Selenopeptides activating tnfr2 and uses thereof

Abstract

This invention relates to a selenopeptide that activates TNFR2 and its applications. The TNFR2-activating selenopeptide comprises a tmTNF-targeting peptide, an oligoethylene glycol hydrophilic chain, a selenoamino acid linker, and an alkyl hydrophobic chain connected in sequence. This invention develops a self-assembled selenopeptide with TNFR2-activating activity, which possesses functions of inflammation-targeted delivery, oxidative response phase transition, and reactive oxygen species (ROS) scavenging. In use, it can be pre-assembled into nanomicelles and delivered intravenously to rheumatoid arthritis (RA) tissues for enrichment. In the microenvironment of highly reactive oxygen species in inflamed tissues, the selenoamino acid linker specifically cleaves in response to the overexpression of ROS in damaged cells. This eliminates ROS and reduces oxidative stress. Furthermore, its oxidative metabolite, the tmTNF-targeting peptide, induces liquid-liquid phase separation of tmTNF on the surface of dendritic cell membranes, inducing oligomerization and efficient activation of TNFR2 on the surface of Treg cell membranes, thereby promoting Treg cell proliferation and improving RA inflammation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a selenopeptide that activates TNFR2 and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease caused by an imbalance of immune tolerance, which is usually characterized by persistent joint inflammation and cartilage destruction.

[0003] Among existing treatment options, biologics targeting tumor necrosis factor (TNF) are widely used in clinical practice. However, systemic blockade of TNF can easily lead to serious adverse reactions during long-term administration, including a significantly increased risk of infection and the induction of other autoimmune-related diseases, limiting its further application.

[0004] In recent years, TNF receptor 2 (TNFR2) agonists have attracted widespread attention because they can expand CD4⁺ Foxp3⁺ regulatory T cells (Tregs) to suppress autoimmune inflammatory responses, showing great potential for application in the treatment of RA and other autoimmune diseases.

[0005] Currently, TNFR2 agonist antibodies are used to treat autoimmune diseases, but problems remain, such as insufficient activation efficiency, low targeting, or safety concerns. Therefore, there is an urgent need to provide a novel molecule or formulation that can efficiently and specifically regulate the TNFR2 signaling pathway to overcome the limitations of existing treatments. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a selenopeptide that activates TNFR2 and its application. This selenopeptide can target and induce tmTNF (the natural ligand of TNFR2) to form liquid-liquid phase separation aggregates, efficiently activate TNFR2, and thereby expand CD4⁺ Foxp3⁺ regulatory T cells (Tregs) and promote immune tolerance.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a selenopeptide that activates TNFR2, wherein the selenopeptide that activates TNFR2 comprises a tmTNF-targeting peptide, an oligoethylene glycol hydrophilic chain, a selenoamino acid linker, and an alkyl hydrophobic chain connected in sequence.

[0009] Preferably, the selenopeptide comprises, from the C-terminus to the N-terminus, a tmTNF-targeting peptide, an oligoethylene glycol hydrophilic chain, a selenoamino acid linker, and an alkyl hydrophobic chain connected in sequence.

[0010] The selenopeptide involved in this invention can be pre-assembled into nanomicelles and delivered intravenously to rheumatoid arthritis inflamed tissues for enrichment. In the microenvironment of highly reactive oxygen species (ROS) in inflamed tissues, selenoamino acid linkers can specifically cleave in response to the overexpression of ROS in damaged cells. On the one hand, this eliminates ROS and reduces oxidative stress; on the other hand, its oxidative metabolite, tmTNF-targeting peptide, can induce liquid-liquid phase separation of tmTNF on the surface of dendritic cell membranes. Furthermore, tmTNF condensates can further induce oligomerization and efficient activation of TNFR2 on the surface of Treg cell membranes, achieving Treg cell proliferation and improving rheumatoid arthritis inflammation. In a collagen-induced rheumatoid arthritis (CIA) animal model, it can significantly inhibit joint swelling and the level of inflammatory factors, increase the percentage of regulatory Treg cells, and promote the remodeling of immune tolerance.

[0011] Preferably, the tmTNF targeting peptide is a polypeptide that targets the transmembrane region of tmTMF, and its sequence includes FLILALFIYWWCLRDE.

[0012] Preferably, the oligoethylene glycol hydrophilic chain is an oligoethylene glycol with amino-carboxyl groups at both ends.

[0013] Preferably, the molecular structure of the oligoethylene glycol hydrophilic chain is NH2-(PEG)m-(CH2)n-COOH, where m is selected from any integer from 4 to 6 (e.g., 4, 5 or 6), and n is selected from any integer from 1 to 4 (e.g., 1, 2, 3 or 4).

[0014] Preferably, the selenoamino acid linker is selenocysteine.

[0015] Preferably, the molar ratio of the tmTNF targeting peptide, the oligoethylene glycol hydrophilic chain, the selenoamino acid linker, and the alkyl hydrophobic chain is 1:1:1:1 or 1:1:2:2.

[0016] Preferably, the selenium amino acid linker is connected via lysine or directly to the oligoethylene glycol hydrophilic chain.

[0017] Because the lysine molecule contains two amino groups, the selenoamino acid linker and the alkyl hydrophobic chain can be attached to one amino group in one molar amount, or to both amino groups in two molar amounts.

[0018] Preferably, the alkyl hydrophobic chain is independently a straight-chain alkane with 6-18 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18) carbons.

[0019] In a second aspect, the present invention provides a selenium peptide nanomicelle, wherein the selenium peptide nanomicelle is formed by self-assembly of the selenium peptide and DMG-PEG described in the first aspect as carrier materials and encapsulating tetrandrine.

[0020] The selenium peptide involved in this invention has the functions of inflammation-targeted delivery, oxidation-responsive phase transition and reactive oxygen species scavenging. It can also deliver tetrandrine for synergistic treatment of rheumatoid arthritis. In the microenvironment of high surface reactive oxygen species in inflamed tissue, tetrandrine is released after the nanomicelles disintegrate. Tetrandrine can inhibit tmTNF from being cleaved into exocrine TNF-α by tumor necrosis factor converting enzyme.

[0021] Preferably, the molecular weight of PEG in the DMG-PEG is 1000-5000, such as 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, etc. Other unlisted values ​​within this range can be selected, and will not be elaborated here.

[0022] Preferably, the mass content of tetrandrine in the selenium peptide nanomicelles is 20-50%, such as 20%, 25%, 28%, 30%, 34%, 35%, 38%, 40%, 45%, 50%, etc. Other unlisted values ​​within this range can be selected, and will not be elaborated here.

[0023] Preferably, the hydrated particle size of the selenium peptide nanomicelles is 100-200 nm, such as 100 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 180 nm, 190 nm, 200 nm, etc. Other unlisted values ​​within this range can be selected, and will not be described in detail here.

[0024] Thirdly, the present invention provides the use of selenopeptide nanomicelles according to the first aspect or the second aspect in the preparation of formulations that induce tmTNF to form aggregates on the surface of dendritic cell membranes.

[0025] Fourthly, the present invention provides the use of the selenopeptide according to the first aspect or the selenopeptide nanomicelles according to the second aspect in the preparation of TNF receptor 2 agonists or formulations that promote Treg cell proliferation.

[0026] Fifthly, the present invention provides the use of the selenopeptide according to the first aspect or the selenopeptide nanomicelles according to the second aspect in the preparation of a medicament for the prevention, relief or treatment of rheumatoid arthritis.

[0027] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0028] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, solubilizers, osmotic pressure regulators, surfactants, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention develops a novel treatment strategy for rheumatoid arthritis, namely, a self-assembled selenium peptide that activates TNFR2. The selenium peptide involved in this invention has the functions of inflammatory targeted delivery, oxidative response phase transition, and reactive oxygen species scavenging.

[0031] In use, it can be pre-assembled into nanomicelles and delivered intravenously to inflamed rheumatoid arthritis tissue for accumulation. In the microenvironment of highly reactive oxygen species (ROS) in inflamed tissue, selenoamino acid linkers can specifically cleave in response to the overexpression of ROS in damaged cells. On the one hand, this eliminates ROS and reduces oxidative stress; on the other hand, its oxidative metabolite, tmTNF-targeting peptide, can induce liquid-liquid phase separation of tmTNF on the surface of dendritic cell membranes. Furthermore, tmTNF condensates can further induce oligomerization and efficient activation of TNFR2 on the surface of Treg cell membranes, achieving Treg cell proliferation and improving rheumatoid arthritis inflammation. In a collagen-induced rheumatoid arthritis (CIA) animal model, it can significantly inhibit joint swelling and the level of inflammatory factors, increase the percentage of regulatory Treg cells, and promote the remodeling of immune tolerance.

[0032] This self-assembled selenopeptide can also deliver tetrandrine for synergistic treatment of rheumatoid arthritis. In the microenvironment of high surface reactive oxygen species in inflamed tissues, tetrandrine is released after the nanomicelles disintegrate. Tetrandrine can inhibit tmTNF from being cleaved into exocrine TNF-α by tumor necrosis factor converting enzyme. Attached Figure Description

[0033] Figure 1A This is the ESI-MS characterization image of the selenopeptide molecule prepared in Example 1;

[0034] Figure 1B This is the ESI-MS characterization image of the selenopeptide molecule prepared in Example 2;

[0035] Figure 1C This is the ESI-MS characterization image of the selenopeptide molecule prepared in Example 3;

[0036] Figure 1D This is the ESI-MS characterization image of the selenopeptide molecule prepared in Example 4;

[0037] Figure 2AThis is an HPLC characterization chromatogram of the selenopeptide molecule obtained in Preparation Example 1;

[0038] Figure 2B This is the HPLC characterization chromatogram of the selenopeptide molecule obtained in Preparation Example 2;

[0039] Figure 2C This is the HPLC characterization chromatogram of the selenopeptide molecule obtained in Preparation Example 3;

[0040] Figure 2D This is the HPLC characterization chromatogram of the selenopeptide molecule obtained in Preparation Example 4;

[0041] Figure 3A This is a graph showing the computer-fitted interaction results between the tmTNF targeting peptide and tmTNF;

[0042] Figure 3B This is a graph showing the affinity test results between the tmTNF targeting peptide and tmTNF;

[0043] Figure 4 This is a characterization result of the selenopeptide oxidation reaction product in Preparation Example 1;

[0044] Figure 5A This is a TEM characterization image of the selenium peptide nanomicelles from Example 4-1;

[0045] Figure 5B This is a TEM characterization image of the selenium peptide nanomicelles from Example 4-2;

[0046] Figure 5C This is a particle size characterization diagram of the selenium peptide nanomicelles in Example 4-1;

[0047] Figure 5D This is a particle size characterization diagram of the selenium peptide nanomicelles in Example 4-2;

[0048] Figure 6A This is a characterization diagram of the liquid-liquid phase separation aggregates formed by the oxidation of selenium peptide nanomicelles and tmTNF transmembrane domain peptides in Example 4-1.

[0049] Figure 6B This is a characterization diagram of the liquid-liquid phase separation aggregates formed by the oxidation of selenium peptide nanomicelles and tmTNF transmembrane domain peptides in Example 4-2.

[0050] Figure 7 This is a characterization diagram of selenopeptide nanomicelles inducing the formation of tmTNF aggregates on the surface of dendritic cell membranes;

[0051] Figure 8A The figure shows the effect of PBS, selenopeptide, tetrandrine, and selenopeptide nanomicelles on the proliferation of Treg cells.

[0052] Figure 8BThe figure shows the effect of BMDC cells treated with BS, selenopeptide, tetrandrine and selenopeptide nanomicelles on the proliferation of Treg cells after co-culturing with T cells.

[0053] Figure 9 This is a diagram showing the enrichment effect of selenium peptide nanomicelles in inflammatory tissues of rheumatoid arthritis;

[0054] Figure 10 This is a graph showing the clinical scoring results of mice in each group;

[0055] Figure 11 This is a characterization graph of the serum inflammatory and anti-inflammatory factor levels in each group of mice;

[0056] Figure 12 This is a graph showing the percentage levels of Treg cells in the spleen of mice in each group. Detailed Implementation

[0057] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0058] Preparation Example 1

[0059] This preparation example provides a selenopeptide ((C) 12 -Sec)2-K-OEG-FLILALFIYWWCLRDE), its molecular structure is shown below:

[0060]

[0061] Its preparation method is as follows:

[0062] (1) Boc-Sec(C 12 Synthesis and preparation of )-OH:

[0063] Weigh 0.32 g of selenium powder and 0.15 g of sodium borohydride into a 100 mL flask. In a fume hood, add 1 mL of pure water. After the reaction stops bubbling, stopper the flask with a rubber stopper. Then, dissolve 0.97 mL of dodecyl bromide in 5 mL of DMF and add it to the reaction flask. React at 50°C for 12 hours, then cool to room temperature. Next, dissolve 0.2 g of sodium borohydride in 0.5 mL of water and add it to the reaction flask, while simultaneously inserting a nitrogen-filled balloon into the rubber stopper using a syringe. After the bubbling subsides, dissolve 1.34 g of Boc-Ser(OTs)-OMe (CAS No.: 56926-94-4) in 5 mL of DMF and add it to the reaction flask, followed by 0.52 mL of triethylamine. React at room temperature for 6 hours. Weigh 0.24 g of lithium hydroxide, dissolve it in 5 mL of water, and add it to the reaction flask. Continue the reaction for 2 hours. The pH of the reaction solution was adjusted to 4.5 using 1 M KHSO4. Extraction was performed with 100 mL ethyl acetate, followed by purification by silica gel column chromatography using dichloromethane / methanol (10:1) as the mobile phase. Finally, 0.57 g of Boc-Sec(C) was obtained. 12 )-OH, yield 48%.

[0064] (2) Selenium peptides were prepared using polypeptide solid-phase synthesis technology:

[0065] The resin used was Wang resin with Fmoc-Glu(Otbu)-OH attached (amino acid loading rate of 0.35 mmol / g). Subsequently, after repeated deprotection of Fmoc and coupling with the next amino acid, Fmoc-Asp(Otbu)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-NH-PEG4-CH2CH2COOH (CAS No. 557756-85-1), Fmoc-Lys(Fmoc)-OH, and Boc-Sec(C 12 Finally, the peptide is cleaved from the resin by OH-, and then purified using preparative liquid chromatography.

[0066] Preparation Example 2

[0067] This preparation example provides a selenopeptide (C6-Sec-OEG-FLILALFIYWWCLRDE), the molecular structure of which is shown below:

[0068]

[0069] The preparation method is the same as in Preparation Example 1.

[0070] Preparation Example 3

[0071] This preparation example provides a selenopeptide (C 12 -Sec-OEG-FLILALFIYWWCLRDE), its molecular structure is shown below:

[0072]

[0073] The preparation method is the same as in Preparation Example 1.

[0074] Preparation Example 4

[0075] This preparation example provides a selenopeptide (C 18 -Sec-OEG-FLILALFIYWWCLRDE), its molecular structure is shown below:

[0076]

[0077] The preparation method is the same as in Preparation Example 1.

[0078] Example 1

[0079] The molecular structures of the selenopeptides obtained in Preparation Examples 1-4 were characterized:

[0080] The molecular weight of selenopeptides was determined using electrospray ionization mass spectrometry (ESI-MS): The selenopeptides were dissolved in ultrapure water at a concentration of 1 μM, and the concentration was measured using ESI-MS. The results are as follows: Figure 1A , Figure 1B , Figure 1C , Figure 1D As shown, the test results match the calculated values.

[0081] The purity of selenopeptide was determined using high-performance preparative liquid chromatography (HPLC): the selenopeptide was dissolved in ultrapure water at a concentration of 1 mg / mL, and the result was analyzed using HPLC. The results are as follows: Figure 2A , Figure 2B , Figure 2C , Figure 2D As shown, the selenopeptides did not have obvious large impurity peaks and exhibited good retention and separation, with purities of 98%, 97%, 95%, and 95%, respectively.

[0082] Example 2

[0083] This embodiment provides a method for de novo calculation and affinity testing of tmTNF-targeting peptides:

[0084] A two-stage computational design method was used to obtain a targeting peptide for the transmembrane domain of tmTNF. First, based on the mouse tmTNF structure (P06804) provided by the AlphaFold database, peptide sequences were designed using the AfDesign workflow targeting its functional hotspot region (amino acids 36-56). While maintaining a fixed receptor structure, PSSM-guided sequence optimization iterations were used to improve structural reliability and interfacial interactions. Subsequently, the binding mode and affinity of the designed peptide sequence were evaluated using GNINA molecular docking software. A candidate peptide library was obtained through automatic docking frames defined by ligands and a comprehensive scoring function. Finally, a 16-amino acid tmTNF-specific binding peptide was obtained, with the specific sequence FLILALFIYWWCLRDE. The computer interaction fitting results are shown below. Figure 3A As shown, the tmTNF targeting peptide can interact with tmTNF with high affinity. The computer simulation value of affinity is in the nM range, and the optimal binding site is located in the transmembrane domain of tmTNF.

[0085] Simultaneously, surface plasmon resonance (SPR) technology was used to evaluate the binding affinity between the tmTNF targeting peptide and the tmTNF transmembrane domain polypeptide fragment. First, the tmTNF transmembrane domain polypeptide fragment (amino acid fragments 27 to 52 of the tmTNF protein, i.e., the polypeptide with the sequence SRRCLCLSLFSFLLVAGATTLFCLLNFGVI) was immobilized on the surface of a 47.5 nm thick gold SPR chip through incubation. The resulting chip was then used for SPR detection in a PlexArray HT system. The binding constant was calculated by fitting the binding-dissociation curves using BIAevaluation software to obtain its dissociation equilibrium constant (K). d Affinity test results are as follows: Figure 3B As shown, the affinity of the tmTNF targeting peptide for the transmembrane domain polypeptide fragment of the tmTNF protein is 330 nM.

[0086] Example 3

[0087] This embodiment characterizes the selenopeptide oxidation product from Preparation Example 1:

[0088] 100 µM selenopeptide was mixed with 200 µM hydrogen peroxide in equal volumes and allowed to stand at 37 °C for 1 h. The oxidation products of the peptide were then analyzed using ESI-MS. The results are as follows: Figure 4 As shown, the products of selenopeptide oxidation include (Dha)2K-OEG-FLILALFIYWWCLRDE (Dha is dehydroalanine) and C 12-SeOOH indicates that 100 µM hydrogen peroxide can cause selenopeptides to break down, with the breakage occurring at the selenoamino acid site.

[0089] Example 4-1

[0090] This embodiment provides a selenium peptide nanomicelle, the preparation method of which is as follows:

[0091] The selenopeptide (3 mM) and tetrandrine (30 mM) prepared in Preparation Example 1 were dissolved in DMSO, and DMG-PEG2000 (0.6 mM) was dissolved in water. The mixtures were then mixed at a volume ratio of 10:8.6:2.6 and vortexed at 3000 rpm for 5 min in PBS buffer (pH 7.4). The resulting mixture was dialyzed in PBS (pH 7.4) using a dialysis membrane with a molecular weight cutoff of 1000 Da, and the system volume was adjusted to the desired tetrandrine concentration with PBS. The tetrandrine content encapsulated in the nanomicelles was obtained by adding 1 N hydrochloric acid to the sample and measuring the UV absorption at 280 nm in a 96-well quartz plate. Drug loading was calculated as (mass of encapsulated drug / mass of nanoparticles) × 100%. The experimental results showed that the tetrandrine drug loading in the selenopeptide nanomicelles was 34%.

[0092] Example 4-2

[0093] This embodiment provides a selenium peptide nanomicelle, the preparation method of which is as follows:

[0094] The selenopeptide (3 mM) and tetrandrine (30 mM) prepared in Preparation Example 2 were dissolved in DMSO, and DMG-PEG2000 (0.6 mM) was dissolved in water. The mixtures were then mixed at a volume ratio of 10:8.6:2.6 and vortexed at 3000 rpm for 5 min in PBS buffer (pH 7.4). The resulting mixture was dialyzed in PBS (pH 7.4) using a dialysis membrane with a molecular weight cutoff of 1000 Da, and the system volume was adjusted to the desired tetrandrine concentration with PBS. The tetrandrine content encapsulated in the nanomicelles was obtained by adding 1 N hydrochloric acid to the sample and measuring the UV absorption at 280 nm in a 96-well quartz plate. Drug loading was calculated as (mass of encapsulated drug / mass of nanoparticles) × 100%. The experimental results showed that the tetrandrine drug loading in the selenopeptide nanomicelles was 28%.

[0095] Example 5

[0096] This embodiment characterizes the morphology and structure of selenium peptide nanomicelles:

[0097] Prepare a 100 µM aqueous solution of selenopeptide nanomicelles from Example 4-1 or Example 4-2.

[0098] On one hand, 10 μL of a solution was dropped onto the copper grid of a transmission electron microscope (TEM) sample, allowed to stand for 1 minute, and then the solution was blotted away with filter paper. Then, 10 μL of a 2% uranium acetate solution was dropped onto the copper grid, allowed to stand for 1 minute to stain, and then the solution was blotted away with filter paper. After standing and drying, the morphology was observed using a transmission electron microscope (TEM), such as... Figure 5A and Figure 5B As shown.

[0099] On the other hand, the hydrated particle size of selenium peptide nanomicelles was measured using a Malvern Zetasizer Nano ZS nanoparticle size potentiometer, and the results are as follows: Figure 5C and Figure 5D As shown, the hydrated particle size is approximately 100 nanometers.

[0100] Then, an equal volume of 200 µM selenium peptide nanomicelle aqueous solution from Example 4-1 or Example 4-2 was mixed with 200 µM hydrogen peroxide and allowed to stand at 37°C for 1 h. Then, the tmTNF transmembrane domain peptide was added and mixed thoroughly at 37°C. The morphology was then observed under a microscope. The results are as follows: Figure 6A and Figure 6B As shown, after oxidation, selenium peptide nanomicelles can form liquid-liquid phase separation aggregates with tmTNF transmembrane domain peptides, and the size of the aggregates is in the range of 10-100 micrometers.

[0101] Example 6

[0102] Characterization of selenopeptide nanomicelles-induced tmTNF aggregation on the surface of dendritic cell membranes:

[0103] Bone marrow-derived dendritic cells (BMDCs) isolated from C57BL / 6 mice were co-incubated with selenopeptide nanomicelles (Example 4-1), with a final concentration of tetrandrine of 2 μM. After 2 hours of incubation, the BMDCs were stained with an APC-labeled antibody against mouse TNF-α (506308, Biolegend). Subsequently, the stained cells were imaged and analyzed using a PerkinElmer UltraVIEW VoX confocal microscope.

[0104] The results are as follows Figure 7 As shown, the green fluorescence on the surface of BMDC cell membrane treated with tetrandrine exhibits a scattered distribution, while the green fluorescence spots on the surface of BMDC cell membrane treated with selenopeptide nanomicelles appear as clusters, indicating that selenopeptide nanomicelles induce tmTNF to form a more complex oligomeric structure.

[0105] Example 7

[0106] Characterization of the effect of selenium peptide nanomicelles on Treg cell proliferation:

[0107] BMDC cells were spaced at 5 × 10⁶ cells per well. 5 BMDC cells were seeded at a density of 2 × 10⁶ cells / well in 24-well plates and pretreated for 24 h with samples including selenopeptide nanomicelles (Example 4-1) (2 μM tetrandrine). Purified CD4⁺ T cells were obtained by magnetic sorting of mouse spleen cells using CD4 microbeads (130-117-043, Miltenyi Biotec). Subsequently, the pretreated BMDC cells and purified CD4⁺ T cells were seeded at a density of 2 × 10⁶ cells / well. 5 Cells were seeded at a density of [number] cells per well in 96-well U-plates and cultured under IL-2 for 72 hours. After culture, cells were collected and stained with PE / Cyanine7 anti-mouse CD4 antibody (100422, Biolegend) for fixation and permeabilization. Intracellular staining was then performed with Alexa Fluor® 647 anti-mouse Foxp3 antibody (126408, Biolegend) to evaluate Treg cell induction.

[0108] Experimental results are as follows Figure 8A As shown, the effects of PBS, selenopeptide, tetrandrine, and selenopeptide nanomicelles on the percentage of Treg cells in BMDC cells were 12.5%, 12.2%, 20.6%, and 30.5%, respectively. Among them, the selenopeptide nanomicelles showed the most significant enhancement effect, indicating a synergistic effect between selenopeptide and tetrandrine.

[0109] To analyze whether the increase in Treg cell levels was primarily due to differentiation or proliferation, mouse CD4⁺ T cells, pre-labeled and purified with CFSE, were co-cultured with pretreated BMDCs for 72 hours. Subsequently, CFSE-labeled T cells were collected, and their proliferation levels were analyzed by flow cytometry to determine the effect of each treatment on Treg cell proliferation.

[0110] Experimental results are as follows Figure 8B As shown, after co-culturing BMDC cells treated with PBS, selenopeptide, tetrandrine, and selenopeptide nanomicelles with T cells, the percentages of proliferating Treg cells were 26.1%, 27.9%, 41.2%, and 53.6%, respectively. This indicates that the increase in the percentage of Treg cells by selenopeptide nanomicelles mainly comes from promoting the proliferation of Treg cells.

[0111] Example 8

[0112] Enrichment effect test of selenium peptide nanomicelles in inflammatory tissues of rheumatoid arthritis:

[0113] An 8-week-old male DBA / 1 mouse model of CIA arthritis was established. Specifically, bovine type II collagen was emulsified with complete Freund's adjuvant and administered subcutaneously as a primary immunization. A booster immunization was performed 21 days later using collagen emulsified with incomplete Freund's adjuvant. On day 42 post-primary immunization, Cy5-labeled selenopeptide nanomicelles (prepared as described in Example 4) or Cy5 dye were injected via the tail vein. The mice were sacrificed 24 hours after administration, and their paws were harvested. The fluorescence signal in the paws was measured using an IVIS imaging system (PerkinElmer) at an excitation wavelength of 640 nm and an emission wavelength of 680 nm to evaluate its enrichment in inflamed tissues.

[0114] Experimental results are as follows Figure 9 As shown, the enrichment of Cy5-labeled selenopeptide nanomicelles in the swollen paws of CIA mice was 3.6 times that of Cy5 dye, indicating that it has a targeted effect on inflammatory tissues.

[0115] Example 9

[0116] Inhibitory effect of selenium peptide nanomicelles on CIA rheumatoid arthritis model mice:

[0117] An arthritis model of CIA was established in 8-week-old male DBA / 1 mice. Starting 28 days after the initial immunization, mice were randomly divided into five groups (saline group, selenopeptide group, tetrandrine group, Example 4-1 selenopeptide nanomicelle group, and Example 4-2 selenopeptide nanomicelle group), with 4 mice in each group. Simultaneously, drug administration began. The administration route was tail vein injection, with a dose of 10 mg / kg tetrandrine, administered every two days for a total of 8 administrations. From day 28 to day 46 after the initial immunization, the degree of hand swelling in the mice was clinically scored every 3 days.

[0118] The clinical scoring rules are as follows: (1) Each mouse is scored for its forelimbs and hindlimbs, and the scores are added together to get the total score, which is 0-16 points. (2) 0 points is no obvious redness or deformity, 1 point is mild redness (visible between toes or metatarsophalangeal joints), no obvious functional limitation; 2 points is moderate redness, obvious joint swelling, and mild limitation of movement; 3 points is severe swelling with obvious deformity / bending, and obvious limitation of movement; 4 points is severe swelling / rigidity with loss of function or stump (unable to stand / grasp).

[0119] Experimental results are as follows Figure 10 As shown, selenopeptide, tetrandrine, and selenopeptide nanomicelles can all inhibit joint swelling in CIA mice. Among them, selenopeptide nanomicelles have the best therapeutic effect, and their clinical score is significantly lower than that of tetrandrine, indicating that selenopeptide nanomicelles have the effect of inhibiting the condition of rheumatoid arthritis.

[0120] On the fourth day after the end of drug administration, i.e., 46 days after the first immunization, the experimental mice were euthanized. Blood was collected from mice in the saline group, selenium peptide group, tetrandrine group, and the selenium peptide nanomicelle group of Example 4-1. The levels of serum inflammatory factors (including TNF-α, IL-1β, IL-6, IL-17, and IFN-γ) and the anti-inflammatory factor IL-10 were measured using Luminex multifactor detection technology. The experimental results are as follows: Figure 11 As shown, compared with the PBS group, selenopeptide, tetrandrine, and selenopeptide nanomicelles can significantly reduce the level of inflammatory factors in the blood of CIA mice and increase the level of anti-inflammatory factor IL-10. Among them, the effect of selenopeptide nanomicelles is significantly higher than that of the other two groups, indicating that selenopeptide nanomicelles have a highly efficient anti-inflammatory ability.

[0121] Spleens from mice in the saline group, selenopeptide group, tetrandrine group, selenopeptide nanomicelle group (Example 4-1), and selenopeptide nanomicelle group (Example 4-2) were collected and prepared into single-cell suspensions. Cells were stained sequentially with PE / Cyanine7 anti-mouse CD4 antibody (100422, Biolegend), fixed and permeabilized, and then intracellularly stained with Alexa Fluor® 647 anti-mouse Foxp3 antibody (126408, Biolegend) to evaluate the percentage level of Treg cells. Results are as follows: Figure 12 As shown, selenopeptide, tetrandrine, and selenopeptide nanomicelles can all significantly increase the percentage of Treg cells in the spleen of CIA mice, with selenopeptide nanomicelles showing a particularly significant effect, indicating that selenopeptide nanomicelles have a highly efficient ability to resist autoimmune inflammation and promote immune tolerance remodeling.

[0122] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0123] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0124] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A selenopeptide that activates TNFR2, characterized in that, The TNFR2-activating selenopeptide comprises, in sequence, a tmTNF-targeting peptide, an oligoethylene glycol hydrophilic chain, a selenoamino acid linker, and an alkyl hydrophobic chain.

2. The selenopeptide according to claim 1, characterized in that, The tmTNF targeting peptide is a polypeptide that targets the transmembrane region of tmTMF, and its sequence includes FLILALFIYWWCLRDE.

3. The selenopeptide according to claim 1 or 2, characterized in that, The oligoethylene glycol hydrophilic chain is an oligoethylene glycol with amino-carboxyl groups at both ends; Preferably, the molecular structure of the oligoethylene glycol hydrophilic chain is NH2-(PEG)m-(CH2)n-COOH, where m is selected from any integer from 4 to 6, and n is selected from any integer from 1 to 4.

4. The selenopeptide according to any one of claims 1-3, characterized in that, The selenoamino acid linker is selenocysteine.

5. The selenopeptide according to any one of claims 1-4, characterized in that, The molar ratio of the tmTNF targeting peptide, oligoethylene glycol hydrophilic chain, selenoamino acid linker and alkyl hydrophobic chain is 1:1:1:1 or 1:1:2:

2. Preferably, the selenized amino acid linker is connected via lysine or directly to the oligoethylene glycol hydrophilic chain; Preferably, the alkyl hydrophobic chain is independently a straight-chain alkane with 6-18 carbons.

6. A selenium peptide nanomicelle, characterized in that, The selenopeptide nanomicelles are formed by self-assembly of selenopeptide and DMG-PEG as carrier materials according to any one of claims 1-5, and encapsulating tetrandrine.

7. The selenium peptide nanomicelles according to claim 6, characterized in that, The molecular weight of PEG in the DMG-PEG is 1000-5000; Preferably, the mass content of tetrandrine in the selenium peptide nanomicelles is 20-50%; Preferably, the hydrated particle size of the selenium peptide nanomicelles is 100-200 nm.

8. The use of the selenopeptide according to any one of claims 1-5 or the selenopeptide nanomicelles according to any one of claims 6-7 in the preparation of formulations that induce tmTNF to form aggregates on the surface of dendritic cell membranes.

9. The use of the selenopeptide according to any one of claims 1-5 or the selenopeptide nanomicelles according to any one of claims 6-7 in the preparation of TNF receptor 2 agonists or preparations that promote Treg cell proliferation.

10. The use of the selenopeptide according to any one of claims 1-5 or the selenopeptide nanomicelles according to any one of claims 6-7 in the preparation of a medicament for the prevention, relief or treatment of rheumatoid arthritis.

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