Preparation method and application of anti-IL-11 antibody modified Treg cell

Abstract

The invention provides a preparation method and application of an anti-IL-11 antibody modified Treg cell, and belongs to the technical field of genetic engineering.The preparation method of the anti-IL-11 antibody modified Treg cell comprises the steps that after the Treg cell and umbilical cord blood pluripotent stem cells are co-cultured, the Treg cell is separated and recycled, an anti-IL-11 antibody is adopted for modification, and the anti-IL-11 antibody modified Treg cell is obtained; the nucleotide sequence of the anti-IL-11 antibody is as shown in SEQ ID NO. 2 in the sequence table. In the co-culture process, stem cells are used for providing an immune microenvironment, promoting Treg cell amplification and enhancing the inhibition ability of Treg cells, and obtaining the specific recognition ability of pancreas islet beta cell antigens, so that autoimmune attacks are inhibited more accurately; the nucleotide sequences of the heavy chain and the light chain of the anti-IL-11 antibody are optimized, and the anti-IL-11 antibody has higher efficacy in treatment of diabetic mice.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method of anti-IL-11 antibody modified Treg cells and its application, belonging to the field of genetic engineering technology. BACKGROUND

[0002] Diabetes, especially type I diabetes (T1D) and some autoimmune type II diabetes (T2D), the core pathogenesis is that the immune system mistakenly attacks and destroys pancreatic beta cells, leading to insufficient insulin secretion and blood glucose metabolism disorder. Traditional treatment methods (such as insulin injection, hypoglycemic drugs, etc.) can alleviate symptoms, but cannot cure the disease or reverse beta cell damage. In recent years, immunosuppressive cell-based therapy has become a new direction for diabetes intervention, among which regulatory T cells (Treg) have attracted attention due to their unique immune regulation ability.

[0003] Treg cells are a subset of CD4+ T cells with immunosuppressive function, which have shown initial success in the treatment of diabetes. It mainly maintains immune tolerance and protects pancreatic beta cells in the following ways. 1. Cytokine-mediated immunosuppression: Treg cells secrete anti-inflammatory factors such as IL-10 and TGF-β, inhibit the activation of effector T cells and autoreactive CD8+ T cells, and reduce the destruction of beta cells by inflammatory microenvironment. 2. Direct contact inhibition between cells: Treg cells bind to antigen-presenting cells or effector T cells through CTLA-4, PD-1 and other immune checkpoint molecules, block their activation signals, and thus inhibit autoimmune reactions. 3. Promote tissue repair and regeneration: Treg cells not only inhibit inflammation, but also promote beta cell survival and islet tissue repair by secreting growth factors.

[0004] However, the efficacy of Treg cells is a key variable that cannot be ignored in the treatment of immune-related diseases, so how to improve the efficacy of Treg cells is a top priority.

[0005] Currently, there are multiple dimensions to improve the efficacy of Treg cells, including genetically engineering Treg cells, such as the preparation of CAR-Treg for the treatment of systemic lupus erythematosus: in a study published in Nature in March 2024, scientists used Treg cells expressing CD19-targeting chimeric antigen receptors (CAR) to treat SLE. The results showed that CAR-Treg cells can effectively inhibit B cell function, reduce autoantibody production, and restore immune homeostasis. There are also methods to improve the efficacy of Treg cells by increasing the proportion of Treg cell differentiation and enhancing Treg cell function, such as CN120249200A provides a composition for increasing the proportion of Treg cell differentiation, including zinc salt, basic cell culture medium, fetal bovine serum, antibiotics, L-glutamine and pH buffer, wherein the highest proportion of Treg cells in lymphocytes is 10.8%, but this is limited to the optimization of the basic culture medium, and lacks functional verification of the cells. CN119799633A provides a method for enhancing the function of Treg cells, by irradiating hUC-MSCs with gamma rays to form feeder cells, and then co-culturing the feeder cells with Treg cells to obtain Treg cells with enhanced cell function, with the highest expression of CD4+CD25+Foxp3+ reaching 69%. However, gamma rays themselves can induce DNA double-strand breaks and chromosomal aberrations, and their residual fragments may be phagocytosed by Treg cells, affecting safety.

[0006] The conventional in vitro culture method of Treg cells is difficult to completely simulate the in vivo immune microenvironment, and is prone to cell culture aging problems, affecting cell viability; Treg cells cultured in vitro also lack pancreatic beta cell antigen specificity and cannot precisely target and inhibit autoimmune attacks, and some cytokines in the body inhibit the development of Treg cells and reduce the function of Treg cells, ultimately resulting in the inability of Treg cells to achieve the expected effect on the treatment of diabetes. SUMMARY

[0007] To solve the above technical problems, the present application provides a preparation method of Treg cells and its application, which achieves the following purposes: improving the survival rate and expression rate of cell markers of Treg cells, obtaining specific recognition ability to pancreatic beta cell antigens, and improving the treatment efficacy of diabetes.

[0008] To solve the above technical problems, the present application adopts the following technical solutions: A preparation method of anti-IL-11 antibody modified Treg cells, after co-culturing Treg cells with umbilical cord blood multipotent stem cells, separating and recovering Treg cells, modifying with anti-IL-11 antibody, obtaining anti-IL-11 antibody modified Treg cells; the nucleotide sequence of the anti-IL-11 antibody is shown as SEQ ID NO. 2 in the sequence listing.

[0009] The starting number ratio of Treg cells to umbilical cord blood multipotent stem cells in the co-culture is 9-11:1.

[0010] The co-culture time is 6 days.

[0011] The co-culture method is that Treg cells and umbilical cord blood multipotent stem cells are inoculated into a culture container, RPMI-1640 culture medium containing 9-11% Vol serum replacement is added, 1 / 2 volume of old culture medium is aspirated and discarded on the 2nd and 4th days of co-culture, an equal amount of fresh RPMI-1640 culture medium containing 98-102 U / mL IL-2 is supplemented, and the co-cultured Treg cells are separated and recovered on the 6th day of co-culture.

[0012] The Treg cells before co-culture are obtained by the following method: Treg cells are isolated from autologous peripheral blood, first expanded and cultured in an anti-CD3 monoclonal antibody coated culture container for 3 days, and then expanded and cultured in an uncoated culture container for 11 days.

[0013] The culture medium used in the expansion culture is RPMI-1640 supplemented with 14.8-15.2% Vol serum replacement, 1.8-2.2 mM L-glutamine, 0.9-1.1% Vol penicillin-streptomycin, 0.9-1.1% Vol insulin-transferrin-selenium, 48-52 μg / mL 2-phospho-ascorbic acid, 9.8-10.2 ng / mL IL-2, 29-31 nM AS2863619, 2.4-2.6 μg / mL TNFR2 antibody, 4.8-5.2 ng / mL TGF-beta, and 98-102 nM rapamycin.

[0014] The fusion degree of the umbilical cord blood multipotent stem cells before co-culture is 89-91%.

[0015] The umbilical cord blood multipotent stem cells before co-culture are obtained by the following method: mononuclear cells are isolated from umbilical cord blood, RPMI-1640 culture medium containing 9.8-10.2% Vol serum replacement is added, and the cells are cultured for 16 days until the cell fusion degree reaches 89-91%.

[0016] The anti-IL-11 antibody modified Treg cells prepared by the preparation method are used in the preparation of a drug for treating diabetes.

[0017] Compared with the prior art, the present application has the following beneficial effects: The present application adopts umbilical cord blood pluripotent stem cells to co-culture with Treg cells, and then adopts anti-IL-11 antibody to transfect the Treg cells after co-culture. In the co-culture process, the immune microenvironment is provided by the stem cells, the Treg cells are promoted to expand and enhance the inhibitory capacity, and the specific recognition capacity to the pancreatic islet beta cell antigen is obtained, so that the autoimmune attack is more accurately inhibited. The Treg cells tamed by the stem cells can activate the pancreatic islet progenitor cells through the paracrine effect, and promote the beta cell regeneration. The anti-IL-11 antibody maintains or restores the function of the Treg cells by blocking the IL-11 / IL-11R signal. The heavy chain and light chain nucleotide sequences of the anti-IL-11 antibody are optimized in the present application, which can improve the survival rate of the Treg cells and the expression rate of the cell markers, improve the effective regulation of the blood sugar of the diabetic mice, significantly improve the secretion capacity of the insulin, and has higher efficacy in the treatment of the diabetic mice. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The flow chart of the CD4+ expression rate of the Treg cells cultured in scheme one; Figure 2 The flow chart of the CD4+ expression rate of the Treg cells cultured in scheme two; Figure 3 The flow chart of the CD4+ expression rate of the IL-11-Treg-1 cells; Figure 4 The flow chart of the CD4+ expression rate of the IL-11-Treg-2 cells; Figure 5 The flow chart of the CD25+FoxP3+ expression rate of the Treg cells cultured in scheme one; Figure 6 The flow chart of the CD25+FoxP3+ expression rate of the Treg cells cultured in scheme two; Figure 7 The flow chart of the CD25+FoxP3+ expression rate of the IL-11-Treg-1 cells; Figure 8 The flow chart of the CD25+FoxP3+ expression rate of the IL-11-Treg-2 cells; Figure 9 The column chart of the body weight change of the mice in each group; Figure 10 The column chart of the blood sugar value change of the mice in each group; Figure 11 The column chart of the cytokine detection value of the mice in each group. DETAILED DESCRIPTION

[0019] Example 1 Culture of Treg cells Scheme 1 is a conventional method for culturing Treg cells, the specific steps are as follows: Take 40 mL of patient's autologous peripheral blood, separate peripheral blood mononuclear cells with Ficoll-Paque lymphocyte separation medium. The separated cells are sorted into CD4+CD25+Treg cells with CD4+CD25+Treg cell sorting kit provided by Germany Meitenyi Biotech Co., Ltd., and cell counting is performed. The obtained Treg cells are cultured in 48-well cell culture plates coated with anti-CD3 monoclonal antibody for 3 days, and the specific steps are as follows: Coat the 48-well plate with anti-CD3 monoclonal antibody at 2 μg / mL, 200 μL / well, incubate at 37°C for 2 h, then wash once with PBS. Resuspend the Treg cells to 1 × 10 8 cells / mL, inoculate into the expansion medium at 0.5 mL / well, wherein the expansion medium is RPMI-1640 supplemented with 15% Vol serum substitute, 2 mM L-glutamine, 1% Vol penicillin-streptomycin, 1% Vol insulin-transferrin-selenium (ITS), 50 μg / mL 2-phospho-ascorbic acid concentration, 10 ng / mL IL-2 concentration, 30 nM AS2863619 (CDK inhibitor), 2.5 μg / mL TNFR2 antibody, 5 ng / mL TGF-beta, 100 nM rapamycin, and incubate in a 37°C, 5% CO2 incubator for 3 days.

[0020] On the 4th day, replace it in a normal T75 bottle, add the same volume of expansion medium every 3 days, and culture to the 14th day. Collect the cells for subsequent experiments.

[0021] Scheme 2 is to co-culture umbilical cord blood pluripotent stem cells with Treg cells, the steps are as follows: 1. Culture of umbilical cord blood pluripotent stem cells (1) The donated umbilical cord blood is separated into mononuclear cells using Ficoll-Paque lymphocyte separation medium, and inoculated in T75 bottles at 1 × 10 6 cells / mL, 10 mL of RPMI-1640 medium containing 10% Vol serum substitute is added to each T75 bottle, and incubated in a 37°C, 5% CO2 incubator.

[0022] (2) Replace the new medium every 3 days, and culture to 16 days, the cell fusion degree reaches 90%, for subsequent experiments.

[0023] 2. Culture of Treg cells Treg cells are collected according to scheme 1 to expand to the 14th day.

[0024] 3. Umbilical cord blood pluripotent stem cells co-cultured with Treg cells A. The collected Treg cells were inoculated into the culture bottle of umbilical cord blood pluripotent stem cells at a ratio of 10:1, 25 mL of RPMI-1640 medium containing 10% Vol serum substitute was added, and the culture bottle was placed back into a 37°C, 5% CO2 incubator to start co-culture, which was recorded as day 0.

[0025] B. On the 2nd and 4th days of co-culture, 1 / 2 of the old medium in the culture bottle was aspirated and discarded, and an equal amount of fresh RPMI-1640 medium containing 100 U / mL IL-2 was supplemented.

[0026] C. On the 6th day of co-culture, the suspended cells in the culture well were gently blown with a pipette to avoid touching the adherent umbilical cord blood pluripotent stem cells, and the cell suspension was transferred to a 15 mL centrifuge tube. Wash twice with 2 mL of 1x PBS, collect the wash and put it into the centrifuge tube to ensure that all residual Treg cells are recovered.

[0027] D. The centrifuge tube was centrifuged at 300g for 5 min, the supernatant was discarded, the cells were resuspended and washed twice with 1x PBS to remove residual medium and cell debris, and then sorted with CD4+CD25+ magnetic beads to harvest the co-cultured Treg cells for subsequent experiments.

[0028] Example 2 Preparation of pLent-EF1α-IL-11 modified Treg cells 1. Preparation of pLent-EF1α-IL-11 plasmid The anti-IL-11 scFv, whose nucleic acid artificial sequence is shown in SEQ ID NO. 1 in the sequence listing, was entrusted to Shandong Honno Biological Technology Co., Ltd. to synthesize and inserted into the BamHI-NotI site of the pLent-EF1α vector (purchased from Vigene Company), transformed into E. coli (Top10), and after sequencing, the plasmid was extracted using the plasmid extraction kit of OMEGA Company to obtain the recombinant expression vector pLent-EF1α-IL-11-1, with a plasmid concentration of 1.2 µg / µL.

[0029] The anti-IL-11 scFv heavy chain and light chain sequences were optimized, whose nucleic acid artificial sequence is shown in SEQ ID NO. 2 in the sequence listing, entrusted to Shandong Honno Biological Technology Co., Ltd. to synthesize and inserted into the BamHI-NotI site of the pLent-EF1α vector (purchased from Vigene Company), transformed into E. coli (Top10), and after sequencing, the plasmid was extracted using the plasmid extraction kit of OMEGA Company to obtain the recombinant expression vector pLent-EF1α-IL-11-2, with a plasmid concentration of 1.1 µg / µL.

[0030] 2. Lentivirus packaging Recover 293T cells, culture for 3 days, and passage according to cell density. When the cell confluence reaches 80% after 1 passage, transfection is performed. Seed 6×10 5 cells / well in a six-well plate, and add 2 mL of DMEM medium (purchased from Gibco Company, item number 11960-044) to each well to prepare for transfection the next day. Before transfection, replace the six-well plate with fresh DMEM medium (purchased from Gibco Company, item number 11960-044) at 2 mL / well, and culture in a 37°C incubator for 1 h.

[0031] Preparation of transfection reagent: In a 5 mL centrifuge tube, prepare Tube A and Tube B reagents respectively. The preparation of Tube A and Tube B is shown in Table 1.

[0032] Table 1 Preparation method of Tube A and Tube B

[0033] After preparation, place for 5 min, then slowly add Tube A to Tube B, mix well, and place at room temperature for 20 min to form a liposome-DNA mixture. Add the mixture to the culture bottle, mix gently, and place in a 37°C, 5% CO2 incubator for culture.

[0034] After 48 h, observe the morphological changes of 293T cells after transfection under a microscope. After 72 h, collect the cell culture supernatant containing virus into a centrifuge tube, centrifuge at 3500 rpm for 10 min to remove cell debris, filter with a 4.5 μm filter, and centrifuge at 70000 g at 4°C for 2 h. Resuspend the precipitate with 100 μL of PBS, aliquot, and store at -80°C. Meanwhile, determine the virus titer. The virus titer of the virus liquid containing pLent-EF1α-IL-11-1 in this application is 2.06×10 8 TU / mL, and the virus titer of the virus liquid containing pLent-EF1α-IL-11-2 is 2.11×10 8 TU / mL.

[0035] 3. Lentivirus infection of Treg cells Take the virus liquid prepared above from -80°C, thaw, and add RPMI-1640 medium to dilute the virus titer to 3×10 7 TU / mL to obtain the diluted virus liquid. Resuspend 1×10 6 Treg cells with 100 μL of the diluted virus liquid to make the ratio of virus particle number to Treg cell number 3:1 to obtain a virus and cell suspension.

[0036] Add the virus and cell suspension to a T75 flask and incubate at 37°C in a 5% CO2 incubator for 24 hours. Collect the cells, centrifuge at 400g for 5 minutes, discard the supernatant, and count the cells at a ratio of 1×10⁻⁶. 6 Treg cells were seeded at a density of 100 cells / mL and added to RPMI-1640 medium containing IL-2 at a final concentration of 1500 IU / mL. The medium was added serially every 3 days, and the cells were cultured at 37°C in a 5% CO2 incubator for 14 days to obtain Treg cells infected with recombinant lentiviruses containing pLent-EF1α-IL-11-1 and pLent-EF1α-IL-11-2. Treg cells cultured under scheme two were transfected with viral solution containing pLent-EF1α-IL-11-1 and named IL-11-Treg-1 cells. Treg cells cultured under scheme two were transfected with viral solution containing pLent-EF1α-IL-11-2 and named IL-11-Treg-2 cells.

[0037] Example 3: Expression of Treg cell markers Treg cells cultured under scheme 1 and scheme 2, as well as IL-11-Treg-1 and IL-11-Treg-2 cells prepared in Example 2, were collected. After sampling and measuring cell viability, the cells were centrifuged at 1000 rpm for 5 min, washed twice with PBS, and the concentration was adjusted to 1×10⁻⁶. 6 cells / mL, dispensed into flow cytometry tubes. The Treg cell marker CD4 was detected by flow cytometry. + CD25 + FoxP3 + The expression levels and results are shown in Table 2 below. Figures 1-8 As shown.

[0038] Table 2 Cell viability and biomarker expression

[0039] As shown in Table 2, the Treg cells cultured under scheme 2 exhibited significantly higher cell viability and biomarker expression rates compared to those cultured under scheme 1, indicating that co-culturing with umbilical cord blood pluripotent stem cells can improve the viability and biomarker expression rates of Treg cells. Among the cell groups mentioned above, IL-11-Treg-2 cells had the highest viability, reaching 98.6%, and CD4+ expression was also high. + CD25 + FoxP3 + The highest expression rate was observed in CD4. + The expression rate reached 98.3%, CD25 + FoxP3 +The expression rate of IL-11-Treg-2 cells was 82.6%, which was significantly higher than that of IL-11-Treg-1 cells, indicating that the optimized nucleotide sequence of the anti-IL-11 antibody can improve the viability and marker expression rate of the modified Treg cells.

[0040] Example 4: In vivo experiment of diabetic mice Fifty 8-10-week-old female NOD mice were purchased as a diabetic mouse model and acclimated for one week (purchased from Changzhou Cavens Experimental Animal Co., Ltd.). The mice were randomly divided into five groups, with 10 mice in each group, and were injected with 100 μL of PBS buffer solution into the abdominal cavity near the pancreas. Experimental group A: collect Treg cells cultured according to scheme one, take 3 x 10 6 cells, centrifuge and suspend in 100 μL of PBS buffer solution, and inject the model mice into the abdominal cavity near the pancreas. On the seventh day, the mice were treated again with 100 μL of PBS buffer solution containing 2 x 10 6 cells of Treg cells cultured according to scheme one, and were also injected into the abdominal cavity near the pancreas.

[0041] Experimental group B: collect Treg cells cultured according to scheme two, take 3 x 10 6 cells, centrifuge and suspend in 100 μL of PBS buffer solution, and inject the model mice into the abdominal cavity near the pancreas. On the seventh day, the mice were treated again with 100 μL of PBS buffer solution containing 2 x 10 6 cells of Treg cells cultured according to scheme two, and were also injected into the abdominal cavity near the pancreas.

[0042] Experimental group C: IL-11-Treg-1 cells prepared in Example 2, take 3 x 10 6 cells, centrifuge and suspend in 100 μL of PBS buffer solution, and inject the model mice into the abdominal cavity near the pancreas. On the seventh day, the mice were treated again with 100 μL of PBS buffer solution containing 2 x 10 6 cells of IL-11-Treg-1 cells, and were also injected into the abdominal cavity near the pancreas.

[0043] Experimental group D: IL-11-Treg-2 cells prepared in Example 2, take 3 x 10 6 cells, centrifuge and suspend in 100 μL of PBS buffer solution, and inject the model mice into the abdominal cavity near the pancreas. On the seventh day, the mice were treated again with 100 μL of PBS buffer solution containing 2 x 10 6 cells of IL-11-Treg-2 cells, and were also injected into the abdominal cavity near the pancreas.

[0044] Control group: inject 100 μL of PBS buffer solution into the abdominal cavity near the pancreas of the model mice on the first and seventh days of treatment.

[0045] The body weight and blood glucose of mice were detected every three days after the second injection, and the growth curve was drawn.

[0046] The results are shown in Tables 3 and 4 and Figures 9-10

[0047] Table 3 Statistics of body weight change of mice (g)

[0048] Table 4 Statistics of blood glucose of mice (mmol / L)

[0049] The results show that in the 21-day treatment experiment of diabetic mice, compared with the control group without Treg cell treatment, the Treg cells prepared by different methods (experimental groups A, B, C and D) all have an improvement effect on the decrease of mouse body weight and the increase of blood glucose, and there is a significant difference in the treatment effect among different experimental groups. According to the mouse body weight change table, it can be seen that Treg cells can significantly delay the decrease of body weight of diabetic mice, and the decrease of body weight of mice in experimental group D is the smallest, and the body weight only decreases by 0.9g after 21 days, close to the normal body weight stable state, and almost completely alleviates the weight loss related to diabetes. According to the mouse blood glucose value change table, it can be seen that Treg cells can significantly inhibit the increase of blood glucose of diabetic mice, and the blood glucose control effect of experimental group D is the most stable, and the blood glucose is successfully reduced to 4.9 mmol / L within 21 days, and the blood glucose level reaches the normal range, realizing the effective regulation of blood glucose of diabetic mice.

[0050] After 3 weeks of treatment, the tail vein blood of mice in experimental and control groups was collected, and the serum insulin level was measured using a super-sensitive mouse insulin enzyme-linked immunosorbent assay kit (purchased from Beijing Solabio Technology Co., Ltd., SEKM-0141) according to the instructions. The results are shown in Table 5.

[0051] Table 5 Serum insulin level of mice

[0052] The serum insulin level of the control group mice was only 0.97 ng / mL, while the insulin levels of all experimental groups (A, B, C and D) receiving Treg cell treatment were significantly higher than that of the control group, proving that Treg cell treatment can effectively improve the serum insulin level of diabetic mice. Among them, the effect of experimental group D is the best, and the insulin level is nearly 5 times that of the control group, which shows that the umbilical cord blood pluripotent stem cells are co-cultured with Treg cells, and the Treg cells are modified by using the anti-IL-11 antibody with optimized nucleotide sequence, which can significantly improve the ability of protecting islet β cells and improving insulin secretion.

[0053] ​Example 5 Cytokine detection experiment After 3 weeks of treatment in Example 4, 1 mL of whole blood of mice in the experimental group and the control group was taken, and was left at room temperature for 30 minutes, and was centrifuged at 4°C and 3000g for 15 minutes, and the supernatant was collected for detection of cytokines IFN-γ, IL-10 and TGF-β. The ELISA kits for detecting cytokines IFN-γ, IL-10 and TGF-β were all purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd., and the item numbers were ml002277, ml037873 and ml057830, respectively. The ELISA results are shown in Table 6. Figure 11 and Table 6.

[0054] Table 6 Cytokine detection results

[0055] It can be seen from the results that the IFN-γ concentration of Treg cells in the experimental group D is the lowest, indicating that the Treg cells prepared in the application can better exert their inhibitory function, reduce the secretion of IFN-γ in the local islet, and reduce the inflammatory damage to β cells; the concentrations of cytokines IL-10 and TGF-β secreted by Treg cells in the experimental group D are up-regulated, and the concentration is the highest, and the Treg cells with stronger efficacy often have a highly activated Foxp3+ phenotype, can secrete a large amount of IL-10 factor, and the IL-10 factor can maintain the stability of Treg cells. The secretion of TGF-β can also inhibit effector T cells and promote β cell repair. This fully shows that the Treg cells prepared in the application have higher efficacy in the treatment of diabetic mice.

Claims

1. A method for preparing Treg cells modified with anti-IL-11 antibody, characterized in that: After co-culturing Treg cells with umbilical cord blood pluripotent stem cells, the Treg cells were isolated and recovered, and modified with anti-IL-11 antibody to obtain anti-IL-11 antibody-modified Treg cells; the nucleotide sequence of the anti-IL-11 antibody is shown in SEQ ID NO.2 in the sequence listing.

2. The method for preparing Treg cells modified with anti-IL-11 antibody according to claim 1, characterized in that: In the co-culture, the initial ratio of Treg cells to umbilical cord blood pluripotent stem cells was 9-11:

1.

3. The method for preparing Treg cells modified with anti-IL-11 antibody according to claim 1, characterized in that: The co-culture period is 6 days.

4. The method for preparing Treg cells modified with anti-IL-11 antibody according to claim 1, characterized in that: The co-culture method involves seeding Treg cells and umbilical cord blood pluripotent stem cells into a culture container, adding RPMI-1640 medium containing 9-11% Vol serum substitute, and co-culturing for 2 and 4 days. Half the volume of the old medium is then discarded, and an equal volume of fresh RPMI-1640 medium containing 98-102 U / mL IL-2 is added. After co-culturing for 6 days, the co-cultured Treg cells are isolated and recovered.

5. The method for preparing Treg cells modified with anti-IL-11 antibody according to claim 1, characterized in that: The Treg cells prior to co-culture were obtained by culturing them as follows: Treg cells were isolated from autologous peripheral blood, expanded and cultured for 3 days in a culture vessel coated with anti-CD3 monoclonal antibody, and then expanded and cultured for 11 days in an uncoated culture vessel.

6. A method for preparing Treg cells modified with anti-IL-11 antibody according to claim 5, characterized in that: The amplification culture medium used was RPMI-1640 supplemented with 14.8-15.2% Vol serum substitute, 1.8-2.2 mM L-glutamine, 0.9-1.1% Vol penicillin-streptomycin, 0.9-1.1% Vol insulin-transferrin-selenium, 48-52 μg / mL ascorbic acid-2-phosphate, 9.8-10.2 ng / mL IL-2, 29-31 nM AS2863619, 2.4-2.6 μg / mL TNFR2 antibody, 4.8-5.2 ng / mL TGF-beta, and 98-102 nM rapamycin.

7. The method for preparing Treg cells modified with anti-IL-11 antibody according to claim 1, characterized in that: The fusion rate of the umbilical cord blood pluripotent stem cells before co-culture was 89-91%.

8. The method for preparing Treg cells modified with anti-IL-11 antibody according to claim 1, characterized in that: The umbilical cord blood pluripotent stem cells before co-culture were obtained by culturing them as follows: mononuclear cells were isolated from umbilical cord blood, added to RPMI-1640 medium containing 9.8-10.2% Vol serum substitute, and cultured for 16 days until the cell confluence reached 89-91%.

9. The use of the anti-IL-11 antibody-modified Treg cells prepared by the method according to any one of claims 1-8 in the preparation of a medicament for treating diabetes.

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