Stem cell composition and application thereof in regulating sub-health

Abstract

The invention discloses a stem cell composition and application thereof in regulating sub-health. The stem cell composition comprises mesenchymal stem cells (MSCs) modified by fusion polypeptide and an anti-IL-6R single domain antibody. The amino acid sequence of the fusion polypeptide is as shown in SEQ ID NO.1, and the fusion polypeptide is modified on the surface of stem cells through click chemistry, so that the chemotactic migration ability, collagen matrix affinity and inflammation tolerance of the stem cells are remarkably improved. The anti-IL-6R single-domain antibody is obtained through phage display screening, the VHH gene of the anti-IL-6R single-domain antibody is shown as SEQ ID NO.2, and the anti-IL-6R single-domain antibody has high affinity and activity of inhibiting an IL-6 / STAT3 signal channel. Animal experiments show that the composition can significantly reduce inflammatory factor (IL-6) and cortisol levels of chronic stress model mice, improve oxidation resistance (SOD) and recover immune homeostasis, and provides an innovative treatment strategy for adjustment of sub-health status.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a stem cell composition and its application in regulating sub-health. Background Art

[0002] As a critical stage between health and disease, the global incidence rate of the sub-health state has reached 35%-50%. Its characteristic manifestations include persistent fatigue, sleep disorders, and low-grade inflammatory responses. Existing intervention methods such as nutritional supplements and psychotherapy have defects such as a single action target and short-lasting curative effects.

[0003] Mesenchymal stem cells (MSCs) are regarded as a potential means for sub-health intervention due to their paracrine and immunomodulatory properties. However, traditional MSCs therapy faces two major technical bottlenecks: ① low homing efficiency, only 18%-23% of the cells can migrate to the target tissue directionally after intravenous injection; ② poor microenvironment adaptability, and the survival time in the chronic inflammatory microenvironment is less than 72 hours. Among existing improvement strategies, although overexpression of the CXCR4 gene can increase the homing rate to 40%, there is a risk of gene insertion mutation; while simply using the SDF-1 chemokine is safe, but its half-life is only 5-7 minutes, making it difficult to maintain an effective concentration.

[0004] On the other hand, the core characteristics of the sub-health related inflammatory microenvironment are the abnormal elevation of pro-inflammatory factors such as IL-6 and TNF-α. Although anti-IL-6R monoclonal antibodies (such as tocilizumab) can block the IL-6 signaling pathway, systemic administration is prone to side effects such as immunosuppression. How to achieve the spatio-temporal synergistic effect between stem cells and antibody drugs has become the key to technological breakthrough. Summary of the Invention

[0005] The purpose of the present invention is to provide a composition of mesenchymal stem cells modified with a fusion polypeptide and an anti-IL-6R single domain antibody, and its application in regulating the sub-health state.

[0006] Therefore, on the one hand, the present invention discloses a stem cell composition, and the stem cell composition comprises the following components:

[0007] (1) Mesenchymal stem cells modified with a fusion polypeptide, and the amino acid sequence of the fusion polypeptide is as shown in SEQ ID NO.1;

[0008] (2) Anti-IL-6R single domain antibody, and the nucleotide sequence of its VHH gene is as shown in SEQ ID NO.2.

[0009] Preferably, the fusion polypeptide in the present invention is covalently modified on the surface of mesenchymal stem cells through click chemistry, and the modification efficiency is ≥95%.

[0010] Preferably, the affinity KD of the anti-IL-6R single-domain antibody of the present invention for IL-6R is 0.81 nM, and its IC50 is 3.1 ± 0.2 nM.

[0011] Preferably, the mesenchymal stem cells of the present invention are human umbilical cord mesenchymal stem cells, which are cultured under hypoxic conditions until the 3rd generation and then used for modification.

[0012] In one aspect, the present invention also discloses a method for preparing the stem cell modified with the fusion polypeptide, and the method includes the following steps:

[0013] (1) Synthesize the fusion polypeptide shown in SEQ ID NO.1 by Fmoc solid-phase synthesis method, and purify it by HPLC to a purity of ≥ 98%;

[0014] (2) Couple the fusion polypeptide with NHS-PEG4-DBCO to generate DBCO-polypeptide;

[0015] (3) Pre-incubate the mesenchymal stem cells with Azide-PEG12-NHS to introduce azide groups;

[0016] (4) Covalently modify the DBCO-polypeptide to the surface of the stem cells through click chemistry reaction.

[0017] In one aspect, the present invention also discloses the application of the stem cell composition in the preparation of a drug for regulating sub-healthy state.

[0018] Preferably, the drug of the present invention is administered by tail vein injection, and the dose is 1×10 6 Mesenchymal stem cells modified with fusion polypeptide combined with 50 μg anti-IL-6R single-domain antibody, once a week for 4 weeks.

[0019] The beneficial effects of the present invention are summarized as follows:

[0020] 1. Enhanced stem cell function: Fusion polypeptide modification increases the migration ability of stem cells by 4.23 times, and the survival rate in the inflammatory environment is increased to 89%.

[0021] 2. High-efficiency anti-inflammatory and immune regulation: The anti-IL-6R single-domain antibody combined with modified stem cells can synergistically inhibit the IL-6 signaling pathway, reduce the level of inflammatory factors, and restore immune homeostasis.

[0022] 3. Comprehensive improvement of sub-health: The composition effectively reverses the physiological imbalance caused by chronic stress through multi-target actions (antioxidation, anti-stress, immune regulation), providing an innovative solution for sub-health intervention. Description of the Drawings

[0023] Figure 1SDS-PAGE detection results of anti-IL-6R single-domain antibody, where 1 is the anti-IL-6R single-domain antibody.

[0024] Figure 2 Detection results of STAT3 phosphorylation in 293T cells of different groups. Detailed implementation methods

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the description of this invention herein are only for the purpose of describing specific embodiments and are not intended to limit the invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0026] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0027] Example 1: Preparation of stem cells modified with fusion polypeptide

[0028] 1. Synthesis of fusion polypeptide

[0029] 1.1 Sequence design: The fusion polypeptide consists of four functional domains:

[0030] (1) CRGDS: An integrin-binding sequence that enhances the adhesion ability of stem cells;

[0031] (2) (Gly4Ser)3: A flexible linker peptide that increases the conformational freedom of the protein;

[0032] (3) CXCR4(1-38): The N-terminal domain of the chemokine receptor CXCR4, which enhances chemotaxis;

[0033] (4) Col1α1(758-793): The type I collagen-binding domain, which increases the affinity of cells for the collagen matrix;

[0034] The amino acid sequence of the fusion polypeptide designed in this way is shown in SEQ ID NO.1.

[0035] 1.2 Synthesis and purification

[0036] (1) Solid-phase synthesis: The Fmoc solid-phase peptide synthesis (SPPS) method is used. The resin used is Rink Amide AM resin (100-200 mesh). Fmoc-amino acids are gradually coupled in 5-fold molar amounts. HBTU / HOBt is used as the coupling reagent, and diisopropylethylamine (DIEA) is used as the base. The coupling efficiency is monitored for each step (>99%).

[0037] (2) Deprotection and cleavage: After stirring with TFA / H2O / TIS / EDT (92.5:2.5:2.5:2.5, v / v) at room temperature for 2 h, precipitation occurred. The polypeptide was precipitated using cold diethyl ether.

[0038] (3) HPLC purification: HPLC was used with a C18 column (250 mm × 4.6 mm, 5 μm), a flow rate of 1.0 mL / min, and gradient elution (10%-60% acetonitrile / 0.1% TFA, 30 min).

[0039] (4) Purity and molecular weight detection: The molecular weight was approximately 11.22 kDa. The HPLC purity was calculated by peak area normalization, and the purity > 98%.

[0040] 2. Stem cell surface modification

[0041] 2.1 Cell culture: Human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultured under hypoxic conditions (5% O 2 , 37 °C) in DMEM / F12 medium (Gibco), containing 10% fetal bovine serum (FBS), supplemented with 1% GlutaMAX TM and 1% penicillin / streptomycin. The cells were passaged to the P3 generation and used for modification when reaching 80%-90% confluence.

[0042] 2.2 Cell surface modification steps

[0043] (1) Preparation of DBCO-polypeptide: In PBS (pH 7.4), 10 μM of the fusion polypeptide was added with 2 mM NHS-PEG4-DBCO, and the reaction was carried out at room temperature for 30 min. The completeness of the reaction was detected by HPLC and purified by dialysis (MWCO 3,500 Da).

[0044] (2) Cell pre-labeling: hUC-MSCs were incubated in 1 mM Azide-PEG12-NHS at 37 °C for 1 h and washed 3 times to remove unbound reagents.

[0045] (3) Click chemistry modification: The cells were suspended in RPMI 1640 medium, and DBCO-polypeptide (10 μM) was added, and the incubation was carried out at 37 °C for 2 h. After washing 3 times, the cells were resuspended in PBS for flow cytometry detection.

[0046] 2.3 Flow cytometry analysis: The FITC-labeled fusion polypeptide was used to detect the modification efficiency. Instrument: BD, excitation wavelength 488 nm, detection wavelength 530 nm. The number of cells was 1 × 10 5 cells per group, and repeated 3 times (n = 3). The results are shown in Table 1: The positive rate of the experimental group was significantly higher than that of the unmodified control group, indicating that the modification of the experimental group was successful.

[0047] Summary of Flow Cytometry Detection Results

[0048]

[0049] (**p < 0.01 vs unmodified group)

[0050] 2.4 Cell Viability Detection (CCK-8): After cell modification, the CCK-8 kit was used to evaluate the viability. 1×10 4 cells / well were seeded in a 96-well plate, and 10 μL of CCK-8 solution was added to each well. Incubate at 37 °C for 2 h. The absorbance (OD) was measured at a wavelength of 450 nm using a microplate reader. The results showed (Table 2) that there was no significant decrease in cell viability after modification (p > 0.05).

[0051] Table 2 Results of Cell Viability Detection

[0052]

[0053] 3. Conclusion: The fusion polypeptide was synthesized by Fmoc-SPPS with a purity of 98%. hUC-MSCs were modified by click chemistry with a modification efficiency of 96.7% (flow cytometry positive rate). The cell viability was not affected after modification (detected by CCK-8), demonstrating that this method has high feasibility and is suitable for further in vivo experiments.

[0054] Example 2: In Vitro Function Verification

[0055] 1. Transwell Migration Assay

[0056] 1.1 Cell Preparation

[0057] (1) Cell source: The experimental group was hUC-MSCs modified with the polypeptide in Example 1, and the control groups were unmodified MSCs and MSCs treated with free fusion polypeptide.

[0058] (2) Cell culture: DMEM / F12 medium containing 10% FBS, cultured at 37 °C in 5% CO 2 culture.

[0059] (3) Cell harvesting: Passage to P3 generation, count after digestion (0.25% trypsin-EDTA), and adjust to 5×10 5 cells / mL.

[0060] 1.2 Transwell Migration Assay: A 24-well plate + 8 μm pore size Transwell chamber was used.

[0061] (1) Lower chamber: Add 600 μL of DMEM / F12 + 100 ng / mL SDF-1α to each well.

[0062] (2) Upper chamber: Inoculate 5×10 4 cells (200 μL serum-free DMEM / F12), and incubate at 37 °C for 12 h.

[0063] (3) After incubation: Use a cotton swab to wipe off the cells in the upper chamber, and gently wash 3 times with PBS. Fix with methanol for 10 min, and wash with PBS. Stain with 0.5% crystal violet for 15 min, and remove the excess stain. Randomly count the cells in 5 fields of view under a microscope, and calculate the average number of migrated cells.

[0064] 1.3 Result analysis: As shown in Table 3, the cell migration in the experimental group was significantly enhanced (p < 0.01), indicating that the modified polypeptide improved the chemotactic ability of hUC-MSCs.

[0065] Table 3 Migration quantitative data (n = 6)

[0066]

[0067] (*p < 0.05, **p < 0.01 vs unmodified group)

[0068] 2. Detection of cell viability

[0069] 2.1 Construction of inflammatory environment: Induce cell inflammatory injury with 10 ng / mL TNF-α to simulate the in vivo inflammatory environment.

[0070] (1) Experimental group: Modified MSCs; Control group: Unmodified MSCs.

[0071] (2) Culture medium: Low-glucose DMEM + 1% FBS + 1% P / S (without additional growth factors).

[0072] 2.2 Detection of cell viability by CCK-8: Seed the cells in a 96-well plate (5×10 3 cells per well, 100 μL). At each time point (0 h, 24 h, 48 h, 72 h): Add 10 μL of CCK-8 solution to each well. Incubate at 37 °C for 2 h. Measure the absorbance (OD value) at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0073] 2.3 Result analysis: As shown in Table 4, the cell viability in the experimental group was significantly higher than that in the control group (p < 0.01), indicating that polypeptide modification enhanced the tolerance of hUC-MSCs to inflammatory injury.

[0074] Table 4 Viability kinetics (%, n = 4)

[0075]

[0076] 3. Summary

[0077] 3.1 Migration experiment: Peptide modification increased the migration ability of hUC-MSCs by 4.23 times, indicating that its chemotactic ability was significantly enhanced.

[0078] 3.2 Survival rate experiment: The survival rate of modified MSCs in the inflammatory environment induced by TNF-α was still 89% at 72h, which was 26% higher than that of the control group (63%).

[0079] 3.3 Conclusion: Fusion peptide modification significantly enhanced the chemotaxis and inflammatory tolerance of hUC-MSCs, demonstrating its potential application value in tissue repair.

[0080] Example 3: Animal experiment (sub-health model)

[0081] 1. Model construction

[0082] 1.1 Animals: 8-week-old male C57BL / 6 mice, weighing 20-25 g. Adapted for 7 days in a standard laboratory environment (temperature 22±2°C, humidity 50±10%, 12h day-night cycle). Free access to food and water, all experiments were in accordance with the ethical requirements of animal experiments.

[0083] 1.2 Chronic stress induction

[0084] (1) Restraint stress: Use a special breathable restraint device to immobilize mice for 3 hours every day (09:00-12:00) for 4 consecutive weeks. During the process, ensure that the animal's head is free, do not affect breathing, and avoid painful stimulation.

[0085] (2) Sleep deprivation: Multiple small platforms with a diameter of 3 cm and a spacing of 3 cm were placed in a water tank (50 cm × 40 cm × 30 cm). The water depth was 1 cm, and the mice were made to fall into the water when they entered REM sleep, thereby depriving them of sleep for 8 h per day (22:00-06:00) for 4 weeks.

[0086] (3) LPS stimulation: 0.5 mg / kg LPS (lipopolysaccharide) was injected intraperitoneally twice a week (Monday and Thursday) for 4 weeks.

[0087] 2. Grouping and Dosing

[0088] 2.1 Grouping (n=10 / group, 80 mice in total)

[0089] (1) Normal control group: normal saline (injected into tail vein, once a week for 4 weeks).

[0090] (2) Model control group: normal saline (injected into tail vein, once a week for 4 weeks).

[0091] (3) Experimental group: 1×10^6 modified MSCs + 50 μg anti-IL-6R single-domain antibody (as shown in Example 4, injected via the tail vein, once a week for 4 weeks).

[0092] (4) MSCs group: 1×10^6 MSCs (injected via the tail vein, once a week for 4 weeks).

[0093] (5) Single-domain antibody group: 50 μg anti-IL-6R single-domain antibody (injected via the tail vein, once a week for 4 weeks).

[0094] (6) MSCs + single-domain antibody group: 1×10^6 MSCs + 50 μg anti-IL-6R single-domain antibody (injected via the tail vein, once a week for 4 weeks).

[0095] (7) Modified MSCs group: 1×10^6 modified MSCs (injected via the tail vein, once a week for 4 weeks).

[0096] 2.2 Administration method

[0097] (1) MSCs and modified MSCs were injected via the tail vein and suspended in 100 μL of PBS. The anti-IL-6R single-domain antibody was dissolved in PBS, and 50 μg was injected via the tail vein each time.

[0098] (2) Administration time: Once a week for 4 weeks.

[0099] 3. Detection indexes and methods (4 weeks after treatment)

[0100] 3.1 Serological detection

[0101] (1) IL-6: The concentration of serum IL-6 was detected using an ELISA kit.

[0102] (2) SOD: The activity of superoxide dismutase was determined by the WST-1 method.

[0103] (3) Cortisol: It was determined using an ELISA kit.

[0104] (4) CD4+ / CD8+ ratio: Analyzed by flow cytometry. 100 μL of mouse peripheral blood was taken, and mononuclear cells were separated by Ficoll density gradient centrifugation. Anti-CD4 antibody labeled with FITC and anti-CD8 antibody labeled with PE were used, and the detection was carried out after incubation at 37°C for 30 min.

[0105] 3.2 Statistical analysis: Statistical analysis was performed using GraphPad Prism 9. The data were expressed as mean ± standard deviation (Mean ± SD). Significance level (*p < 0.05, **p < 0.01).

[0106] 4. Analysis and summary of experimental results. The specific results are shown in Table 5.

[0107] (1) Changes in IL-6: IL-6 in the model group was significantly increased (51.2 ± 6.7 pg / mL). In the experimental group (modified MSCs + single-domain antibody), IL-6 was significantly decreased (16.5 ± 2.3 pg / mL, p < 0.01). The therapeutic effects of MSCs alone or single-domain antibody alone were inferior to those of the experimental group (MSCs group: 45.8 ± 5.6 pg / mL; single-domain antibody group: 40.2 ± 4.9 pg / mL). This indicates that combination therapy can more effectively reduce the inflammatory response.

[0108] (2) SOD activity: SOD in the model group was significantly decreased (79 ± 8 U / mL). In the experimental group, it was restored to 129 ± 10 U / mL (p < 0.01), approaching the normal group (138 ± 11 U / mL). This indicates that modified MSCs + single-domain antibody can enhance antioxidant capacity.

[0109] (3) Cortisol level: Cortisol in the model group was significantly increased (148 ± 13 nmol / L), indicating a chronic stress state. In the experimental group, it was significantly decreased to 88 ± 7 nmol / L (p < 0.01), approaching the normal group (75 ± 6 nmol / L). This indicates that the treatment can improve the endocrine disorders caused by chronic stress.

[0110] (4) CD4+ / CD8+ ratio: The immune balance was disrupted in the model group (CD4+ / CD8+ = 1.2 ± 0.2). In the experimental group, it was significantly restored to 1.9 ± 0.2 (p < 0.01), approaching the normal level (2.1 ± 0.3). This indicates that the treatment can improve immune homeostasis.

[0111] Table 5 Serological indicators (4 weeks after treatment, n = 10)

[0112]

[0113] 5. Conclusion: The combination of modified MSCs and anti-IL-6R single-domain antibody can effectively reduce the inflammatory level, enhance antioxidant capacity, improve stress-induced cortisol elevation, and restore immune homeostasis. This treatment strategy may provide new ideas for chronic stress-related diseases.

[0114] Example 4: Preparation of anti-IL-6R single-domain antibody

[0115] 1. Materials and methods

[0116] 1.1 Shark immunization and cDNA library construction

[0117] 1.1.1 Shark immunization protocol: Healthy adult sharks weighing approximately 3.5 kg were selected. The immunizing antigen was recombinant human IL-6R protein (ab302448, 10 μg / μL). The specific immunization protocol is as follows:

[0118] (1) Day 0: Recombinant human IL-6R protein 100 μg + Freund's complete adjuvant (CFA, Sigma), immunized by intramuscular injection.

[0119] (2) Days 14, 28, and 42: Recombinant human IL-6R protein 100 μg + Freund's incomplete adjuvant (IFA, Sigma), immunized by intramuscular injection.

[0120] (3) Day 56: Recombinant human IL-6R protein 200 μg, immunized by intramuscular injection without adjuvant.

[0121] (4) On Day 60, 30 mL of peripheral blood was collected and lymphocytes were isolated for cDNA library construction.

[0122] 1.2 Screening, expression, and purification of single-domain antibodies

[0123] 1.2.1 Construction of phage display library: Total RNA of lymphocytes was extracted and cDNA was synthesized by reverse transcription using SuperScript III. The VHH gene was amplified using VHH-specific primers. A pComb3X phage display library was constructed. Three rounds of phage panning were performed using ELISA plates coated with recombinant human IL-6R protein to obtain high-affinity clones.

[0124] 1.2.2 Cloning and expression of single-domain antibodies: The high-affinity clone VHH gene (whose nucleotide sequence is shown in SEQ ID NO. 2) was selected, cloned into the pET-22b(+) vector, and a 6His tag was added to its C-terminus for purification. Then it was expressed in E. coli BL21(DE3). The induction conditions during expression were IPTG 0.5 mM, 16 °C, 12 h. After collecting the bacterial cells and lysing them, the supernatant was purified using Ni-NTA affinity chromatography (His-tag). After purification, SDS-PAGE detection was performed, and the results were as Figure 1 shown. Its molecular weight was approximately 12 kDa, the purity was >95%, and the calculated expression yield was approximately 1.5 mg / mL of the culture medium.

[0125] 1.3 Determination of binding activity and affinity

[0126] 1.3.1 Detection of binding activity by ELISA: Recombinant human IL-6R protein (100 ng / well) was immobilized on the solid phase. Anti-IL-6R single-domain antibody or commercial monoclonal antibody (ab271042) was added for binding detection. The results showed (Table 6) that the binding ability of the anti-IL-6R single-domain antibody to recombinant human IL-6R protein was significantly higher than that of the commercial monoclonal antibody.

[0127] Table 6 Results of binding activity detection

[0128]

[0129] 1.3.2 Surface plasmon resonance (SPR) assay for affinity: The Biacore T200 was used to detect the antibody-antigen binding kinetic parameters. The results are shown in Table 7. The KD value of the anti-IL-6R single-domain antibody was lower (0.81 nM vs. 1.22 nM), indicating higher affinity.

[0130] Table 7 Results of affinity detection

[0131]

[0132] 1.4 Functional experiments at the cellular level

[0133] 1.4.1 Detection of STAT3 phosphorylation in 293T cells

[0134] (1) Cells: 293T-IL-6R cells (293T stably expressing recombinant human IL-6R protein) were taken, counted and adjusted to a density of 6×10 5 cells / well, seeded in 6-well plates, and 2 mL of medium was cultured in each well. Incubated at 37 °C for 24 h. Changed to serum-free DMEM for 6 h to synchronize the cell state.

[0135] (2) Treatment protocol: Add shark-derived anti-IL-6R single-domain antibody (10 nM) or commercial monoclonal antibody (10 nM), incubate at 37 °C for 30 min. Add IL-6 (final concentration 10 ng / mL), incubate for 5 min, and gently wash the cells twice with PBS. After cell lysis, total cell protein was extracted, and after quantifying the total protein, Western blot was used to detect p-STAT3, with GAPDH as an internal reference, and the ratio of the two was calculated.

[0136] (3) The results showed (Table 8 and Figure 2 ), the effect of the anti-IL-6R single-domain antibody in inhibiting STAT3 phosphorylation was better than that of the commercial monoclonal antibody (p<0.05).

[0137] Table 8 Results of phosphorylation detection

[0138]

[0139] 1.4.2 U937 cell proliferation inhibition assay

[0140] (1) Cell culture: After digestion, human monocytes U937 were made into single-cell suspensions, counted, and the cell density was adjusted to 5×104 cells / well, 200 μL per well, and inoculated into 96-well plates. Cultured overnight at 37 °C and 5% CO2 to allow the cells to adhere and adapt to growth. The next day, after changing the medium, IL-6 with a final concentration of 10 ng / mL was added to stimulate cell proliferation (IL-6 was not added to the control wells).

[0141] (2) Treatment protocol: Anti-IL-6R single-domain antibody was added at final concentrations of 0.1, 1, 10, 100 nM, and 6 replicate wells were set for each group. The control groups were respectively added with PBS (blank control) and equimolar commercial monoclonal antibody. The cells were cultured for another 48 h.

[0142] (3) CCK-8 assay for cell proliferation: After 48 h, 10 μL of CCK-8 solution was added to each well. Incubated at 37 °C for 2 h, avoiding light during the period. The OD450nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The cell proliferation rate (%) and IC50 (half maximal inhibitory concentration) were calculated.

[0143]

[0144] (4) The results showed (Tables 9 and 10) that the inhibition rate of the anti-IL-6R single-domain antibody was higher than that of the commercial monoclonal antibody, and its IC50 was significantly lower than that of the commercial monoclonal antibody, indicating that the anti-IL-6R single-domain antibody had higher inhibitory activity. At the same time, the anti-IL-6R single-domain antibody had a small molecular weight and was more likely to penetrate tissue barriers, making it suitable for the development of new drugs.

[0145] Table 9 Detection results of cell proliferation rate

[0146]

[0147] Table 10 Calculation results of IC50 values (GraphPad Prism 9)

[0148]

[0149] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A stem cell composition, characterized in that: The stem cell composition comprises the following components: (1) mesenchymal stem cells modified with a fusion polypeptide, wherein the amino acid sequence of the fusion polypeptide is shown in SEQ ID NO.1; (2) An anti-IL-6R single domain antibody, the nucleotide sequence of its VHH gene is shown in SEQ ID NO.

2.

2. The stem cell composition according to claim 1, characterized in that The fusion polypeptide is covalently modified on the surface of mesenchymal stem cells by click chemistry, and the modification efficiency is ≥95%.

3. The stem cell composition according to claim 1, characterized in that The affinity KD of the anti-IL-6R single domain antibody to IL-6R is 0.81 nM, and its IC50 is 3.1±0.2 nM.

4. The stem cell composition according to claim 1, characterized in that The mesenchymal stem cells are human umbilical cord mesenchymal stem cells, which are cultured under hypoxic conditions to the third generation and then used for modification.

5. A method for preparing the fusion polypeptide modified stem cell according to claim 1, characterized in that: The method comprises the following steps: (1) synthesizing the fusion polypeptide shown in SEQ ID NO.1 by Fmoc solid phase synthesis method, and purifying it by HPLC to a purity of ≥98%; (2) coupling the fusion polypeptide with NHS-PEG4-DBCO to generate DBCO-peptide; (3) pre-incubating mesenchymal stem cells with Azide-PEG12-NHS to introduce azide groups; (4) DBCO-peptide was covalently modified onto the stem cell surface through click chemistry reaction.

6. Use of the stem cell composition according to any one of claims 1 to 4 in the preparation of a drug for regulating sub-health conditions.

7. The use according to claim 6, characterized in that: The drug was administered via tail vein injection at a dose of 1×10 6 Fusion polypeptide-modified mesenchymal stem cells were combined with 50 μg of anti-IL-6R single domain antibody once a week for 4 weeks.

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