A stem cell exosome composition and its application in preparing a drug for treating osteoporosis

Abstract

The present invention relates to a stem cell exosome composition and its application in the preparation of a drug for treating osteoporosis. The stem cell exosome composition comprises stem cell exosomes and an anti-RANKL antibody, wherein the anti-RANKL antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 2. The anti-RANKL antibody has a new structure and can efficiently bind to the target antigen; the preparation method of stem cell exosomes is optimized, and osteoporosis can be effectively treated and bone strengthened; the combination of stem cell exosomes and anti-RANKL antibodies can improve bone metabolism, reduce the expression of inflammatory factors, and activate the Wnt3 signaling pathway.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides a stem cell exosome composition and its application in the preparation of a drug for treating osteoporosis. Background Art

[0002] Osteoporosis is a systemic bone disease that seriously endangers the health of middle-aged and elderly people. Its serious clinical outcomes mainly include brittle fractures and subsequent disability and death. With the acceleration of the aging process of the global population, osteoporosis, including chronic diseases such as hypertension, coronary heart disease, and diabetes, has become a key health problem that needs to be faced by the world. It is reported that approximately 50% of women and 20% of men worldwide suffer from primary osteoporotic fractures after the age of 50, and 50% of patients suffer from osteoporotic fractures (see Zhang YW, Lu PP, Li YJ, et al. Low dietary choline intake is associated with the risk of osteoporosis in elderly individuals: a population-based study. Food Funct. 2021; 12(14): 6442–51). Therefore, the prevention, treatment and prevention of osteoporosis have gradually become the focus of health attention for middle-aged and elderly people. Reducing the prevalence of osteoporosis is of great significance for reducing the medical burden and relieving the pressure of residents.

[0003] Current drugs for the treatment of osteoporosis can be divided into anti-resorptive modulators (including estrogen, estrogen receptor modulators, calcitonin and bisphosphonates) and anabolic therapies (including teriparatide). These drugs often cause adverse reactions, which leads to decreased compliance. In the case of teriparatide, studies have shown that 77% of participants taking 20 μg and 78% of participants taking 40 μg experienced adverse side effects, including joint pain, muscle cramps, fatigue, etc. (see Tsai J.N., Lee H., David N. et al. Combination denosumab and high dose teriparatide for postmenopausal osteoporosis (DATA-HD): a randomised, controlled phase 4 trial. Lancet Diabetes Endocrinol. 2019; 7767–775). Estrogen therapy has known side effects on the breast and uterus, especially with long-term use, and calcitonin therapy can lead to tolerance due to the production of circulating antibodies to calcitonin (see Wada S., Udagawa N., Nagata N., et al. Physiological levels of calcitonin regulate the mouse osteoclast calcitonin receptor by a protein kinase Alpha-mediated mechanism. Endocrinolog. 1996; 137 312–320). Bisphosphonates are the first-line drug for the treatment of osteoporosis and are usually taken for at least 3-5 years, but they have various adverse side effects, including muscle pain and jaw necrosis (see Black DM, Rosen CJ. Clinical practice. Postmenopausal osteoporosis. N Engl J Med. 2016; 374: 254–262). Denosumab is an effective antiresorptive drug that significantly increases bone mineral density (BMD), however it is associated with increased rebound bone resorption, leading to a sharp decrease in bone density and an increased risk of multiple vertebral fractures (see Tsourdi E, Langdahl B, Cohen-Solal M, et al. Discontinuation of denosumab therapy for osteoporosis: a systematic review and position statement by ECTS. Bone. 2017; 105: 11–17).Therefore, it is imperative to develop therapeutic approaches with high targeting ability and low toxicity. Exosomes, with fewer safety concerns and strong osteogenic capacity, are a promising approach to reverse osteoporosis.

[0004] Exosomes are nanoscale extracellular vesicles secreted by cells that encapsulate a variety of bioactive substances, including microRNA, lipids, and proteins. Exosomes are important vesicles that regulate or mediate cellular processes in intercellular communication and can guide MSCs to differentiate into osteoblastic lineages. For example, exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPSCs, hiPSC-MSC-Exos) can effectively promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells in vitro (see Qi X., Zhang J., Yuan H., et al.. Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells repair critical-sized bone defects through enhanced angiogenesis and osteogenesis in osteoporoticrats. Int. J. Biol. Sci. 2016; 12836–849). Exosomes may play an important role in regulating cell differentiation through their non-coding RNA (miRNA and lncRNA), thereby affecting the expression of some transcription factors, such as Runt-related transcription factor 2 (Runx2), osterix, β-catenin and bone morphogenetic protein (BMP), etc. (see Komori T. Regulation of osteoblast differentiation by transcription factors. J. Cell. Biochem. 2006; 991233–1239).

[0005] Compared with traditional therapies, exosome therapy has several advantages. First, exosomes have low immunogenicity. Human bone marrow-derived MSCs express MHC I molecules on their cell surface, and animal studies have shown that mesenchymal stem cell-exosomes can promote bone formation without any adverse events (see He XY, Yu HM, Lin S, et al. Advances in the application of mesenchymal stem cells, exosomes, biomimetic materials, and 3D printing in osteoporosis treatment. Cell Mol Biol Lett. 2021; 26: 47). Second, exosomes have low toxicity. In recent years, drug carriers, especially nano drug carriers, have received extensive attention, but their use is limited due to the inevitable toxicity caused by their synthetic lipid membranes; exosomes, as natural endogenous nano-microvesicles, have not shown induced toxicity in either in vivo or in vitro tests (see Narayanan R., Huang CC, Ravindran S. Hijacking the cellular mail: exosome mediated differentiation of mesenchymalstem cells. Stem Cells Int. 2016: 3808674). Third, exosomes have excellent targeting. For example, osteoclast exosomes can target osteoblasts through EphrinA2 (see Sun W., Zhao C., Li Y., et al. Osteoclast-derived microRNA-containing exosomes selectively inhibitosteoblast activity. Cell Discov. 2016; 2: 16015). Fourth, exosomes also exhibit good permeability, and studies have shown that exosomes can penetrate cell membranes and biological barriers (see Janas T., Janas MM, Sapon K., et al. Mechanisms of RNA loading into exosomes. FEBS Lett. 2015; 5891391–1398).

[0006] However, stem cell exosomes are slightly insufficient in immune regulation. Studies have shown that the key to preventing bone loss is to inhibit the function of osteoclasts. The control and regulation of osteoclast activation and differentiation are achieved through the biologically relevant TNF receptor (TNFR) / TNF-like protein family, including osteoprotegerin (OPG), receptor activator of NF-κB (RANK) and RANK ligand (RANKL). Early animal experiments and clinical trials have shown that RANKL-targeted therapy has the potential to treat diseases related to osteoclast-mediated bone loss. RANKL inhibition leads to reduced osteoclastogenesis and osteoclast activity, thereby reducing bone resorption (see Bekker PJ, Holloway DL, Rasmussen AS, et al. A single-dose placebo-controlled study of AMG 162, a fully human monoclonal antibody to RANKL, in postmenopausal women. J. Bone Miner. Res. 2004; 191059–1066). Denosumab is a targeted antibody that binds to and inhibits RANKL to reduce bone resorption. It has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of osteoporosis in postmenopausal women with a high risk of fractures (see Dore RK The RANKL pathway and denosumab. Rheum. Dis. Clin. 2011; 37433–452). However, some adverse reactions have also occurred in clinical applications of denosumab, such as affecting the normal physiological function of the immune system, inducing skin eczema, flatulence, skin allergies, and hypocalcemia (see Anastasilakis AD, Toulis KA, Polyzos SA, et al. Long-term treatment of osteoporosis: safety and efficacy appraisal of denosumab. Ther. Clin. Risk Manag. 2012; 8295–306).

[0007] In order to overcome the adverse reactions or insufficient efficiency caused by the use of stem cell exosomes or anti-RANKL antibodies alone, the present invention provides a new type of RANKL antibody, which is used in combination with stem cell exosomes to effectively alleviate osteoporosis symptoms and maintain normal bone metabolism. Summary of the invention

[0008] The first aspect of the present invention provides a pharmaceutical composition comprising stem cell exosomes and an anti-RANKL antibody, wherein the anti-RANKL antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 1 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 2.

[0009] Stem cell exosomes have many advantages in reducing immunogenicity, reducing toxicity, and improving targeting, and their efficacy in immune regulation is insufficient. Studies have shown that osteoporosis is related to many immune regulation pathways. Therefore, the present invention combines mesenchymal stem cell exosomes with anti-RANKL antibodies, which can not only improve the symptoms of osteoporosis, but also exert a powerful immune regulation ability, thereby achieving both symptomatic and root cause treatment.

[0010] Furthermore, the stem cell exosomes are mesenchymal stem cell exosomes.

[0011] Furthermore, the mesenchymal stem cells are derived from umbilical cord, umbilical cord blood, fat and / or bone marrow.

[0012] Furthermore, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0013] Furthermore, the preparation method of the mesenchymal stem cells includes: culturing umbilical cord mesenchymal stem cells; culturing until the cell confluence is about 70%, washing three times with exosome-free culture medium, and adding human bFGF with a final concentration of 50 ng / mL and human IL-2 with 100 ng / mL, culturing for 48 hours at 37°C and 5% CO2, and collecting the cell supernatant; separating exosomes by gradient centrifugation.

[0014] Furthermore, the gradient centrifugation method includes: centrifugation at 300×g and 4°C for 10 min, taking the supernatant; centrifugation at 10,000×g and 4°C for 30 min, taking the supernatant; centrifugation at 30,000×g and 4°C for 30 min, taking the supernatant, and filtering the supernatant through a 0.22 μm filter; centrifugation at 120,000×g and 4°C for 90 min, removing the supernatant, and retaining the precipitate; after resuspending in PBS, centrifuging at 120,000×g and 4°C for 90 min, removing the supernatant, and collecting umbilical cord mesenchymal stem cell exosomes.

[0015] The second aspect of the present invention provides a use of the pharmaceutical composition in the preparation of a drug for treating osteoporosis.

[0016] Furthermore, the low bone mass disease is osteoporosis.

[0017] Beneficial Effects

[0018] The present invention provides a stem cell exosome composition and its application in the preparation of a drug for treating osteoporosis, with the following specific beneficial effects:

[0019] (1) Provides a novel anti-RANKL antibody that can efficiently bind to the target antigen;

[0020] (2) The preparation method of stem cell exosomes has been optimized, which can effectively treat osteoporosis and strengthen bones;

[0021] (3) The combination of stem cell exosomes and anti-RANKL antibodies can improve bone metabolism, reduce the expression of inflammatory factors, and activate the Wnt3 signaling pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Bone density test;

[0023] Figure 2 : ALP expression level in rat serum;

[0024] Figure 3 : TRAP expression level in rat serum;

[0025] Figure 4 : Expression levels of inflammatory factors;

[0026] Figure 5 :Activation of Wnt3 signaling pathway. DETAILED DESCRIPTION

[0027] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, biological materials and detection kits are all commercially available unless otherwise specified.

[0028] Example 1 Preparation of umbilical cord mesenchymal stem cell exosomes

[0029] 1.1 Primary culture of umbilical cord mesenchymal stem cells

[0030] Umbilical cord tissue of healthy volunteers was obtained, placed in a clean culture dish, and repeatedly rinsed 3 to 5 times with PBS containing 1% penicillin-streptomycin to remove residual umbilical cord blood; sterile tweezers were used to peel off the umbilical vein and umbilical artery to obtain Wharton's jelly tissue; sterile surgical scissors were used to cut the Wharton's jelly tissue into small pieces of 1 to 3 mm square, and evenly placed in the culture dish; DMEM / F-12 culture medium containing 1% penicillin-streptomycin and 10% FBS was added, and cultured for about 7 to 10 days, and long spindle-like cells could be seen growing around the tissue blocks; the confluence was about 70 to 80%, rinsed once with PBS, and an appropriate amount of trypsin-0.25% EDTA was added to digest in an incubator for 3 to 5 minutes, and the cells were collected by centrifugation, and then subcultured; a total of 3 generations were passed, and the cells were placed in a -80°C refrigerator for cryopreservation.

[0031] 1.2 Preparation of umbilical cord mesenchymal stem cell exosomes

[0032] Umbilical cord mesenchymal stem cells were revived and cultured until the cell confluence was about 70%, and then washed three times with exosome-free medium; exosome-free medium was added, and human bFGF and human IL-2 with a final concentration of 50 ng / mL and 100 ng / mL were added, and cultured at 37°C, 5% CO2 for 48 h, and the cell supernatant was collected; centrifuged at 300×g for 10 min at 4°C, the living cells were removed, and the supernatant was taken; centrifuged at 10000×g for 30 min at 4°C, the cell debris were removed, and the supernatant was taken; centrifuged at 30000×g for 30 min at 4°C, the supernatant was taken, and the supernatant was filtered through a 0.22 μm filter to remove organelles such as apoptotic bodies; centrifuged at 120000×g at 4°C for 90 min, the supernatant was removed, and the precipitate was retained; after resuspending in PBS, centrifuged at 120000×g at 4°C for 90 min, the supernatant was removed, and the umbilical cord mesenchymal stem cell exosomes (Exos) were collected and stored in a -80°C refrigerator for use.

[0033] 1.3 Characterization of umbilical cord mesenchymal stem cell exosomes

[0034] The morphology of Exos was observed under a transmission electron microscope, and the results showed that the Exos were spherical, had a double-layer membrane structure, and had a diameter of about 50-150 nm.

[0035] Exos markers were detected by flow cytometry, and the Exos suspension was stained with CD63 and CD81 antibodies before being tested on the machine. The results showed that the positive expression rates of CD63 and CD81 were 75.3% and 81.5%, respectively.

[0036] After testing, the exosomes meet the experimental requirements and can be used for subsequent experiments.

[0037] Example 2 Preparation of humanized anti-receptor activator of nuclear factor-B ligand (RANKL) antibody 2.1 Preparation of mouse anti-RANKL antibody

[0038] Mice were immunized with human RANKL protein, and mouse anti-RANKL antibodies were obtained by hybridoma technology. After protein sequencing, bioinformatics identification and IMGT database sequence analysis, the CDR regions of the heavy chain and light chain were determined, as shown in Table 1.

[0039] Table 1 Analysis of CDR region structure of mouse anti-RANKL antibody

[0040] CDR1 CDR2 CDR3 Heavy chain DTLFSE RGLVDKEH EYQEGRARLYYE Light chain WGYSSFN LPGSF YVLGHQGSAVSS

[0041] 2.2 Antibody humanization

[0042] The CDR on the mouse antibody was transferred to the human antibody framework to obtain a chimeric CDR humanized antibody gene, wherein the humanized antibody light chain variable region (VL) gene and heavy chain variable region (VH) gene were synthesized by Shanghai Sangon Biotechnology Technology Service Co., Ltd. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2. The humanized antibody VL was directly spliced ​​with the human antibody light chain constant region (CL) by Overlapping PCR, and transferred into the expression vector pcDNA3.4 to obtain plasmids pcDNA3.4-VL-CL and pcDNA3.4-VH-CH.

[0043] The heavy chain and light chain genes of the humanized antibody were cloned into the expression vector pATX-GS2, and then the vector was transfected into CHO cells. The CHO cells were cultured under the conditions of 37° C., 5% CO 2 , and 130 r / min, and the cell supernatant was collected and separated and purified.

[0044] The affinity between the antibody and the target antigen was detected using a Biacore X100 molecular interaction instrument. The Kd value of the target antigen was determined to be 3.68 nM, which has a high affinity and meets the requirements of subsequent experiments.

[0045] Example 3 Animal Model Preparation and Drug Administration

[0046] Healthy 2-3 month old nulliparous female SD rats with a body weight of 220-250 g were selected and housed in an SPF animal room at a temperature of 25±2°C, a humidity of 45±5%, a 12h / 12h alternating light-dark cycle, free access to water and food, and adaptive feeding for 1 week.

[0047] The rats were fasted for 24 h but not water, anesthetized with intraperitoneal injection of sodium pentobarbital (45 mg / kg), and fixed on the operating table in a prone position. A longitudinal incision was made in the middle of the lower abdomen, and the ovarian tissue was peeled off layer by layer to expose it. The bilateral ovaries were removed and the wounds were sutured. 50×10 4 U of penicillin was used to prevent infection. The bone mineral density (BMD) of the rats was measured by X-ray at 6 weeks. If the BMD was significantly lower than that before modeling, the model was successfully established.

[0048] Forty rats with successful modeling were randomly divided into four groups, including: EXOs group, 100 μg / kg EXOs were injected into the tail vein every week for 4 weeks; antibody group, 10 mg / kg anti-RANKL antibody was injected into the tail vein twice a week (on the 1st and 4th day of each week) for 4 weeks; EXOs+antibody group: 100 μg / kg EXOs were injected into the tail vein every week, and 10 mg / kg anti-RANKL antibody was injected into the tail vein twice a week (on the 1st and 4th day of each week); control group, the same amount of normal saline was injected every week. Another 10 rats were taken as the sham operation group, and the abdominal cavity was opened in the same way as above, but the ovaries were not removed.

[0049] Example 4 Improving bone metabolism

[0050] After the administration, blood was collected from the eye sockets, and serum was collected by centrifugation at 4°C and 3000g for 5 minutes and stored in a -80°C refrigerator. The rats were killed, and bilateral femurs were dissected and washed with PBS three times. Bone density was measured using a dual-energy X-ray absorption scanner. Bone density reflects the degree of osteoporosis. The results are as follows Figure 1 As shown in the data, the bone density value of the control group decreased significantly, and osteoporosis was obvious; after treatment, the degree of osteoporosis was alleviated, but the relief was poor when anti-RANKL antibodies were used alone, while after using EXOs and anti-RANKL antibodies, BMD showed a significant improvement.

[0051] ELISA kits were used to detect the levels of tartaric acid phosphatase (TRAP) and alkaline phosphatase (ALP) in serum. The kits were purchased from Wuhan Yipu Biotechnology Co., Ltd. The specific method was carried out according to the instructions of the kits. Figure 2 and Figure 3 As described above, compared with the sham operation group, serum ALP in the control group decreased significantly, while TRAP increased significantly, indicating bone metabolic disorder and inflammation. After treatment, the expression levels of ALP and TRAP improved to varying degrees. Among them, the greatest improvement was achieved in ALP and TRAP in rats using both EXOs and anti-RANKL antibodies, although this trend was slightly inferior in TRAP detection.

[0052] Example 5 Regulation of inflammatory factor expression

[0053] Inflammatory factors are important factors affecting osteoporosis, and are often accompanied by abnormal metabolism of inflammatory factors in osteoporotic individuals. In the present invention, ELISA kits were used to detect the expression of TNF-α and IL-1β (the kits were purchased from Wuhan Huamei Biotechnology Co., Ltd.), and the detection steps were carried out according to the kit instructions.

[0054] like Figure 4 As shown in the data, compared with the sham operation group, the expression of inflammatory factors such as TNF-α and IL-1β in the control group was significantly increased. Treatment with stem cell exosomes can significantly reduce the expression level of inflammatory factors, and the combination with anti-RANKL antibodies seems to be able to further reduce the levels of TNF-α and IL-1β, especially in IL-1β.

[0055] Example 6 Activation of signaling pathways

[0056] Osteoporosis is regulated by a variety of signaling pathways, among which Wnt3 / β-catenin / runx2 is the most widely studied physiological signaling pathway in vivo. In the present invention, the Western blot method is used to detect the expression of Wnt3. Take 100 mg of the right tibia of each group of rats, add 1 mL of RIPA lysate, extract total protein, and measure the total protein content by BCA method. Take 20 μg of protein sample for SDS-PAGE electrophoresis, transfer to PVDF membrane after electrophoresis, and block with 5% skim milk powder. After washing the membrane with TBST, add Wnt3 and β-actin antibodies diluted 2000 times (purchased from Abcam, USA) and place them at 4 ° C overnight. After washing the membrane with TBST, add IgG secondary antibody diluted 5000 times and place it at room temperature for 2 hours. Use chemiluminescence to develop color.

[0057] The results are as follows Figure 5 As mentioned above, the expression of Wnt3 protein in the control group was significantly reduced, and after treatment with EXOs and anti-RANKL antibodies, the expression of Wnt3 was restored, but the effect was not significant. In the combined treatment group, the expression of Wnt3 showed a significant difference, indicating that anti-RANKL antibodies combined with stem cell exosomes therapy can effectively activate the Wnt3 / β-catenin / runx2 pathway, thereby alleviating osteoporosis symptoms.

[0058] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the spirit or basic features of the present invention, the present invention can be implemented in other specific forms and various changes and improvements can be made. These changes and improvements all fall within the scope of the present invention to be protected. Therefore, the scope of protection of the present invention is defined by the attached claims and their equivalents, rather than the above description.

[0059] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An anti-RANKL antibody, characterized in that , the anti-RANKL antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

2.

2. A pharmaceutical composition comprising stem cell exosomes and the anti-RANKL antibody according to claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that The stem cell exosomes are mesenchymal stem cell exosomes.

4. The pharmaceutical composition according to claim 3, characterized in that The mesenchymal stem cells are derived from umbilical cord, umbilical cord blood, fat and / or bone marrow.

5. The pharmaceutical composition according to claim 4, characterized in that The mesenchymal stem cells are umbilical cord mesenchymal stem cells.

6. The pharmaceutical composition according to claim 5, characterized in that The preparation method of the mesenchymal stem cells comprises: culturing umbilical cord mesenchymal stem cells; culturing until the cell confluence is 70%, washing three times with an exosome-free culture medium, adding human bFGF with a final concentration of 50 ng / mL and human IL-2 with a final concentration of 100 ng / mL, culturing for 48 hours at 37° C. and 5% CO2, collecting the cell supernatant; and separating the exosomes by gradient centrifugation.

7. The pharmaceutical composition according to claim 6, characterized in that The gradient centrifugation method includes: centrifugation at 300×g and 4°C for 10 min, taking the supernatant; centrifugation at 10,000×g and 4°C for 30 min, taking the supernatant; centrifugation at 30,000×g and 4°C for 30 min, taking the supernatant, and filtering the supernatant through a 0.22μm filter; centrifugation at 120,000×g and 4°C for 90 min, removing the supernatant, and retaining the precipitate; after resuspending in PBS, centrifuging at 120,000×g and 4°C for 90 min, removing the supernatant, and collecting umbilical cord mesenchymal stem cell exosomes.

8. Use of the pharmaceutical composition according to any one of claims 2 to 7 in the preparation of a drug for treating osteoporosis.

9. The use according to claim 8, characterized in that: The low bone mass disease is osteoporosis.

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