first antigenic peptide with an amino acid sequence as shown in SEQ ID NO.2, a second antigenic peptide with an amino acid sequence as shown in SEQ ID NO.4, and a third antigenic peptide with an amino acid sequence as shown in SEQ ID NO.

9.

2. The dendritic cell vaccine according to claim 1, characterized in that, The ratio of dendritic cells to peptides is 1×10. 6 cells: 0.1~200μg.

3. The dendritic cell vaccine according to claim 1, characterized in that, The dendritic cells were induced to differentiate from peripheral blood mononuclear cells; Preferably, the induction of differentiation includes culturing the peripheral blood mononuclear cells in a culture medium containing GM-CSF, IL-4, TNF-α, TLR agonist and IL-12; Preferably, the TLR agonist comprises poly(I:C).

4. The dendritic cell vaccine according to claim 3, characterized in that, The induced differentiation process includes: first culturing the peripheral blood mononuclear cells in a medium containing the GM-CSF, IL-4, TLR agonist, and IL-12, and then culturing them in a medium containing TNF-α to obtain the cells.

5. The dendritic cell vaccine according to claim 3, characterized in that, The concentration of GM-CSF is 100~200 ng / mL; And / or, the concentration of the IL-4 is 50~100 ng / mL; And / or, the concentration of the TNF-α is 15~35 ng / mL; And / or, the concentration of the TLR agonist is 15~25 μg / mL; And / or, the concentration of the IL-12 is 5~15 ng / mL.

6. The dendritic cell vaccine according to any one of claims 1 to 5, characterized in that, The dendritic cell vaccine also contains pharmaceutically acceptable excipients.

7. The method for preparing a dendritic cell vaccine loaded with a novel antigenic peptide as described in any one of claims 1 to 6, characterized in that, include: The peptides were contacted with the dendritic cells and then screened to obtain the final product.

8. A method for constructing a DC-T co-culture model, characterized in that, This includes obtaining dendritic cells loaded with novel antigenic peptides as described in any one of claims 1 to 6 by co-culturing them with T cells.

9. The use of the dendritic cell vaccine loaded with the novel antigenic peptide as described in any one of claims 1 to 6 in the preparation of products that promote T cell proliferation and / or induce T cell differentiation into Th1 types.

10. The use of the dendritic cell vaccine loaded with a novel antigenic peptide as described in any one of claims 1 to 6 in the preparation of an antitumor drug.

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