Preparation method and application of anti-il-11 antibody modified treg cell

Abstract

This invention provides a method for preparing Treg cells modified with anti-IL-11 antibody and its application, belonging to the field of genetic engineering technology. The method involves co-culturing Treg cells with umbilical cord blood pluripotent stem cells, separating and recovering the Treg cells, and then modifying them with an 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. During co-culture, stem cells provide an immune microenvironment, promoting Treg cell proliferation and enhancing their inhibitory ability, and acquiring the ability to specifically recognize pancreatic β-cell antigens, thereby more precisely suppressing autoimmune attacks. This application optimizes the heavy and light chain nucleotide sequences of the anti-IL-11 antibody, resulting in higher efficacy in the treatment of diabetic mice.

Description

A method for preparing anti-IL-11 antibody-modified Treg cells and its application Technical Field

[0001] This invention relates to a method for preparing Treg cells modified with anti-IL-11 antibody and its application, belonging to the field of genetic engineering technology. Background Technology

[0002] Diabetes, especially type 1 diabetes (T1D) and some autoimmune type 2 diabetes (T2D), is fundamentally caused by the immune system mistakenly attacking and destroying pancreatic beta cells, leading to insufficient insulin secretion and disordered glucose metabolism. Traditional treatments (such as insulin injections and hypoglycemic drugs) can alleviate symptoms but cannot cure the disease or reverse beta cell damage. In recent years, immunosuppressive cell-based therapies have emerged as a new direction for diabetes intervention, with regulatory T cells (Tregs) attracting significant attention due to their unique immunomodulatory capabilities.

[0003] Treg cells are a subset of CD4+ T cells with immunosuppressive functions, showing promising results in diabetes treatment. They primarily maintain immune tolerance and protect pancreatic β cells through the following mechanisms: 1. Cytokine-mediated immunosuppression: Treg cells secrete anti-inflammatory factors such as IL-10 and TGF-β, inhibiting the activation of effector T cells and autoreactive CD8+ T cells, reducing the damage to β cells caused by the inflammatory microenvironment. 2. Inhibition of direct cell-to-cell contact: Treg cells bind to antigen-presenting cells or effector T cells through immune checkpoint molecules such as CTLA-4 and PD-1, blocking their activation signals and thus suppressing autoimmune responses. 3. Promotion of tissue repair and regeneration: Treg cells not only suppress inflammation but also promote β cell survival and pancreatic tissue repair by secreting growth factors.

[0004] However, the efficacy of Treg cells is a crucial variable that cannot be ignored in the treatment of immune-related diseases, so improving the efficacy of Treg cells is an urgent task.

[0005] Currently, there are multiple approaches to improving Treg cell efficacy, including genetically engineered Treg cells, such as the preparation of CAR-Tregs for the treatment of systemic lupus erythematosus (SLE). A study published in *Nature* in March 2024 used Treg cells expressing CD19-targeting chimeric antigen receptors (CARs) to treat SLE. The results showed that CAR-Treg cells effectively suppressed B cell function, reduced autoantibody production, and restored immune homeostasis. Other methods to improve cell efficacy include increasing the Treg cell differentiation ratio and enhancing Treg cell function. For example, CN120249200A provides a composition to increase the Treg cell differentiation ratio, including zinc salts, basal cell culture medium, fetal bovine serum, antibiotics, L-glutamine, and a pH buffer. The highest proportion of Treg cells in lymphocytes was 10.8%, but this is limited to optimization of the basal culture medium and lacks functional validation of the cells. CN119799633A provides a method for enhancing Treg cell function. This method involves irradiating hUC-MSCs with gamma rays to form a feeder cell layer, which is then co-cultured with Treg cells to obtain Treg cells with enhanced cell function. The expression level of CD4+CD25+Foxp3+ reaches a maximum of 69%. However, gamma rays themselves may induce DNA double-strand breaks and chromosomal aberrations, and residual fragments may be phagocytosed by Treg cells, affecting safety.

[0006] Conventional in vitro culture methods for Treg cells cannot fully simulate the in vivo immune microenvironment, easily leading to cell senescence and affecting cell viability. Furthermore, conventionally cultured Treg cells lack pancreatic β-cell antigen specificity, making it impossible to precisely target and suppress autoimmune attacks. At the same time, certain cytokines in vivo inhibit Treg cell development and reduce Treg cell function, ultimately resulting in Treg cells failing to achieve the expected therapeutic effect on diabetes. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for preparing Treg cells and its application, achieving the following objectives: improving the survival rate of Treg cells and the expression rate of cell markers, obtaining specific recognition ability for pancreatic β-cell antigens, and improving the therapeutic efficacy for diabetes.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A method for preparing Treg cells modified with anti-IL-11 antibody involves co-culturing Treg cells with umbilical cord blood pluripotent stem cells, separating and recovering the Treg cells, and modifying them with anti-IL-11 antibody to obtain Treg cells modified with anti-IL-11 antibody; the nucleotide sequence of the anti-IL-11 antibody is shown in SEQ ID NO.2 in the sequence listing.

[0010] In the co-culture, the initial ratio of Treg cells to umbilical cord blood pluripotent stem cells was 9-11:1.

[0011] The co-culture period is 6 days.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] The fusion rate of the umbilical cord blood pluripotent stem cells before co-culture was 89-91%.

[0016] 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%.

[0017] The application of the anti-IL-11 antibody-modified Treg cells prepared by the above method in the preparation of drugs for treating diabetes.

[0018] Compared with the prior art, the present invention achieves the following beneficial effects:

[0019] This application utilizes umbilical cord blood pluripotent stem cells to co-culture Treg cells, followed by transfection of the co-cultured Treg cells with a vector constructed using an anti-IL-11 antibody. During co-culture, stem cells provide an immune microenvironment, promoting Treg cell proliferation and enhancing their inhibitory capacity. These cells acquire the ability to specifically recognize pancreatic β-cell antigens, thus more precisely suppressing autoimmune attacks. The stem cell-"dominated" Treg cells can activate pancreatic progenitor cells through paracrine signaling, promoting β-cell regeneration. The anti-IL-11 antibody maintains or restores Treg cell function by blocking IL-11 / IL-11R signaling. This application optimizes the heavy and light chain nucleotide sequences of the anti-IL-11 antibody, which can improve Treg cell viability and the expression rate of cell markers, enhance effective regulation of blood glucose in diabetic mice, significantly improve insulin secretion, and demonstrate higher efficacy in the treatment of diabetic mice. Attached Figure Description

[0020] Figure 1 is a flow cytometry plot of CD4+ expression rate in Treg cells cultured under scheme 1;

[0021] Figure 2 is a flow cytometry plot of CD4+ expression rate in Treg cells cultured under scheme 2;

[0022] Figure 3 shows a flow cytometry plot of CD4+ expression rate in IL-11-Treg-1 cells;

[0023] Figure 4 shows a flow cytometry plot of CD4+ expression rate in IL-11-Treg-2 cells;

[0024] Figure 5 is a flow cytometry plot of CD25+FoxP3+ expression rate in Treg cells cultured under scheme 1;

[0025] Figure 6 is a flow cytometry plot of CD25+FoxP3+ expression rate in Treg cells cultured under scheme 2;

[0026] Figure 7 shows a flow cytometry plot of CD25+FoxP3+ expression rate in IL-11-Treg-1 cells;

[0027] Figure 8 shows a flow cytometry plot of CD25+FoxP3+ expression rate in IL-11-Treg-2 cells;

[0028] Figure 9 is a bar chart showing the changes in body weight of mice in each group;

[0029] Figure 10 is a bar chart showing the changes in blood glucose levels in each group of mice;

[0030] Figure 11 is a bar chart showing the cytokine detection values ​​of each group of mice. Detailed Implementation

[0031] Example 1: Culture of Treg cells

[0032] Option 1 is the conventional Treg cell culture method, with the following specific steps:

[0033] 40 mL of autologous peripheral blood was collected from the patient, and peripheral blood mononuclear cells were separated using Ficoll-Paque lymphocyte separation medium. The separated cells were then sorted into CD4+CD25+Treg cells using a CD4+CD25+Treg cell sorting kit provided by Miltenyi Biotechnology Co., Ltd., Germany, and cell counting was performed. The obtained Treg cells were cultured in 48-well cell culture plates coated with anti-CD3 monoclonal antibody for 3 days, as detailed below:

[0034] Anti-CD3 monoclonal antibody was coated into 48-well plates at 2 µg / mL, 200 µL / well, and incubated at 37°C for 2 h, followed by washing once with PBS. Treg cells were resuspended to 1×10⁻⁶. 8 Cells / mL were seeded at a rate of 0.5 mL / well into amplification medium consisting of 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-phosphate-ascorbic acid, 10 ng / mL IL-2, 30 nM AS2863619 (CDK inhibitor), 2.5 μg / mL TNFR2 antibody, 5 ng / mL TGF-beta, and 100 nM rapamycin. The culture was incubated at 37°C in a 5% CO2 incubator for 3 days.

[0035] On day 4, the cells were transferred to a regular T75 flask, and the same volume of amplification medium was added every 3 days. The cells were cultured until day 14, at which point they were collected for subsequent experiments.

[0036] Option two involves co-culturing umbilical cord blood pluripotent stem cells with Treg cells, with the following steps:

[0037] 1. Umbilical cord blood pluripotent stem cell culture

[0038] (1) Mononuclear cells were separated from the donated umbilical cord blood using Ficoll-Paque lymphocyte separation medium, and then analyzed at a concentration of 1×10⁻⁶ cells. 6 Inoculate cells / mL into T75 bottles, add 10 mL of RPMI-1640 medium containing 10% Vol serum substitute to each T75 bottle, and incubate at 37°C in a 5% CO2 incubator.

[0039] (2) Replace the culture medium every 3 days and culture for 16 days until the cell fusion reaches 90% for subsequent experiments.

[0040] 2. Culture of Treg cells

[0041] Treg cells were collected according to Protocol 1, after expansion to day 14.

[0042] 3. Co-culture of umbilical cord blood pluripotent stem cells and Treg cells

[0043] A. The collected Treg cells were seeded into the umbilical cord blood pluripotent stem cell culture flask at a ratio of 10:1. 25 mL of RPMI-1640 medium containing 10% Vol serum substitute was added. The culture flask was then placed back into a 37°C, 5% CO2 incubator to begin co-culture, which was recorded as day 0.

[0044] B. On the 2nd and 4th days of co-culture, discard half the volume of the old culture medium in the culture flask and replenish with an equal volume of fresh RPMI-1640 culture medium containing 100 U / mL IL-2.

[0045] C. On day 6 of co-culture, gently pipette the suspended cells in the culture wells, avoiding contact with the adherent umbilical cord blood pluripotent stem cells, and transfer the cell suspension to a 15mL centrifuge tube. Wash twice with 2mL of 1×PBS, collect the wash buffer and add it to the centrifuge tube, ensuring that all remaining Treg cells are recovered.

[0046] D. Centrifuge the centrifuge tubes at 300g for 5 minutes, discard the supernatant, resuspend the cells in 1×PBS and wash twice to remove residual culture medium and cell debris, then sort with CD4+CD25+ magnetic beads and harvest the co-cultured Treg cells for subsequent experiments.

[0047] Example 2: Preparation of pLent-EF1α-IL-11 modified Treg cells

[0048] 1. Preparation of pLent-EF1α-IL-11 plasmid

[0049] The anti-IL-11 scFv, whose artificial nucleic acid sequence is shown in SEQ ID NO.1 in the sequence listing, was synthesized by Shandong Hongnuo Biotechnology Co., Ltd., inserted into the BamHI-NotI site of the pLent-EF1α vector (purchased from Vigene), and transformed into E. coli (Top10). After the sequence was confirmed to be correct, the plasmid was extracted using the plasmid extraction kit from OMEGA to obtain the recombinant expression vector pLent-EF1α-IL-11-1, with a plasmid concentration of 1.2 µg / µL.

[0050] The heavy and light chain sequences of the anti-IL-11 scFv were optimized, and its artificial nucleic acid sequence is shown in SEQ ID NO.2 in the sequence listing. It was synthesized by Shandong Hongnuo Biotechnology Co., Ltd., inserted into the BamHI-NotI site of the pLent-EF1α vector (purchased from Vigene), transformed into E. coli (Top10), and after correct sequencing, the plasmid was extracted using the plasmid extraction kit from OMEGA to obtain the recombinant expression vector pLent-EF1α-IL-11-2 with a plasmid concentration of 1.1 µg / µL.

[0051] 2. Lentiviral Packaging

[0052] Resuscitate 293T cells, culture for 3 days, and passage according to cell density. Transfect when cell confluence reaches 80% after one passage. In a six-well plate, use 6 × 10⁶ cells / well. 5 Seed cells per well, with 2 mL of DMEM medium (Gibco, catalog number 11960-044) added to each well to prepare for transfection the next day. Before transfection, replace the 6-well plate with fresh DMEM medium (Gibco, catalog number 11960-044) at 2 mL / well and incubate at 37°C for 1 hour.

[0053] Preparation of transfection reagents: Prepare reagents for tube A and tube B separately in 5mL centrifuge tubes.

[0054] The preparation of tubes A and B is shown in Table 1.

[0055] Table 1 Preparation methods for tubes A and B

[0056]

[0057] After preparation, let stand for 5 minutes, then slowly add the mixture from tube A to tube B, mix thoroughly, and let stand at room temperature for 20 minutes to form a liposome-DNA mixture. Add the mixture to a culture flask, mix gently, and incubate at 37°C in a 5% CO2 incubator.

[0058] Forty-eight hours later, the morphological changes of 293T cells after transfection were observed under a microscope. After 72 hours, the cell culture supernatant containing the virus was collected into centrifuge tubes, centrifuged at 3500 rpm for 10 min to remove cell debris, filtered through a 4.5 μm filter, and centrifuged at 70000 g at 4 °C for 2 h. The precipitate was resuspended in 100 μL of PBS, aliquoted, and stored at -80 °C, while the viral titer was measured. The viral titer of the viral solution containing pLent-EF1α-IL-11-1 in this application was 2.06 × 10⁻⁶. 8The viral titer of the viral solution containing pLent-EF1α-IL-11-2 was 2.11 × 10⁻⁶ TU / mL. 8 TU / mL.

[0059] 3. Lentiviral infection of Treg cells

[0060] Remove the prepared virus solution from -80℃, thaw it, and add RPMI-1640 medium to dilute the virus titer to 3×10⁻⁶. 7 The virus concentration was increased to TU / mL to obtain a diluted viral solution. The virus was then resuspended in 100 µL of the diluted viral solution at a concentration of 1×10⁻⁶ TU / mL. 6 Using Treg cells, the ratio of virus particles to Treg cells was 3:1, resulting in a virus and cell suspension.

[0061] 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.

[0062] Example 3: Expression of Treg cell markers

[0063] 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 are shown in Table 2 and Figure 1-8 below.

[0064] Table 2 Cell viability and biomarker expression

[0065]

[0066] 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 was 82.6%, which was significantly higher than that of IL-11-Treg-1 cells, indicating that the optimization of the nucleotide sequence of the anti-IL-11 antibody can improve the viability and biomarker expression rate of the modified Treg cells.

[0067] Example 4: In vivo experiment in diabetic mice

[0068] Fifty female NOD mice (8-10 weeks old) acclimatizing to diabetes (purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) were purchased and randomly divided into 5 groups of 10 mice each:

[0069] Experimental Group A: Collect Treg cells cultured in Protocol 1, and take 3 × 10⁻⁶ cells. 6 Cells were centrifuged and resuspended in 100 μL of PBS buffer. The cells were injected intraperitoneally into the pancreas region of the mouse model. A second treatment was administered on day 7, with 2 × 10⁶ cells resuspended in 100 μL of PBS buffer. 6 Treg cells cultured according to scheme one were injected into model mice via intraperitoneal injection near the pancreas.

[0070] Experimental Group B: Collect Treg cells cultured under Protocol 2, and take 3 × 10⁻⁶ cells. 6 Cells were centrifuged and resuspended in 100 μL of PBS buffer. The cells were injected intraperitoneally into the pancreas region of the mouse model. A second treatment was administered on day 7, with 2 × 10⁶ cells resuspended in 100 μL of PBS buffer. 6 The Treg cells cultured in Option 2 were also injected intraperitoneally into model mice near the pancreas.

[0071] Experimental Group C: IL-11-Treg-1 cells prepared in Example 2, 3 × 10⁻⁶ cells were used. 6Cells were centrifuged and resuspended in 100 μL of PBS buffer. The cells were injected intraperitoneally into the pancreas region of the mouse model. A second treatment was administered on day 7, with 2 × 10⁶ cells resuspended in 100 μL of PBS buffer. 6 IL-11-Treg-1 cells were injected intraperitoneally into the pancreas region of the mouse model.

[0072] Experimental Group D: IL-11-Treg-2 cells prepared in Example 2, 3 × 10⁻⁶ cells were used. 6 Cells were centrifuged and resuspended in 100 μL of PBS buffer. The cells were injected intraperitoneally into the pancreas region of the mouse model. A second treatment was administered on day 7, with 2 × 10⁶ cells resuspended in 100 μL of PBS buffer. 6 IL-11-Treg-2 cells were injected intraperitoneally into the pancreas region of the mouse model.

[0073] Control group: 100 μL of PBS buffer was injected into the peritoneal cavity of model mice near the pancreas on the first and seventh days of treatment.

[0074] The day of the second injection was recorded as day 0. The weight of the mice and the blood glucose level after fasting for 5 hours were measured every three days, and a growth curve was plotted.

[0075] The results are shown in Tables 3 and 4 and Figures 9-10.

[0076] Table 3. Statistics on mouse body weight changes (g)

[0077]

[0078] Table 4. Blood glucose levels in mice (mmol / L)

[0079]

[0080] The results showed that in a 21-day treatment experiment on diabetic mice, compared with the control group that did not receive Treg cell treatment, Treg cells prepared by different methods (experimental groups A, B, C, and D) all improved weight loss and blood glucose elevation in mice, and there were significant differences in treatment effects among different experimental groups. According to the mouse weight change table, Treg cells significantly delayed weight loss in diabetic mice. Group D mice experienced the smallest weight loss, with a decrease of only 0.9g after 21 days, approaching a stable normal weight, almost completely alleviating diabetes-related weight loss. According to the mouse blood glucose level change table, Treg cells significantly inhibited blood glucose elevation in diabetic mice. Group D showed the most stable blood glucose control effect, successfully reducing blood glucose to 4.9 mmol / L within 21 days, reaching the normal range, achieving effective regulation of blood glucose in diabetic mice.

[0081] Three weeks after treatment, tail vein blood was collected from mice in the experimental and control groups. Serum insulin levels were measured using an ultrasensitive mouse insulin enzyme-linked immunosorbent assay kit (purchased from Beijing Solarbio Science & Technology Co., Ltd., SEKM-0141) according to the instructions. The results are shown in Table 5.

[0082] Table 5. Serum insulin levels in mice

[0083]

[0084] The serum insulin level in the control group mice was only 0.97 ng / mL, while the insulin levels in all experimental groups (A, B, C, and D) that received Treg cell therapy were significantly higher than those in the control group, demonstrating that Treg cell therapy can effectively increase serum insulin levels in diabetic mice. Experimental group D showed the best results, with insulin levels nearly five times that of the control group. This indicates that co-culturing umbilical cord blood pluripotent stem cells with Treg cells and modifying Treg cells with an anti-IL-11 antibody with optimized nucleotide sequences can significantly enhance their ability to protect pancreatic β cells and improve insulin secretion.

[0085] Example 5 Cytokine Detection Experiment

[0086] Three weeks after treatment as described in Example 4, 1 mL of whole blood was collected from both the experimental and control groups of mice. The blood was allowed to stand at room temperature for 30 minutes, then centrifuged at 3000g for 15 minutes at 4°C. The supernatant was collected for the detection of cytokines IFN-γ, IL-10, and TGF-β. The ELISA kits for detecting cytokines IFN-γ, IL-10, and TGF-β were purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., with catalog numbers ml002277, ml037873, and ml057830, respectively. The ELISA results are shown in Figure 11 and Table 6.

[0087] Table 6. Cytokine Detection Results

[0088]

[0089] The results showed that the Treg cells in experimental group D had the lowest IFN-γ concentration, indicating that the Treg cells prepared in this application could better exert their inhibitory function, reduce local IFN-γ secretion in the pancreatic islets, and reduce β-cell inflammatory damage. The concentrations of the cytokines IL-10 and TGF-β secreted by the Treg cells in experimental group D were both upregulated and the highest. Treg cells with stronger efficacy often exhibit a highly activated Foxp3+ phenotype and can secrete large amounts of IL-10, which in turn maintains Treg cell stability. TGF-β secretion can also inhibit effector T cells and promote β-cell repair. This fully demonstrates that the Treg cells prepared in this 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 then transfected and co-cultured with a vector constructed from anti-IL-11 antibody to prepare 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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