Application of recombinant protein in preparation of medicine for treating multiple sclerosis diseases
Through the HSP65-6MOG35-55 fusion protein, it regulates immune balance, inhibits inflammatory response and promotes myelin repair, solving the problems of immune disorders and myelin repair in the existing treatment of multiple sclerosis, and achieving effective disease relief and myelin repair effects.
Patent Information
- Application Number
- CN202510478889.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-16
- Publication Date
- 2025-08-01
AI Technical Summary
The existing treatment methods for multiple sclerosis mainly rely on immunosuppression and anti-inflammatory drugs, which have failed to effectively solve the problems of immune disorders and myelin repair, and have side effects. Developing new therapeutic strategies with strong targeting and high biosafety has become the focus of research.
The HSP65-6MOG35-55 fusion protein was used to inhibit the overexpression of TNF-α, IL-17A, and IL-1β, and the levels of IL-4, IL-10, and TGF-β were increased, and the immune balance of Th1/Th2 and Th17/Treg were regulated, the inflammatory response was inhibited and myelin repair was promoted.
Significantly reduce the incidence and severity of multiple sclerosis diseases, reduce inflammatory damage and demyelinating lesions, achieve immune homeostasis, and promote myelin repair.
Smart Images
Figure CN120392968A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biopharmaceuticals, and particularly to the application of a recombinant protein in the preparation of a medicament for treating multiple sclerosis. Background Art
[0002] Multiple Sclerosis (MS) is a chronic central nervous system (CNS) autoimmune disease characterized by inflammatory infiltration, demyelination, and axonal damage. More than 2 million people worldwide are affected by this disease, and its significant disability and complex etiology pose a huge challenge to disease prevention and treatment. Experimental Autoimmune Encephalomyelitis (EAE) is an important animal model for studying MS. Because of its high similarity to MS in immunological and pathological characteristics, it is widely used to explore the pathogenesis and treatment strategies of MS. In the immunological mechanisms of MS and EAE, the activation of CD4+ T cells plays a key role. They trigger tissue damage in the central nervous system by releasing a variety of inflammatory factors. However, the current treatment methods targeting these mechanisms still have significant limitations.
[0003] The existing MS treatment methods mainly include immunomodulators and disease-modifying therapies (DMTs), such as interferon β and fingolimod. Although these therapies have shown certain effects in slowing down the disease progression and alleviating symptoms, their treatment mechanisms mainly target immunosuppression and anti-inflammation, and they have not fundamentally solved the problems of immune dysregulation and myelin repair. In addition, their potential side effects (such as increased infection risk and long-term dependence) further limit their clinical application. Based on this, the development of new treatment methods targeting immunomodulation has become the research focus. In recent years, antigen-specific therapies have gradually become a research hotspot for treating MS because of their ability to regulate specific immune pathways without causing general immunosuppression. For example, studies on IFN-β1a and peptide-loaded cells have shown that they have a certain role in inhibiting inflammatory responses and inducing regulatory T cells (Tregs). However, these methods still cannot completely solve the problem of re-establishing immune tolerance. Therefore, the development of a new treatment strategy with strong targeting and biological safety is an important direction in MS research. Summary of the Invention
[0004] The purpose of the present invention is to propose the application of a recombinant protein in the preparation of a medicament for treating multiple sclerosis, HSP65-6MOG 35-55 The fusion protein has strong targeting and has a good therapeutic effect on multiple sclerosis.
[0005] To achieve this purpose, the present invention adopts the following technical solutions:
[0006] Use of a recombinant protein in the preparation of a drug for treating multiple sclerosis, wherein the recombinant protein is HSP65-6MOG 35-55 fusion protein;
[0007] The recombinant protein can inhibit the overexpression of TNF-α, IL-17A, and IL-1β and increase the levels of IL-4, IL-10, and TGF-β.
[0008] Furthermore, the drug for treating multiple sclerosis is a pharmaceutical composition prepared from a reagent of HSP65-6MOG 35-55 fusion protein and a pharmaceutical carrier.
[0009] Furthermore, the drug for treating multiple sclerosis can inhibit the inflammatory response, reduce the progression of spinal cord demyelination, and promote myelin repair.
[0010] Furthermore, the drug for treating multiple sclerosis is administered orally or by the mucosal route.
[0011] Furthermore, the pharmaceutical composition is a capsule, granule, aerosol spray, powder spray, drop, suppository, or perfusion agent.
[0012] The technical solution provided by the present invention may include the following beneficial effects:
[0013] The reagent of HSP65-6MOG 35-55 fusion protein can effectively reduce the incidence and severity of the disease course of EAE, reduce the cumulative nerve damage of EAE mice, reduce the inflammatory damage and demyelination lesions of EAE mice. It can be detected that the HSP65-6MOG 35-55 fusion protein can inhibit the overexpression of TNF-α, IL-17A, and IL-1β and increase the levels of IL-4, IL-10, and TGF-β, indicating that the HSP65-6MOG 35-55 fusion protein can regulate the Th1 / Th2 and Th17 / Treg immune balance, inhibit the inflammatory response, and promote immune homeostasis, thereby alleviating the pathological symptoms of EAE and making it applicable to the preparation of drugs for treating multiple sclerosis. Description of the Drawings
[0014] Figure 1 is the incidence of EAE in each group of mice;
[0015] Figure 2 is the clinical score of each group of mice;
[0016] Figure 3 is the highest clinical score and cumulative clinical score of each group of mice;
[0017] Figure 4 is the result of inflammatory infiltration and demyelination in each group of mice;
[0018] Figure 5 are the cytokine levels in mouse spleen cells;
[0019] Figure 6 are the cytokine levels in mouse peripheral blood cells. Detailed implementation manners
[0020] The present invention will be further clarified below in conjunction with the specific implementation manners. It should be understood that the specific implementation manners described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.
[0021] Heat Shock Protein (HSP)65, as an important member of the heat shock protein family, has become a potential therapeutic factor due to its prominent role in immune regulation. Research has shown that HSP65 can inhibit inflammation and maintain immune tolerance by inducing regulatory T cells, reducing the release of inflammatory factors, and regulating T cell differentiation. Myelin Oligodendrocyte Glycoprotein (MOG) is one of the key autoantigens in MS, which can induce the activation of peripheral T cells and participate in the occurrence of CNS myelin damage. Research on antigen vaccines based on MOG has shown certain immunomodulatory effects, but its inflammatory risk still needs to be addressed. Combining HSP65 and MOG to design a fusion protein may integrate the advantages of both and achieve more precise immune regulation. In order to enhance its antigen specificity and immunomodulatory effect, the research designed a fusion protein of HSP65-6MOG35-55 in which the myelin oligodendrocyte glycoprotein (MOG) 35-55 short peptide was tandemly repeated six times and fused with HSP65. Previous research by the inventors of this patent has shown that the HSP65-6MOG35-55 fusion protein has a preventive effect on EAE mice, and the research of the present invention has shown that the HSP65-6MOG 35-55 fusion protein can inhibit the inflammatory response and promote immune homeostasis by regulating the Th1 / Th2 and Th17 / Treg immune balance, thereby alleviating the pathological symptoms of EAE. Making HSP65-6MOG 35-55 can be applied to the preparation of drugs for treating multiple sclerosis.
[0022] The present invention provides an application of a recombinant protein in the preparation of a drug for treating multiple sclerosis, and the recombinant protein is an HSP65-6MOG35-55 fusion protein;
[0023] The recombinant protein can inhibit the overexpression of TNF-α, IL-17A, and IL-1β and increase the levels of IL-4, IL-10, and TGF-β.
[0024] Furthermore, the drug for treating multiple sclerosis is a pharmaceutical composition prepared from a reagent of HSP65-6MOG35-55 fusion protein and a pharmaceutical carrier.
[0025] Furthermore, the drug for treating multiple sclerosis can inhibit inflammatory responses, reduce the progression of spinal cord demyelination, and promote myelin repair.
[0026] Furthermore, the drug for treating multiple sclerosis is administered orally or by the mucosal route. The ways of mucosal route administration include, but are not limited to, oral mucosal administration, nasal mucosal administration, ocular mucosal administration, pulmonary mucosal administration, and rectal mucosal administration.
[0027] Furthermore, the pharmaceutical composition is a capsule, granule, aerosol spray, powder spray, drop, suppository, or perfusion agent. The following
[0028] Combined with experiments below, the present invention will be further elaborated. It should be understood that the following experiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following experiments, they are usually carried out according to conventional methods and conditions or selected according to the product instructions. The reagents, unless otherwise specified, can be obtained from commercial sources; and the performance of products from different sources does not have a significant impact.
[0029] The animals and reagents used in the following experiments are as follows.
[0030] Fifty 6-8-week-old, 18-20 g SPF-grade healthy female C57BL / 6 mice (Zhuhai BestBio) were used. The mice were housed in a clean-grade animal room with a constant experimental environmental temperature (24 ± 1°C), 12 h of light / dark each day, and drinking water and food available for self-taking. Myelin oligodendrocyte glycoprotein polypeptide (MOG 35-55 , purity > 98%), keyhole limpet hemocyanin-conjugated myelin oligodendrocyte glycoprotein (KLH+MOG 35-55 ), (both from Shanghai Gil Biotech); HSP65-6MOG 35-55 recombinant protein was constructed and purified by the inventors themselves; complete Freund's adjuvant (CFA, Sigma) and MOG 35-55 were emulsified on ice at a ratio of 1:1; pertussis toxin (PTX, List Labs) was dissolved in sterile PBS at a final concentration of 200 ng / μL;. Nucleic acid extraction and synthesis reagents included TransZol, One Step gDNA Removal and cDNA Synthesis SuperMix, and GreenqPCR SuperMix (all from Beijing TransGen Biotech). ELISA kits (Shanghai Jianglai Biotech).
[0031] Experiment 1: Construction and treatment of EAE mouse model
[0032] Randomly select 10 mice as the blank control group, and establish an experimental autoimmune encephalomyelitis (EAE) model for the remaining 40 mice according to the method in the literature: Each mouse is immunized with 200 μL of MOG 35-55 emulsified mixture with CFA (volume ratio 1:1), and the injection site is subcutaneous injection at four points in the axilla and groin; on the day of immunization (day 0) and day 2, inject PTX (200 ng / mouse) intraperitoneally. The control group is not treated. The EAE model mice are randomly divided into the following four groups (10 mice in each group): HSP63-6MOG 35-55 (100 μg), HSP63-6MOG 35-55 (200 μg), KLH+MOG 35-55 , and PBS group. Starting from the 12th day after immunization (disease onset period), intranasal administration is carried out continuously for 5 days, and then administration is carried out every other day for 5 times.
[0033] Experiment 2: Neurological function scoring and pathological observation of mice
[0034] Monitor the clinical symptoms of EAE mice daily, and use the double-blind method to perform neurological deficit scoring (continuous scoring for 60 days). The scoring criteria refer to the literature: 0 points, asymptomatic; 1 point, loss of tail tension and clumsy gait; 2 points, weakness in both hind limbs and can recover by itself; 3 points, paralysis of both hind limbs and cannot recover; 4 points, quadriplegia and cannot be reset; 5 points, near-death state or death. On the 61st day, randomly select 5 mice from each group for histological observation of the brain and spinal cord. After removing the brain and spinal cord, soak them in 4% paraformaldehyde, embed them in paraffin, and make 4-μm-thick sections. HE staining is used to observe inflammatory infiltration in the brain tissue, and LFB staining is used to observe demyelination in the spinal cord. Inflammatory infiltration scoring: 0 points, no inflammatory cells; 1 point, a small number of scattered inflammatory cells; 2 points, inflammatory cells aggregated around blood vessels; 3 points, extensive perivascular cuffing formation extending to the adjacent parenchyma. Demyelination scoring: 0 points, no demyelination; 1 point, a small amount of demyelination under the leptomeninges; 2 points, obvious demyelination under the leptomeninges and around blood vessels; 3 points, fusion of demyelination plaques under the leptomeninges or around blood vessels; 4 points, extensive demyelination involving half of the spinal cord; 5 points, involving the entire cross-section of the spinal cord.
[0035] ① The HSP65-6MOG 35-55 fusion protein reduces the incidence of EAE mice.
[0036] In the experimental autoimmune encephalomyelitis (EAE) model, we evaluated the effects of different treatment groups on the incidence of EAE as Figure 1A - B, the results showed significant differences in the cumulative incidence of EAE among different treatment groups. The PBS group rapidly reached a 100% incidence after immunization and maintained a high level throughout the experiment. In contrast, the HSP65 - 6MOG 35-55 groups (100 μg and 200 μg) showed a significant delay in onset, and the incidence stabilized at approximately 40% and 50% respectively during days 20 to 40. The KLH - MOG 35-55 group had a progressive increase in incidence to 60% in the first 20 days and then gradually decreased, showing a trend of spontaneous remission after disease onset. The experimental results indicated that the HSP65 - 6MOG 35-55 fusion protein and the KLH - MOG 35-55 groups could significantly delay the onset of EAE in mice, reduce the incidence, and the low - dose 100 - μg group had a better effect.
[0037] ② The HSP65 - 6MOG 35-55 fusion protein treatment could reduce the maximum clinical score and cumulative score of EAE mice.
[0038] The mean clinical scores of mice in each group were as Figure 2 shown. The mice in the PBS group had a rapid increase in clinical score after immunization, and the score was significantly higher than that of the HSP65 - 6MOG 35-55给 drug group on day 14 (P < 0.05), significantly higher than that of the KLH + MOG35 - 55 group on day 16 (P < 0.05). The clinical score on day 22 was 2.08 ± 0.076 and then remained at a high level, indicating severe EAE symptoms. In contrast, the mice in the HSP65 - 6MOG 35-55 (100 μg and 200 μg) groups showed an obvious delay in symptoms, and the clinical scores remained at a low level throughout the experiment. The mice in the KLH + MOG35 - 55 group had a gradually increasing clinical score and stabilized at approximately 1.0 ± 0.042, and the clinical symptoms were milder compared to those of the PBS group. The mice in the blank control group did not show obvious EAE symptoms throughout the experiment, and the clinical score was zero. Therefore, the HSP65 - 6MOG 35-55 treatment could effectively reduce the onset and severity of the disease course of EAE. From the perspective of clinical scores, the effect of the 200 - μg dose was equivalent to that of the 100 - μg dose. The clinical scores of the KLH + MOG 35-55 group also indicated that it could alleviate the onset of EAE to a certain extent, but the effect was not as significant as that of the HSP65 - 6MOG 35-55 group.
[0039] ③ The HSP65 - 6MOG 35-55 fusion protein reduced the degree of inflammation and demyelination in EAE mice.
[0040] Figure 3 For the highest clinical score and cumulative score of EAE mice, the highest score in the PBS group was significantly higher than that in other groups, indicating that the EAE symptoms were the most severe. HSP65-6MOG 35-55 (100 μg and 200 μg) groups had maximum scores of (0.69 ± 0.028, 0.75 ± 0.042), which were significantly lower than that in the PBS group (1.94 ± 0.036) (P < 0.01), showing a significant protective effect on the disease. KLH+MOG 35-55 The maximum score of the KLH+MOG group was relatively high (1.097 ± 0.037), but still lower than that of the PBS control group (P < 0.05). The cumulative score of the PBS control group was the highest, indicating the most severe disease condition. HSP65-6MOG 35-55 (100 μg and 200 μg) groups had cumulative scores significantly lower than that in the PBS group (P < 0.01), suggesting that this dose significantly reduced the cumulative nerve damage in EAE mice. KLH+MOG 35-55 The cumulative score of the KLH+MOG group was relatively high, but still lower than that of the PBS group. These results indicate that HSP65-6MOG 35-55 at different doses (100 μg and 200 μg) could effectively reduce the maximum score and cumulative score of EAE mice, thus alleviating the severity of disease progression.
[0041] ④ HSP65-6MOG 35-55 The fusion protein reduces the degree of inflammation and demyelination in EAE mice.
[0042] As Figure 4 shown in A, for the HE staining of brain tissue, obvious inflammatory cell infiltration could be seen in the PBS group, and a large perivascular cuff was formed around the blood vessels, indicating severe inflammatory damage; in the HSP65-6MOG 35-55 (100 μg) group, the inflammatory damage was significantly reduced, with less inflammatory cell infiltration and a significantly reduced formation of perivascular cuff; in the HSP65-6MOG 35-55 (200 μg) group, the degree of inflammatory cell infiltration was further reduced, showing no significant difference compared with the 100 μg group; in the KLH+MOG 35-55 group, the situation of inflammatory cell infiltration was between that of the HSP65-treated group and the PBS group. For the LFB staining, obvious demyelinated areas were present in the PBS group, with a large amount of blue myelin missing, indicating severe demyelination lesions; in the HSP65-6MOG 35-55 (100 μg) group, the demyelination damage was significantly reduced, and the myelin remained relatively intact; in the HSP65-6MOG 35-55 (200 μg) group, the demyelination damage was further reduced, but showed no significant difference compared with the 100 μg group. In the KLH+MOG 35-55 group: The demyelinated area was smaller, but still clearly visible. The summary of the inflammation and demyelination scores of mice in each group is shown in Figure 4B-C. Thus, it can be seen that HSP65-6MOG 35-55 fusion protein vaccine can significantly reduce the inflammatory damage and demyelination lesions in EAE mice, and the effect is more significant at a low dose (100 μg).
[0043] ⑤ Quantitative analysis of cytokine expression levels in mouse spleen cells.
[0044] The expression levels of related cytokines in the spleen of mice in each group were detected by RT-PCR, and the results are as Figure 5 shown. The PBS group showed a significant increase in the expression of pro-inflammatory cytokines (TNF-α, IL-17A, IL-1β) (P < 0.01), while the expression of anti-inflammatory cytokines (IL-4, IL-10, TGF-β) was significantly decreased (P < 0.05). This result indicates that the immune microenvironment in the PBS group presented a significant pro-inflammatory state, which may exacerbate the inflammatory damage of the central nervous system. In contrast, HSP65-6MOG 35-55 group effectively reversed this imbalance, significantly inhibited the overexpression of pro-inflammatory cytokines (TNF-α, IL-17A, IL-1β), and at the same time significantly increased the levels of anti-inflammatory cytokines (IL-4, IL-10, TGF-β). This indicates that HSP65-6MOG 35-55 can inhibit the inflammatory response and promote immune homeostasis by regulating the Th1 / Th2 and Th17 / Treg immune balance, thereby alleviating the pathological symptoms of EAE. The KLH+MOG 35-55 group also showed a certain regulatory effect, which could partially improve the imbalance in the expression of pro-inflammatory and anti-inflammatory cytokines, but its regulatory effect was weaker than that of the HSP65-6MOG 35-55 group.
[0045] ⑥ Fusion protein reduces the expression of inflammatory factors in the peripheral blood of EAE mice.
[0046] The cytokine levels in the sera of mice in each group were detected by ELISA, and the results are as Figure 6 shown. The PBS group showed a significant increase in the levels of pro-inflammatory cytokines (TNF-α, IL-17A) in the peripheral blood (P < 0.01), while the levels of anti-inflammatory cytokines (IL-4, TGF-β) were significantly decreased (P < 0.01). This result further verified the pro-inflammatory state in the peripheral immune environment of the PBS group, suggesting that the overactivation of Th1 and Th17 cells and the impaired functions of Th2 and Treg may be the key mechanisms triggering inflammation. In contrast, the HSP65-6MOG 35-55 group and the KLH+MOG 35-55Both groups could significantly reduce the levels of pro-inflammatory cytokines (TNF-α, IL-17A), while significantly increasing the levels of anti-inflammatory cytokines (IL-4, TGF-β) (P<0.01), indicating that both interventions regulated peripheral immune balance to a certain extent. However, the HSP65-6MOG 35-55 (100 μg) group had the most significant effect, further reducing the expression of pro-inflammatory cytokines and significantly increasing the levels of anti-inflammatory factors, showing the best immunomodulatory effect.
[0047] Based on the above experiments, HSP65-6MOG 35-55 The fusion protein could significantly reduce the incidence and clinical score of EAE mice, alleviate inflammatory infiltration in the brain tissue and the progression of spinal cord demyelination. And by inhibiting the release of pro-inflammatory factors (such as IL-1β, IL-17 and TNF-α) in the spleen and peripheral blood, while promoting the secretion of the anti-inflammatory factor IL-10, and increasing the proportion of Treg cells, inhibiting the activation of Th1 and Th17. These results indicate that HSP65-6MOG 35-55 The fusion protein can effectively alleviate the symptoms of EAE mice and has potential value in the treatment of multiple sclerosis (MS). Therefore, HSP65-6MOG 35-55 The fusion protein can effectively treat the EAE mouse model. It can achieve antigen-specific immunomodulation and broad-spectrum anti-inflammatory effects, significantly reducing demyelination and inflammatory responses.
[0048] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, any one of the two endpoints of each numerical range and any value between the two endpoints can be selected. All those within the ratio range of the present invention can achieve the stated performance. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.
[0049] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purposes of the present invention. The scope of the present invention is defined by the claims and their equivalents.
Claims
1. Use of a recombinant protein in the preparation of a medicament for treating multiple sclerosis, characterized in that, The recombinant protein is HSP65-6MOG 35-55 fusion protein; The recombinant protein can inhibit the overexpression of TNF-α, IL-17A, and IL-1β and increase the levels of IL-4, IL-10, and TGF-β.
2. The application according to claim 1, characterized in that, The drug for treating multiple sclerosis is prepared from an HSP65-6MOG 35-55 fusion protein reagent and a pharmaceutical carrier to form a pharmaceutical composition.
3. The application according to claim 1, characterized in that, The drug for treating multiple sclerosis can inhibit the inflammatory response, reduce the progression of spinal cord demyelination, and promote myelin repair.
4. The application according to claim 1, wherein The drug for treating multiple sclerosis is administered orally or by the mucosal route.
5. The application according to claim 1, characterized in that, The pharmaceutical composition is a capsule, granule, aerosol spray, powder spray, drop, suppository, or perfusion agent.