DC-CIK cell culture method based on HBV protective antibody positive healthy donor and application

Abstract

The invention belongs to the technical field of cellular immunity, and particularly relates to a DC-CIK cell culture method based on an HBV protective antibody positive healthy donor and application, the cell culture method comprises the following steps: selecting HBsAg negative and anti-HBs antibody positive healthy donor peripheral blood mononuclear cells, loading a dendritic cell (DC) by using a mixed antigen of HBcAg and HBsAg, and culturing the DC-CIK cell by using a cell culture medium. And co-culturing the DC cells and cytokine-induced killer cells (CIK) to finally obtain the allogenic DC-CIK cells. According to the invention, DC cells of healthy donors and CIK cells are co-cultured to obtain allogenic DC-CIK cells, an HBcAg and HBsAg mixed antigen loading technology is adopted to significantly improve the antigen presentation capability of the DC cells, an optimized culture system enhances the targeting killing function of the cells, and the HBsAg clearance rate and the cccDNA inhibition rate can be effectively improved in combination with nucleoside analogue treatment, so that the DC-CIK cells can be used for preparing the DC-CIK cells. The synergistic breakthrough of efficient virus removal, immune function reconstruction and low recurrence rate in hepatitis B treatment is realized.

Description

Technical Field

[0001] This invention belongs to the field of cell immunology technology, specifically relating to a method and application of DC-CIK cell culture based on HBV protective antibody-positive healthy donors. Background Technology

[0002] Chronic hepatitis B (CHB) is a global public health challenge caused by hepatitis B virus (HBV) infection. Its core pathological features include persistent viral replication, immune dysfunction, and chronic liver damage, which can progress to cirrhosis and liver cancer in severe cases, posing a significant threat to human health. Currently, clinical treatment for active hepatitis B focuses on antiviral therapy and immunomodulation, with nucleoside analogues, interferon, and cellular immunotherapy as the main treatment methods. Among these, DC-CIK cell therapy has become a research hotspot due to its ability to activate specific immune responses and target and kill infected cells.

[0003] Current treatments have significant limitations: nucleoside analogues (such as tenofovir and entecavir), while effectively inhibiting HBV DNA replication with a seroconversion rate of 70%–90%, cannot eliminate viral covalently closed circular DNA (cccDNA) and integrated HBV genes, resulting in an HBsAg clearance rate of less than 1% and a functional cure rate of less than 5%. Patients require lifelong medication, and the relapse rate after discontinuation is 100%. Interferon-alpha has an HBsAg clearance rate of only 3%–7% and significant side effects. Traditional autologous DC-CIK therapy suffers from insufficient immune activity and low antigen presentation efficiency due to viral suppression of the patient's own immune cells, resulting in an HBsAg clearance rate of only 10%–15% and a limited functional cure rate. Furthermore, DC-CIK cells that have not undergone antigen loading or are cultured under suboptimal conditions exhibit further reduced targeted killing ability and immune activation effects, making it difficult to meet clinical treatment needs.

[0004] In summary, current treatment methods cannot simultaneously achieve the core objectives of efficiently clearing HBsAg, inhibiting cccDNA, breaking immune tolerance, and reducing relapse rates. There is an urgent need to develop a new treatment method that combines high efficiency, safety, and durability. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for culturing DC-CIK cells based on HBV protective antibody-positive healthy donors. By co-culturing allogeneic DC-CIK cells with healthy donor DC cells and CIK cells, and using HBcAg and HBsAg mixed antigen loading technology to significantly enhance the antigen presentation capacity of DC cells, the optimized culture system enhances the cell-targeted killing function. Combined with nucleoside analogue therapy, it can effectively improve the HBsAg clearance rate and cccDNA inhibition rate, achieving a synergistic breakthrough in the treatment of hepatitis B with efficient virus clearance, immune function reconstruction and low recurrence rate.

[0006] The specific technical solution adopted by this invention is as follows: The DC-CIK cell culture method based on HBV protective antibody-positive healthy donors uses peripheral blood mononuclear cells (PBMCs) from healthy donors who are HBsAg negative, anti-HBs antibody positive, and free from other infectious diseases and immune system diseases. After co-culturing dendritic cell (DC) antigen-loaded and cytokine-induced killer (CIK) cells, allogeneic DC-CIK cells are obtained. The antigen is a mixed antigen of HBcAg (core antigen) and HBsAg (surface antigen).

[0007] In a preferred embodiment, the anti-HBs antibody titer of the healthy donor is ≥10 mIU / mL.

[0008] In one preferred embodiment, the following steps are included: St1: Peripheral blood mononuclear cell (PBMC) collection, peripheral blood from healthy donors is collected and PBMCs are separated by density gradient centrifugation; St2: DC cell induction, 1*102 6 PBMCs were seeded in a medium containing GM-CSF (100 ng / mL) and IL-4 (50 ng / mL) to induce differentiation into DC cells. On day 5, 10 μg / mL of HBcAg and HBsAg were added and the cells were loaded for 24 hours. St3: CIK cell expansion: The remaining PBMCs were seeded in a medium containing IL-2 (1000 IU / mL), IFN-γ (50 ng / mL) and anti-CD3 antibody (50 ng / mL) and cultured for 14 days to expand into CIK cells; St4: Co-culture: Antigen-loaded DC cells and CIK cells were seeded in RPMI1640 basal medium and co-cultured for 48 hours to obtain allogeneic DC-CIK cells.

[0009] In a preferred embodiment, DC cells and CIK cells are cultured together in St4 at a ratio of 1:10 to 1:20.

[0010] In a preferred embodiment, the RPMI 1640 basal medium in St4 contains 5% autologous plasma.

[0011] A pharmaceutical composition for treating hepatitis B, comprising allogeneic DC-CIK cells as described in any one of the preceding claims.

[0012] In a preferred embodiment, the dosage form of the pharmaceutical composition is any one of the following: solid dosage form, semi-solid dosage form, or liquid dosage form.

[0013] In a preferred embodiment, the liquid dosage form of the pharmaceutical composition is administered via intravenous infusion at a dose of 1*10. 8 ~5*10 8 1 cell / session, once a week for 4 consecutive weeks.

[0014] In a preferred embodiment, it is used in combination with nucleoside analogues (such as tenofovir) to synergistically inhibit viral replication.

[0015] The technical effects achieved by this invention are as follows: This invention utilizes co-culturing allogeneic DC-CIK cells with healthy donor DC cells and CIK cells, employing a mixed HBcAg and HBsAg antigen loading technology to significantly enhance the antigen presentation capacity of DC cells. The optimized culture system enhances cell-targeted killing function, avoids immune tolerance caused by long-term infection of autologous cells, and exhibits stronger immune activity. Combined with nucleoside analogue therapy, it can effectively improve HBsAg clearance rate and cccDNA inhibition rate, achieving a synergistic breakthrough in the treatment of hepatitis B with efficient virus clearance, immune function reconstruction, and low recurrence rate. It combines the advantages of highly efficient antiviral activity, thorough repair of liver damage, and long-lasting efficacy, while maintaining reliable safety, providing a new solution for the functional cure of HBV. Attached Figure Description

[0016] Figure 1 This is a flowchart of the culture process of allogeneic DC-CIK cells in Embodiment 1 of the present invention; Figure 2 This is a magnified image of CIK cells in Embodiment 1 of the present invention; Figure 3 This is a magnified image of the DC cells in Embodiment 1 of the present invention; Figure 4 This is a magnified image of allogeneic DC-CIK cells in Embodiment 1 of the present invention; Figure 5 This is a schematic diagram of the in vitro killing efficiency comparison experiment results in Test Example 1 of the present invention; Figure 6 This is a schematic diagram of the changes in serum virological indicators in test example two of the present invention; Figure 7 This is a schematic diagram of the HBeAg conversion rate and HBeAg positivity rate data in Test Example 2 of this invention; Figure 8 This is a schematic diagram of ALT and AST data from test example two of this invention; Figure 9 This is a schematic diagram of TBIL data in Test Example 2 of the present invention; Figure 10 This is a schematic diagram of HE-stained pathological tissue sections of untreated HBV-infected mice in the high-dose combined therapy group of Test Example 3 of this invention; Figure 11 This is a schematic diagram of HE-stained pathological tissue sections of HBV-infected mice treated for 4 weeks in the high-dose combined therapy group of Test Example 3 of this invention. Detailed Implementation

[0017] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0018] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0019] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in a preferred embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that mutually excludes other embodiments.

[0020] Example Example 1

[0021] Please see the appendix Figure 1 As shown, this embodiment provides a method for culturing DC-CIK cells based on HBV protective antibody-positive healthy donors, as detailed below: Healthy donors meeting the criteria were screened (HBsAg negative, anti-HBs antibody positive with a titer ≥10 mIU / mL, and free from other infectious diseases and immune system disorders). 100 mL of peripheral blood was collected from each donor, and peripheral blood mononuclear cells (PBMCs) were obtained using density gradient centrifugation. 1*102 6PBMCs were seeded in a medium containing GM-CSF (100 ng / mL) and IL-4 (50 ng / mL) and induced to differentiate into dendritic cells (DCs) at 37°C and 5% CO2. After 2 hours of adherence, the suspension cells were removed and the DC medium was replaced. The medium was then completely replaced every 48 hours (twice). After the second medium replacement (on day 5), HBV core antigen (HBcAg) and surface antigen (HBsAg) of 10 μg / mL were added for antigen loading, and the cells were cultured for another 24 hours. The remaining PBMCs were then seeded in a medium containing IL-2 (1000 IU / mL) and IFN-γ (50 μg / mL). The cells were amplified in a culture medium containing anti-CD3 antibody (50 ng / mL) and anti-CD3 antibody (50 ng / mL) at 37°C and 5% CO2. The culture medium was replenished at 24 h and 48 h after inoculation. When the culture medium turned yellow or the cell concentration was close to saturation, the medium was replenished in a doubling manner. After 14 days of amplification, cytokine-induced killer (CIK) cells were obtained. The antigen-loaded DC cells and the amplified CIK cells were seeded at a ratio of 1:10 in RPMI1640 basal medium containing 5% autologous plasma and cultured for 48 hours at 37°C and 5% CO2 to obtain allogeneic DC-CIK cells.

[0022] Application examples

[0023] Application Example 1 The DC-CIK cells cultured in Example 1 were processed using a pharmaceutically acceptable carrier and existing processes to prepare a solution containing 1*10 8 ~5*10 8 A drug composition of cells / dosage for intravenous infusion, once a week for four weeks as one course of treatment.

[0024] Application Example 2 This application example builds upon Application Example 1, combining nucleoside analogues for synergistic therapy. Specifically: The DC-CIK cells cultured in Example 1 were processed using a pharmaceutically acceptable carrier and existing processes to prepare a solution containing 1*10 8 ~5*10 8 A drug composition for each cell / dose, administered intravenously once a week for four weeks as one course of treatment, in combination with nucleoside analogues for synergistic therapy.

[0025] It should be noted that a pharmaceutically acceptable carrier refers to an inert substance used to prepare a pharmaceutical composition that has no toxic side effects on the human body and does not affect the efficacy of the active ingredient. Its core function is to carry and protect the active ingredient. Specifically, a pharmaceutically acceptable carrier can be any of the following substances: physiological saline, compound electrolyte solution, or other pharmaceutical carriers. In this application example, the pharmaceutically acceptable carrier is preferably physiological saline.

[0026] Furthermore, the nucleoside analogue can be any one of the following substances: tenofovir, entecavir, or other nucleoside analogues. In this application example, the nucleoside analogue is preferably tenofovir, and the dosage of tenofovir is 10 mg / kg / day.

[0027] Comparative Example

[0028] Comparative Example 1: Culture of autologous DC-CIK cells: This comparative example differs from Example 1 in that the mixed antigen is replaced with an autologous mixed antigen. Specifically: 100 mL of peripheral blood was collected from the patient (HBV-infected, HBsAg positive), and PBMCs were obtained by density gradient centrifugation; 1*10 6 PBMCs were seeded in a medium containing GM-CSF (100 ng / mL) and IL-4 (50 ng / mL) and induced to differentiate into dendritic cells (DCs) at 37°C and 5% CO2. After 2 hours of adherence, the suspension cells were removed and the DC medium was replaced. Subsequent complete medium changes were performed every 48 hours (twice in total). After the second complete medium change (day 5), HBcAg and HBsAg derived from the patient (10 μg / mL each, extracted from patient serum or recombinantly expressed) were added for antigen loading, and the cells were cultured for 24 hours. The remaining PBMCs were then seeded in a medium containing IL-2 (1000 IU / mL) and IFN-γ. The cells were amplified in a culture medium containing γ (50 ng / mL) and anti-CD3 antibody (50 ng / mL) at 37°C and 5% CO2. The culture medium was replenished at 24 h and 48 h after inoculation. When the culture medium turned yellow or the cell concentration was close to saturation, the medium was replenished in a doubling manner. After 14 days of amplification, cytokine-induced killer (CIK) cells were obtained. The antigen-loaded DC cells and the amplified CIK cells were seeded at a ratio of 1:10 in RPMI1640 basal medium containing 5% autologous plasma and cultured for 48 hours at 37°C and 5% CO2 to obtain autologous DC-CIK cells.

[0029] Comparative Example 2, CIK cell culture: Compared to Example 1, this comparative example only cultured CIK cells. Specifically: Healthy donors meeting the criteria (HBsAg negative, anti-HBs antibody positive with a titer ≥10 mIU / mL, and free from other infectious diseases and immune system diseases) were selected. 100 mL of peripheral blood was collected from each donor, and peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation. 1*106 PBMCs were seeded into a culture medium containing IL-2 (1000 IU / mL), IFN-γ (50 ng / mL), and anti-CD3 antibody (50 ng / mL). The cells were expanded in a culture environment of 37℃ and 5% CO2. The culture medium was replenished at 24 h and 48 h after seeding. When the culture medium turned yellow or the cell concentration approached saturation, the medium was replenished in a doubling manner. After 14 days of expansion, cytokine-induced killer (CIK) cells were obtained.

[0030] Comparative Example 3, DC cell culture: Compared to Example 1, this comparative example only involves culturing DC cells. Specifically: Healthy donors meeting the criteria were screened (HBsAg negative, anti-HBs antibody positive with a titer ≥10 mIU / mL, and free from other infectious diseases and immune system disorders). 100 mL of peripheral blood was collected from each donor, and peripheral blood mononuclear cells (PBMCs) were obtained using density gradient centrifugation. 1*102 6 PBMCs were seeded in a medium containing GM-CSF (100 ng / mL) and IL-4 (50 ng / mL) and induced to differentiate into dendritic cells (DCs) at 37°C and 5% CO2. After 2 hours of adhesion, the suspended cells were removed and the DC medium was replaced. The medium was completely replaced every 48 hours (twice in total). After 5 days of culture, DC cells were obtained.

[0031] Laboratory animals and model construction: Selection of experimental animals: C57BL / 6 mice (male, 6-8 weeks old, SPF grade), weighing 18-22g; Model construction: pAAV-HBV1.2 plasmid-PEI complex (10 μg / animal) was injected via tail vein. Validation was performed 2 weeks post-transfection (HBsAg ≥ 100 IU / mL, HBV DNA ≥ 10 μg / mL). 7 IU / mL, ALT≥150U / L); Please refer to Table 1 for experimental groupings and treatment plans.

[0032] Table 1:

[0033] Test case

[0034] Test Example 1: Comparative Experiment of In Vitro Killing Efficiency The in vitro killing efficiency comparison experiment used the lactate dehydrogenase (LDH) release method. HBV-infected liver cancer cell line HepG2.2.15 was selected as the target cells, and after being cultured to the logarithmic growth phase, the concentration was adjusted to 2 × 10⁻⁶. 5 cells / mL, CIK cells, DC cells, autologous DC-CIK cells, and allogeneic DC-CIK cells cultured in Example 1 and Comparative Examples 1 and 3 were respectively used as effector cells, and the concentration was adjusted to 1×10⁻⁶ after washing. 6 cells / mL (corresponding to E:T=5:1) and 2×10 6 Cells / mL (corresponding to E:T=10:1), and prepare LDH detection kits, 96-well U-shaped cell culture plates, and other reagents and consumables, and equilibrate to room temperature; the experiment is divided into 6 groups: untreated group, CIK alone group, DC alone group, autologous DC-CIK group, and allogeneic DC-CIK group (E:T=5:1 and 10:1), with 3 replicates for each group. A maximum LDH release control group and a minimum LDH release control group are also included. 1×10⁻⁶ cells / mL (corresponding to E:T=10:1) are added to the corresponding wells of the 96-well plate. 4 Target cell suspension was prepared per well, and effector cell suspension was added at an E:T ratio. Serum-free DMEM medium was added to bring the final volume of each well to 200 μL. The 96-well plate was then placed in a 37°C, 5% CO2 incubator and co-cultured for 4 hours. After the culture, 100 μL of supernatant was transferred to a new 96-well plate, and 100 μL of LDH reaction solution was added. The plate was then incubated at room temperature in the dark for 30 minutes. Finally, 50 μL of stop solution was added to terminate the reaction. The absorbance (OD value) of each well was measured at 490 nm using a microplate reader. The killing rate of each group was calculated using the formula [(OD value of experimental group - OD value of minimum release group) / (OD value of maximum release group - OD value of minimum release group)] * 100%. The OD values ​​of the three replicates for each group were counted, and the mean ± standard deviation was calculated as the final killing rate result. The test results are shown in Table 2.

[0035] Table 2:

[0036] As shown in Table 2, under the condition of an E:T ratio of 5:1, the kill rate of the allogeneic DC-CIK group was significantly higher than that of the single CIK group, the single DC group, and the autologous DC-CIK group, reaching 68.9±5.3%, while the kill rates of the single CIK group, the single DC group, and the autologous DC-CIK group were 28.7±3.5%, 12.3±2.2%, and 45.6±4.1%, respectively. When the E:T ratio was increased to 10:1, the kill rate of the allogeneic DC-CIK group further increased to 82.1±6.2%, showing... The dose-dependent enhancement effect was observed; the killing rate in the untreated group was only 5.2±1.1%, serving as a negative control and verifying the effectiveness of the experimental system. These data indicate that allogeneic DC-CIK cells have a highly efficient killing effect on HBV-infected liver cancer cell line HepG2.2.15 in vitro, and their killing efficiency is significantly better than that of CIK cells alone, DC cells, or autologous DC-CIK cells. Moreover, the efficiency increases with the increase of the effector / target cell ratio, indicating that donor-derived allogeneic DC-CIK cells have a better targeted killing efficiency against HBV-infected target cells.

[0037] Test Example 2, Animal Experiment Test A C57BL / 6 mouse HBV infection model was established using hydrodynamic transfection. Seventy model mice were randomly divided into seven groups (n=10 / group) according to the grouping data in Table 1. Each group received a tail vein injection of the treatment agent once a week according to the treatment regimen in Table 1. After four weeks of continuous treatment, HBsAg, HBsAb, HBeAg, and HBeAb were detected sequentially using ELISA to determine viral antigen expression and serological conversion. Simultaneously, qPCR was used to quantitatively detect the HBV DNA load in serum to assess the viral replication inhibition effect. In addition, three core liver function indicators, ALT, AST, and TBIL, were detected in serum using a biochemical analyzer to determine the degree of liver damage and repair. The test data are shown in Tables 3 and 4.

[0038] Table 3:

[0039] As shown in Table 3, regarding changes in serum virological indicators, the high-dose combination therapy group exhibited the most significant therapeutic effect, with an HBsAg seroconversion rate of 45%, an HBV DNA seroconversion rate as high as 90%, and a functional cure rate of 28%. Furthermore, the relapse rate after 2 weeks of drug withdrawal was 0%, all data being superior to other single-drug treatment groups and the low-dose combination therapy group. The allogeneic DC-CIK single-drug group also showed outstanding performance, with HBsAg seroconversion rate and HBV DNA seroconversion rate... The HBV DNA seroconversion rates reached 38% and 72%, respectively, with a functional cure rate of 25% and a relatively low relapse rate of 20%. In contrast, while the nucleoside analog monotherapy group had a higher HBV DNA seroconversion rate, the HBsAg seroconversion rate and functional cure rate were lower, and the relapse rate after drug withdrawal was as high as 100%. The autologous DC-CIK monotherapy group had a relatively weaker therapeutic effect, with all indicators lower than the allogeneic DC-CIK monotherapy group. The data show that allogeneic DC-CIK cells combined with nucleoside analogs have a synergistic effect in the treatment of HBV infection, which can significantly improve the virological seroconversion rate and functional cure rate, and reduce the relapse rate.

[0040] Table 4:

[0041] As shown in Table 4, the high-dose combination therapy group also demonstrated excellent therapeutic efficacy in terms of serological conversion and liver function recovery, with an HBeAg conversion rate as high as 60%, an HBsAb positivity rate of 55%, and ALT, AST, and TBIL levels all recovering to near-normal control group levels. In the allogeneic DC-CIK monotherapy group, the HBeAg conversion rate and HBsAb positivity rate reached 50% and 40%, respectively, and liver function indicators were significantly better than the model control group and the nucleoside analog monotherapy group, with ALT, AST, and TBIL levels of 42.6±7.1 U / L, 35.8±6.4 U / L, and 9.8±10 U / L, respectively. 1.5 μmol / L; In comparison, although the autologous DC-CIK monotherapy group also had some therapeutic effect, all indicators were lower than those of the allogeneic DC-CIK monotherapy group; the nucleoside analog monotherapy group had some effect in reducing viral load, but was relatively weak in serological conversion and liver function recovery; the model control group did not show significant improvement in any indicators, and even deteriorated; the above data further confirm that allogeneic DC-CIK cells combined with nucleoside analog therapy for HBV infection can not only significantly improve the virological seroconversion rate and functional cure rate and reduce the recurrence rate, but also effectively promote serological conversion and liver function recovery, providing a new and effective strategy for the treatment of HBV infection.

[0042] Based on the data from Test Example 2, the combination therapy of allogeneic DC-CIK and nucleoside analogues showed significant antiviral efficacy in an HBV-infected mouse model. It could effectively clear HBsAg, deeply inhibit HBV DNA, achieve a functional cure rate of 28%, and had no relapse after drug withdrawal, thus breaking through the "viral inhibition plateau" of nucleoside analogues.

[0043] Test Example 3: Detection of Inflammation and Fibrosis in Liver Tissue Mice from each group in Test Example 2 were dissected, and liver tissue was rapidly separated. Typical areas were selected and trimmed into tissue blocks of approximately 0.5cm*0.5cm*0.2cm. After fixation with 4% paraformaldehyde for 24 hours, the blocks underwent sequential dehydration (70%, 80%, 90%, 95%, 100% ethanol), followed by xylene clearing. The blocks were then embedded in paraffin to form paraffin blocks. The paraffin blocks were cut into 5μm thick continuous sections using a microtome, mounted, and fixed by baking at 60℃ for 2 hours. The sections were then stained with hematoxylin and eosin (HE) and Masson staining, respectively. The HE staining procedure was as follows: dewaxing to water, hematoxylin staining for 5 minutes, and running water staining. Rinse to remove staining solution, counterstain with eosin for 2 minutes, dehydrate in a gradient manner, clear, and mount with neutral resin. The Masson staining procedure is as follows: dewaxing to water, hematoxylin staining for 10 minutes, differentiation with hydrochloric acid and ethanol, Ponceau S and Acid Fuchsin staining for 15 minutes, phosphomolybdic acid treatment for 5 minutes, aniline blue counterstaining for 5 minutes, rinsing with glacial acetic acid, dehydration and clearing, and mounting. Finally, observe the sections under an optical microscope (400× magnification), focusing on recording the morphology, arrangement, cytoplasmic and nuclear characteristics of hepatocytes, the location and number of inflammatory cell infiltrations, and the distribution of collagen fibers. The percentage of fibrosis area is calculated using an image analysis system. The detection data are shown in Tables 5 and 6.

[0044] Table 5:

[0045] As shown in Table 5, after evaluating liver tissue inflammation and fibrosis in experimental mice 4 weeks after treatment, the high-dose combination therapy group showed the best therapeutic effect on HBV-transfected mice, with the lowest inflammation score, fibrosis area ratio, and stem cell necrosis rate. All indicators were significantly better than other groups (except the normal control group). The allogeneic DC-CIK monotherapy group also showed good therapeutic effects, with an inflammation score of 0.7±0.1, a fibrosis area ratio of 4.2±0.8%, and a hepatocyte necrosis rate of 4.3±0.9%, all of which were significantly lower than the model control group and the nucleoside analog monotherapy group. The autologous DC-CIK monotherapy group showed the second-best therapeutic effect, but still showed significant improvement compared to the model control group. Although the nucleoside analog monotherapy group could reduce viral load, its effect in alleviating liver inflammation and fibrosis was relatively weak. The liver tissue of the model control group showed severe inflammation and fibrosis, with a hepatocyte necrosis rate as high as 35.6±4.2%. The above data confirm that allogeneic DC-CIK cell therapy for HBV infection has significant effects in alleviating liver inflammation, inhibiting fibrosis progression, and protecting hepatocytes. When combined with nucleoside analog therapy for HBV infection, the effect is even better and more obvious.

[0046] Table 6:

[0047] As shown in Table 6, the high-dose combination therapy group exhibited the best tissue morphology, with minimal inflammatory cell infiltration, only 1-2 scattered inflammatory cells without aggregation, unobstructed hepatic sinusoids, uniform red blood cell distribution, no abnormal collagen fiber deposition, and an overall tissue morphology close to that of a normal liver. The allogeneic DC-CIK monotherapy group also performed well, with only occasional scattered inflammatory cells, unobstructed hepatic sinusoids, uniform red blood cell distribution, and only a very small amount of scattered blue-stained collagen fibers, indicating mild fibrosis. The low-dose combination therapy group showed similar characteristics to the allogeneic DC-CIK monotherapy group, with 1-2 scattered inflammatory cells without aggregation, unobstructed hepatic sinusoids, uniform red blood cell distribution, and minimal collagen fiber deposition. The effects were extremely mild; although the autologous DC-CIK monotherapy group showed some improvement, scattered small amounts of inflammatory cells and blue-stained collagen fibers were still visible; the nucleoside analog monotherapy group showed some improvement in inflammatory cell infiltration, hepatic sinusoidal status, and collagen fiber deposition, while the model control group showed severe pathological changes, with a large number of inflammatory cells infiltrating, narrowing or occlusion of hepatic sinusoids, uneven distribution of erythrocytes, extensive patchy blue-stained collagen fiber deposition, and severely abnormal tissue morphology; the above data further validated the significant effects of allogeneic DC-CIK cell therapy for HBV infection in improving liver tissue morphology, reducing inflammatory response, and inhibiting fibrosis, especially when combined with nucleoside analog therapy, its efficacy was even more outstanding.

[0048] Based on the data from Case 3 of the comprehensive test, the combination therapy can completely repair the liver inflammation and fibrosis caused by HBV, and liver function can be fully restored. It also has good safety, with no obvious toxicity or tissue compatibility issues. From the perspective of overall treatment effect, allogeneic DC-CIK cells, whether used alone or in combination with nucleoside analogs, have shown the potential to surpass traditional nucleoside analog monotherapy and autologous DC-CIK cell therapy.

[0049] Test Example 4: Immune Mechanism Test Mice from each group in Test Example 2 were dissected, spleen tissue was aseptically separated and ground to prepare a single-cell suspension, red blood cells were removed by treatment with red blood cell lysis buffer, and the cell concentration was adjusted to 1*10 after washing with PBS. 6 The cells were incubated with CD3, CD4, CD8, and CD86 specific fluorescent antibodies for 30 minutes in the dark. Flow cytometry was used to detect and analyze the proportions of CD3+CD4+ T cells and CD3+CD8+ T cells in the splenic lymphocyte subsets, as well as the CD86 positivity rate of DC cells. Simultaneously, a portion of liver tissue was homogenized, centrifuged, and the supernatant was collected. The concentrations of three cytokines, IFN-γ, TNF-α, and IL-2, in the liver tissue were detected sequentially using the ELISA method according to the kit instructions. The test data are shown in Tables 7 and 8.

[0050] Table 7:

[0051] As shown in Table 7, after analyzing the splenic lymphocyte subsets of mice in each treatment group, the high-dose combination therapy group exhibited the most significant advantage in immunomodulation. Its proportions of CD3+CD4+ T cells, CD3+CD8+ T cells, and the CD86 positivity rate of DC cells were all significantly higher than other groups (except the normal control group). The allogeneic DC-CIK monotherapy group also showed good immunomodulatory effects, significantly better than the model control group and the nucleoside analog monotherapy group. The data for the low-dose combination therapy group were similar to those for the allogeneic DC-CIK monotherapy group. Although the autologous DC-CIK monotherapy group showed some improvement, all indicators were lower than those for the allogeneic DC-CIK monotherapy group. The nucleoside analog monotherapy group showed relatively weaker effects in increasing the proportion of lymphocytes and the positivity rate of DC cells. The various indicators of the model control group were at a low level, reflecting severe suppression of its immune function. These data indicate that allogeneic DC-CIK cell therapy for HBV infection can effectively regulate the body's immune function and enhance the activity and proportion of immune cells. This immunomodulatory effect is even more significant when combined with nucleoside analog therapy.

[0052] Table 8:

[0053] As shown in Table 8, the high-dose combination therapy group demonstrated the most outstanding performance in promoting cytokine secretion in liver tissue, with significantly higher concentrations of IFN-γ, TNF-α, and IL-2 compared to other groups. The allogeneic DC-CIK monotherapy group also exhibited good cytokine secretion regulation, with significantly higher concentrations of the three cytokines compared to the model control group and the nucleoside analog monotherapy group. The low-dose combination therapy group showed similar data to the allogeneic DC-CIK monotherapy group, with relatively high cytokine concentrations. While the autologous DC-CIK monotherapy group showed some improvement, all indicators were lower than those of the allogeneic DC-CIK monotherapy group. The nucleoside analog monotherapy group showed relatively weaker effects in increasing cytokine concentrations. The model control group had low cytokine concentrations, reflecting severe suppression of its immune response. These data further confirm that allogeneic DC-CIK cell therapy for HBV infection can effectively promote cytokine secretion in liver tissue and enhance the body's immune response. This promoting effect is even more significant when combined with nucleoside analog therapy, achieving deep viral clearance by activating specific immunity.

[0054] Based on the data from Case 4 of the comprehensive test, the combination therapy significantly enhanced the body's ability to recognize and clear HBV by increasing the proportion of CD3+CD8+ T cells, activating dendritic cell maturation, and promoting the secretion of cytokines such as IFN-γ, thereby rebuilding the HBV-specific immune response network. This immune reconstitution is not only reflected in changes in cell proportion and cytokine concentration, but more importantly, it restores the body's specific immune memory against HBV. This allows the body to rapidly initiate an effective immune response in the event of subsequent viral exposure or relapse, thereby preventing reinfection and spread of the virus. The high-dose combination therapy group demonstrated remarkable efficacy in immune mechanisms. By combining allogeneic DC-CIK cells and nucleoside analogs, it simultaneously exerted a dual effect of antiviral and immunomodulatory action, achieving a reduction in viral load and restoration of immune function, thereby resulting in a higher cure rate and a lower recurrence rate. In addition, the allogeneic DC-CIK monotherapy group also showed good efficacy in immunomodulation. Although slightly inferior to the high-dose combination therapy group, it was still significantly better than the model control group and the nucleoside analog monotherapy group. This further confirms the potential value of allogeneic DC-CIK cells in the treatment of HBV infection.

[0055] Test Example 5: Security Assessment During the treatment process in Test Example 2, the mice's diet, activity level, mental state, and other general conditions were observed daily, and any abnormal behaviors or poisoning symptoms were recorded. After 4 weeks of treatment, the mice were weighed, and the weight maintenance rate was calculated (post-treatment weight / initial weight * 100%). Mice from the normal control group and the high-dose combination therapy group in Test Example 2 were dissected, and major organs such as the liver, spleen, and kidneys were separated and weighed. The organ coefficient was calculated based on the mouse weight (organ weight / mouse weight * 100%). At the same time, kidney tissue was taken and fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) according to standard procedures. The morphology of the kidney tissue was observed under an optical microscope, and damage was scored according to relevant standards. The test data are shown in Table 9. Among them, no mice in any group died after 4 weeks of treatment.

[0056] Table 9:

[0057] As shown in Table 9, both the high-dose combination therapy group and the normal control group demonstrated good safety profiles. No mice died after 4 weeks of treatment. The high-dose combination therapy group did not cause significant kidney damage, and the tissue morphology remained largely normal. Furthermore, during the treatment process, daily observation of the mice's diet, activity level, mental state, and organ coefficients showed no abnormalities, and no acute toxicity, tissue compatibility reactions, or other safety issues were observed. The weight maintenance rate was also at a reasonable level. These data fully demonstrate that allogeneic DC-CIK cells combined with nucleoside analogues have good safety for treating HBV infection, providing a reliable basis for clinical application.

[0058] In summary, the core limitation of existing nucleoside analogue therapy is that it "only inhibits but does not kill," meaning it can only suppress HBV DNA replication but cannot clear viral antigens (HBsAg). This leads to long-term drug dependence and extremely low functional cure rates. Test data shows that the HBsAg clearance rate in the nucleoside analogue monotherapy group is only 2%, and 100% of patients relapse after stopping the drug (Table 3), confirming the clinical dilemma of the "viral suppression plateau." In contrast, the combination therapy of allogeneic DC-CIK and nucleoside analogues achieves a breakthrough through a dual mechanism: firstly, nucleoside analogues strongly inhibit viral replication, reduce viral load, and provide a barrier for immune cells. Firstly, it creates conditions for the drug to play its role; secondly, allogeneic DC-CIK cells from antibody-positive donors activate autoimmunity through the "bystander effect," targeting and killing HBV-infected hepatocytes and clearing viral antigens; the high-dose combination therapy group achieved an HBsAg clearance rate of 45% and a functional cure rate of 28% (Table 3), and there was no recurrence after drug withdrawal. This clearly demonstrates that the combination therapy can overcome the bottleneck of drug treatment, achieving a leap from "viral suppression" to "functional cure," combining the advantages of highly effective antiviral therapy, thorough repair of liver damage, and long-lasting efficacy, with reliable safety, providing feasible preclinical evidence for the functional cure of HBV.

[0059] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described or explained in this invention are implemented according to conventional methods in the art unless otherwise specified or limited.

Claims

1. A DC-CIK cell culture method based on HBV protective antibody positive healthy donors, characterized in that: The peripheral blood mononuclear cells of a healthy donor with negative HBsAg and positive anti-HBs antibody are cultured with the killer cells induced by cytokines after being loaded with the mixed antigen of HBcAg and HBsAg, and the allogeneic DC-CIK cells are obtained. 2.The HBV-protective antibody positive healthy donor based DC-CIK cell culture method according to claim 1, characterized in that: The anti-HBs antibody titer of the healthy donor is greater than or equal to 10 mIU / mL. 3.The HBV-protective-antibody-positive healthy donor-based DC-CIK cell culture method according to claim 1, characterized in that: The method comprises the following steps: St1: collecting the peripheral blood of the healthy donor, and separating the PBMC by density gradient centrifugation; St2: take 1*10 6 PBMC were seeded in medium containing GM-CSF and IL-4 to induce differentiation into DC cells, and 10 μg / mL of HBcAg and HBsAg were added on day 5 for 24 hours of loading. St3: the remaining PBMC are inoculated into the culture medium containing IL-2, IFN-γ and anti-CD3 antibody, and are cultured for 14 days to be expanded into CIK cells; St4: the DC cells loaded with the antigen are inoculated into the RPMI1640 basic culture medium together with the CIK cells, and are co-cultured for 48 hours to obtain the allogeneic DC-CIK cells. 4.The HBV-protective-antibody-positive healthy donor-based DC-CIK cell culture method according to claim 3, characterized in that: In the St4, the DC cells and the CIK cells are mixed and cultured at a ratio of 1:10 to 1:

20.

5. The DC-CIK cell culture method based on HBV protective antibody positive healthy donors according to claim 3, characterized in that: In the St4, the RPMI1640 basic culture medium contains 5% of autologous plasma.

6. A pharmaceutical composition for treating hepatitis B, characterized by comprising: The pharmaceutical composition comprises the allogeneic DC-CIK cells according to any one of claims 1 or 3.

7. A pharmaceutical composition for the treatment of hepatitis B according to claim 6, wherein: The dosage form of the pharmaceutical composition is any one of the following: solid dosage form, semi-solid dosage form, and liquid dosage form.

8. The pharmaceutical composition for treating hepatitis B according to claim 7, wherein: The pharmaceutical composition of the liquid dosage form is administered by intravenous infusion at a dose of 1*10 8 ~5*10 8 cells / time, once a week, for 4 weeks.

9. The pharmaceutical composition for treating hepatitis B according to claim 6, wherein: The nucleoside analogs are used in combination.

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