Polypeptide targeting phosphorylated PGAM1 and composition and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a targeted phosphorylated PGAM1 polypeptide and a composition and application thereof, and the amino acid sequence of the targeted phosphorylated PGAM1 polypeptide is as shown in SEQ ID NO.2 and / or SEQ ID NO.4. The positive correlation between the phosphorylation level of PGAM1 S23 and the activation degree of CD4 + T cells is disclosed for the first time, polypeptide drugs capable of specifically and competitively inhibiting phosphorylated PGAM1 and PGK1 are developed based on the positive correlation, over-activation of the CD4 + T cells can be effectively blocked, and a novel treatment strategy is provided for type 1 diabetes. In view of the universality of a CD4 + T cell metabolism mechanism, the technology can also be widely applied to treatment of multiple sclerosis, rheumatoid arthritis and other CD4 + T cell abnormal activation mediated autoimmune diseases, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a polypeptide targeting phosphorylated PGAM1, a composition thereof, and an application thereof. Background Art

[0002] The core pathological hallmark of autoimmune diseases is a chronic inflammatory response driven by abnormal CD4+ T cell activation. In type 1 diabetes (T1D), autoreactive CD4+ T cells recognize pancreatic β-cell antigens and secrete proinflammatory cytokines (such as IFN-γ and IL-17), leading to β-cell destruction and defective insulin secretion. Similarly, the same immune activation mechanism recurs in other autoimmune diseases mediated by excessive CD4+ T cell activation, such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease, suggesting that universal therapeutic strategies targeting CD4+ T cells have significant clinical value.

[0003] Current treatments for these diseases primarily include: 1. Immunosuppressive therapies (e.g., glucocorticoids, anti-CD3 monoclonal antibodies): While they can alleviate symptoms, they are associated with systemic side effects (e.g., increased risk of infection). 2. Antigen-specific immunomodulation (e.g., insulin peptides for T1D): These are only effective for specific conditions and have limited efficacy. 3. Metabolic interventions (e.g., rapamycin to inhibit mTOR): These lack targeted therapy and may interfere with normal immune function. Existing approaches fail to address the fundamental issue of metabolic dependence of CD4+ T cell overactivation, necessitating the development of novel targeted therapies.

[0004] Activated CD4+ T cells exhibit a significant metabolic reprogramming, becoming reliant on glycolysis rather than oxidative phosphorylation for energy and biosynthetic precursors. Studies have shown that inhibiting glycolysis selectively suppresses the differentiation of pro-inflammatory Th1 / Th17 cells while preserving regulatory T cell (Treg) function. Experiments conducted in this study confirmed that phosphorylation levels of PGAM1 S23 and PGK1 S203 are significantly elevated in activated CD4+ T cells. In the peripheral blood of patients with T1D, PGAM1 and PGK1 phosphorylation levels are also significantly elevated in autoreactive CD4+ T cells compared to healthy controls.

[0005] However, existing small-molecule glycolysis inhibitors (such as 2-deoxyglucose) are difficult to clinically apply due to their nonspecific targeting and toxicity. Peptide drugs, with their high specificity and favorable safety profile, have become an emerging tool for treating autoimmune diseases. However, existing peptide therapies primarily target antigen presentation or T cell receptor signaling, and there has been no research on regulating T cell metabolism through competitive inhibition of key glycolytic enzyme phosphorylation sites by peptides.

[0006] Therefore, the present invention has developed a peptide-based glycolysis inhibition strategy that selectively blocks CD4+ T cell metabolic reprogramming by targeting the PGAM1 S23 or / and PGK1 S203 phosphorylation sites. The peptide sequences are derived from the phosphorylation regulatory regions of PGAM1 (amino acids 18-28) and PGK1 (amino acids 198-208), respectively, and can be linked to a cell-penetrating peptide (such as TAT) to enhance delivery efficiency. Experimental studies have demonstrated that this strategy significantly inhibits CD4+ T cell activation and differentiation of pro-inflammatory subsets while preserving regulatory T cell function, providing a safer and more precise treatment option for autoimmune diseases. Summary of the Invention

[0007] To address the above issues, the present invention provides a therapeutic peptide and peptide composition that targets CD4+ T cell metabolic reprogramming, and their applications. These peptides and peptide compositions effectively block abnormal CD4+ T cell activation by specifically inhibiting phosphorylation at the S23 and / or S203 sites of key glycolytic enzymes, PGAM1. The therapeutic peptides provided by this invention offer the following significant advantages: 1) high targeting, specifically binding to PGAM1 / PGK1 phosphorylation sites; 2) excellent penetrance, enabling efficient intracellular delivery via TAT transmembrane peptides; 3) extremely low immunogenicity, minimizing adverse reactions; 4) broad applicability, enabling the treatment of a variety of CD4+ T cell-mediated autoimmune diseases; and 5) industrial advantages, with mature synthesis processes and manageable costs. This innovative therapy overcomes the limitations of existing treatments, combining efficacy and safety, and possesses significant clinical value and promising applications.

[0008] In order to achieve the above objectives, this application adopts the following technical solutions: In a first aspect, the present invention provides a polypeptide or a polypeptide composition, wherein the amino acid sequence of the polypeptide or the polypeptide composition is shown as SEQ ID NO.2 and / or SEQ ID NO.4.

[0009] In the above technical solution, the first amino acid of the polypeptide or polypeptide composition is connected to a cell-penetrating peptide to improve its delivery efficiency.

[0010] In the above technical solution, the amino acid sequence of the cell-penetrating peptide is shown as SEQ ID NO.7, and the amino acid sequence of the polypeptide or polypeptide composition after connection is shown as SEQ ID NO.5 and / or SEQ ID NO.6.

[0011] In a second aspect, the present invention provides the use of the above polypeptide or polypeptide combination in the preparation of a drug for treating autoimmune diseases.

[0012] In the above technical solution, the autoimmune disease is at least one of type 1 diabetes, inflammatory bowel disease, multiple sclerosis, and rheumatoid arthritis mediated by abnormal activation of CD4+ T cells.

[0013] In the above technical solution, the polypeptide or polypeptide combination achieves the treatment of autoimmune diseases by inhibiting effector CD4+ T cells.

[0014] The exogenous polypeptide sequence provided by the present invention is consistent with the phosphorylation target sequence of endogenous proteins PGAM and PGK1, and can be used as a "bait" to competitively bind to protein kinase, thereby reducing the interaction between endogenous proteins and kinases and inhibiting their phosphorylation.

[0015] In a third aspect, the present invention provides a drug for treating autoimmune diseases, comprising the above-mentioned polypeptide or polypeptide combination.

[0016] In the above technical solution, the drug also includes pharmaceutically acceptable excipients.

[0017] In the above technical solution, the auxiliary materials include fillers, diluents, adhesives, disintegrants, and emulsifiers.

[0018] The beneficial effects of the present invention are: (1) The polypeptide P1 of the present invention can inhibit phosphorylated PGAM1, and the polypeptide P2 can inhibit phosphorylated PGK1. Both can reduce cellular glycolysis levels and significantly inhibit effector CD4+ T cells when used alone or in combination. This group of polypeptides has been shown to significantly inhibit the activation of autoreactive CD4+ T cells in a type 1 diabetes model, delaying the onset of disease and reducing the incidence of disease.

[0019] (2) Compared with the full-length protein, the polypeptide of the present invention has a small molecular weight, is easy to process and synthesize, has weak immunogenicity, and has relatively weak side effects; and it has better absorption with cell-penetrating peptides, which provides a reference for the clinical treatment of autoimmune diseases.

[0020] (3) Based on the universality of CD4+ T cell metabolic mechanisms, this strategy can be extended to other autoimmune diseases mediated by abnormal CD4+ T cell activation (such as multiple sclerosis, rheumatoid arthritis, etc.).

[0021] The embodiments of the present invention will be further described through specific examples, but the protection scope is not limited to these examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Expression of PGAM1 S23 and PGK1 S203 phosphorylated proteins in CD4+ T cells.

[0023] Figure 2: Synthesis of cell-penetrating peptides, polypeptides P1 and P2.

[0024] Figure 3 : Mass spectrometry and high performance liquid chromatography detection results of cell-penetrating peptides. Figure 3 A is the mass spectrometry detection result of phosphorylated peptides. Figure 3 B is the high performance liquid chromatography detection result of the cell-penetrating peptide.

[0025] Figure 4 : Mass spectrometry and high performance liquid chromatography detection results of polypeptide P1. Figure 4 A is the mass spectrometry detection result of peptide P1. Figure 4 B is the high performance liquid chromatography detection result of polypeptide P1.

[0026] Figure 5 : Mass spectrometry and high performance liquid chromatography detection results of polypeptide P2. Figure 5 A is the mass spectrometry detection result of peptide P2. Figure 5 B is the high performance liquid chromatography detection result of polypeptide P2.

[0027] Figure 6 : Targeted inhibitory effects of polypeptides P1 and P2 on the expression of phosphorylated PGAM1 and PGK1, respectively.

[0028] Figure 7 : Inhibitory effect of peptides on glycolysis in CD4+ T cells.

[0029] Figure 8 : The inhibitory effect of peptide combination on effector CD4+ T cells.

[0030] Figure 9 : The peptide composition reduces the incidence and improves the condition of type 1 diabetic (NOD) mice. DETAILED DESCRIPTION

[0031] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the present invention to those skilled in the art. The present invention will be limited only by the claims.

[0032] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0033] The present invention provides a group of polypeptides or polypeptide compositions capable of targeted inhibition of glycolysis, comprising polypeptide 1 and / or polypeptide 2. Polypeptide 1 comprises at least amino acid residues 18-28 derived from the N-terminus of the PGAM1 protein. Polypeptide 2 comprises at least amino acid residues 198-208 derived from the N-terminus of the PGK1 protein.

[0034] PGAM1 protein amino acid sequence SEQ ID NO.1: MAAYKLVLIRHGESAWNLENRFSGWYDADLSPAGHEEAKRGGQALRDAGYEFDICFTSVQKRAIRTLWTVLDAIDQMWLPVVRTWRLNERHYGGLTGLNKAETAAKHGEAQVKIWRRSYDVPPPPME PDHPFYSNISKDRRYADLTEDQLPSCESLKDTIARALPFWNEEIVPQIKEGKRVLIAAHGNSLRGIVKHLEGLSEEAIMELNLPTGIPIVYELDKNLKPIKPMQFLGDEETVRKAMEAVAAQGKVKK.

[0035] The amino acid sequence of polypeptide 1 is SEQ ID NO. 2: LENRFSGWYDA.

[0036] PGK1 protein amino acid sequence SEQ ID NO.3: MSLSNKLTLDKLDVKGKRVVMRVDFNVPMKNNQITNNQRIKAAVPSIKFCLDNGAKSVVLMSHLGRPDGVPMPDKYSLEPVAAELKSLLGKDVLFLKDCVGPEV ENACANPAAGTVILLENLRFHVEEEGKGKDASGNKVKAEPAKIDAFRASLSKLGDVYVNDAFGTAHRAHSSMVGVNLPQKAGGFLMKKELNYFAKALESPERPFL AILGGAKVADKIQLINNMLDKVNEMIIGGGMAFTFLKVLNNMEIGTSLYDEEGAKIVKDLMSKAEKNGVKITLPVDFVTADKFDENAKTGQATVASGIPAGWMG LDCGTESSKKYAEAVGRAKQIVWNGPVGVFEWEAFARGTKSLMDEVVKATSRGCITIIGGGDTATCCAKWNTEDKVSHVSTGGGASLELLEGKVLPGVDALSNV.

[0037] The amino acid sequence of polypeptide 1 is SEQ ID NO. 4: AKALESPERPF.

[0038] Preferably, a cell-penetrating peptide sequence is connected before the first amino acid of polypeptides 1 and 2 to synthesize polypeptide P1 (sequence shown in SEQ ID NO. 5) and polypeptide P2 (sequence shown in SEQ ID NO. 6).

[0039] SEQ ID NO.5:YGRKKRRQRRRALENRFSGWYDA SEQ ID NO.6: YGRKKRRQRRRAAKALESPERPF.

[0040] Cell-penetrating peptides are short peptides capable of carrying macromolecules into cells. They include naturally occurring and synthetic cell-penetrating peptides. The most commonly used cell-penetrating peptide is derived from amino acid residues 47-57 (YGRKKRRQRRR) of the human immunodeficiency virus (HIV)-1. In a specific embodiment of the present invention, to enhance binding of the cell-penetrating sequence to polypeptides 1 and 2, an alanine (A) was added to the C-terminus of the YGRKKRRQRRR sequence, resulting in the cell-penetrating peptide sequence YGRKKRRQRRRA (SEQ ID NO. 7).

[0041] The present invention also provides the use of the polypeptide or polypeptide combination in the preparation of drugs for treating autoimmune diseases.

[0042] Preferably, the autoimmune disease is at least one of type 1 diabetes, inflammatory bowel disease, multiple sclerosis, and rheumatoid arthritis mediated by abnormal activation of CD4+ T cells.

[0043] That is, the peptides, alone or in combination, can inhibit effector CD4+ T cells. Effector CD4+ T cells are the main pathogenic cells in autoimmune diseases, mainly including Th1 cells and Th17 cells.

[0044] The present invention also provides a medicine for treating autoimmune diseases, which contains at least one or two of the above polypeptides.

[0045] Preferably, the drug further comprises pharmaceutically acceptable excipients, including fillers, diluents, binders, disintegrants, and emulsifiers.

[0046] In a specific embodiment of the present invention, the polypeptides are all polypeptides linked to a cell-penetrating peptide. Preferably, they are synthesized by Sangon Biotech (Shanghai) Co., Ltd., and are positively identified by MS and have a purity greater than 95% as determined by high-performance liquid chromatography (HPLC).

[0047] Example 1: Expression of PGAM1 S23 and PGK1 S203 Phosphorylated Proteins in CD4+ T Cells 1.1. Detection of changes in PGAM1 and PGK1 phosphorylation proteins before and after activation of mouse naive CD4+ T cells (1) Eight-week-old C57BL / 6 wild-type mice were killed by cervical dislocation and immersed in 75% alcohol for 5 minutes. The spleens were removed.

[0048] (2) Gently grind the spleen using the end of a 1 mL syringe. While grinding, add 1640 basal medium to rinse the cells through a 70 μm filter and collect the spleen cell suspension in a 50 mL centrifuge tube.

[0049] (3) Centrifuge at 300 g for 5 minutes to allow spleen cells to settle at the bottom of the tube.

[0050] (4) Discard the supernatant, add 2 mL of red blood cell lysis buffer, gently pipette to mix the cells, and lyse on ice for 5 minutes.

[0051] (5) Add approximately 8 mL of D-PBS to terminate lysis.

[0052] (6) Centrifuge at 400 g for 5 minutes at room temperature.

[0053] (7) The cells settle at the bottom of the tube and the supernatant is discarded.

[0054] (8) Using mouse initial CD4 + T cell sorting kit (BioLegend, 480040) was used for subsequent sorting: a) Every 1×10 7 Add 100 μL of D-PBS to each cell and resuspend the cells.

[0055] b) Add 10 μL of mixed antibodies, mix well, and incubate on ice for 15 minutes.

[0056] c) Add an equal amount of streptavidin nanoparticles to the antibody and continue incubating on ice for 15 minutes.

[0057] d) Add D-PBS to expand the volume of the cell suspension to approximately 3.5 mL and transfer to a sterile flow cytometry tube.

[0058] e) Place the flow cytometry tube containing the cell suspension in a magnetic adsorption rack and let it sit for 1 minute.

[0059] f) Tilt the magnetic stand and flow cytometry tube as a whole and transfer the cell suspension that has not bound to the antibody and magnetic beads to a sterile 15 mL centrifuge tube.

[0060] g) Centrifuge at 300 g for 5 minutes. The pellet at the bottom of the tube is the initial CD4+ T cells obtained by sorting.

[0061] (9) The initial CD4+ T cells were divided into two parts. One part was directly used to extract protein; the other part was plated with 200 μL / well of cell suspension in culture wells coated with anti-mouse CD3 / CD28 antibodies. After 48 hours of culture, the cells were collected and their total protein was extracted.

[0062] (10) Protein expression in the two groups of cells was analyzed by western blotting.

[0063] 1.2 Analysis of PGAM1 and PGK1 phosphorylation protein expression levels in CD4+ T cells of healthy controls and T1D patients (1) 3–5 mL of venous blood was collected from T1D patients and healthy volunteers using heparin anticoagulant tubes.

[0064] (2) Take an amount of lymphocyte separation fluid equal to the volume of blood and transfer it to a centrifuge tube.

[0065] (3) Tilt the centrifuge tube 45° and slowly spread the blood cells on top of the lymphocyte fluid.

[0066] (4) Centrifuge the gradient at 600 g (speed 3, speed 1) for 25 min.

[0067] (5) After centrifugation, the liquid is divided into 4 layers. Carefully aspirate the cells in the white mist layer.

[0068] (6) Add 10 mL of PBS to wash the cells.

[0069] (7) Centrifuge at 300 g for 5 minutes. The cell pellet at the bottom of the tube is peripheral blood mononuclear cells (PBMCs).

[0070] (8) Add D-PBS and wash twice.

[0071] (9) Resuspend the cells in D-PBS and adjust the cell density to approximately 1×10 8 / mL. Using human CD4 + T cell isolation kit was used for subsequent isolation.

[0072] a) Every 1×10 7 Add 10 μL of antibody to each cell, mix well, and incubate on ice for 15 minutes.

[0073] b) Secure the LS column to the magnetic adsorption stand and rinse the column with 2 mL of D-PBS.

[0074] c) Add 1 mL of D-PBS to dilute the cell-antibody mixture and transfer to the LS column, allowing it to flow out naturally.

[0075] d) After all the cell suspension has entered the column, add 1.5-2 mL of D-PBS to rinse.

[0076] e) After the last drop of liquid flows out naturally, remove the LS column and place it in a 15 mL centrifuge tube.

[0077] f) Add 2 mL of 1640 basal medium to the LS column and use a pusher to quickly flush out the cells in the column. These cells are CD4+ T cells.

[0078] (10) Total protein was extracted from the cells and analyzed by immunoblotting.

[0079] 3. Experimental results Figure 1 A: The expression of PGAM1 S23 and PGK1 S203 phosphorylated proteins increased significantly after activation and stimulation of α-CD3 / CD28 in naive CD4+ T cells.

[0080] Figure 1 B: The phosphorylated protein levels of PGAM1 S23 and PGK1 S203 in CD4+ T cells of T1D patients were significantly higher than those in healthy controls.

[0081] Example 2: Synthesis of cell-penetrating peptides, polypeptides P1 and P2 The base sequences encoding the cell-penetrating peptide (YGRKKRRQRRRA), polypeptide P1 (YGRKKRRQRRRALENRFSGWYDA), and polypeptide P2 (YGRKKRRQRRRAAKALESPERPF) were designed. A variant of alanine (A) was added to the carboxyl terminus of amino acid residues 47-57 (YGRKKRRQRRR) of human immunodeficiency virus (HIV)-1 to form the cell-penetrating peptide sequence (YGRKKRRQRRRA). The sequences of polypeptides P1 and P2 were derived from the phosphorylation regulatory regions of PGAM1 (amino acids 18-28) and PGK1 (amino acids 198-208), respectively. The amino termini were linked to cell-penetrating peptides to form cell-penetrating peptide-polypeptide fusion sequences (YGRKKRRQRRRALENRFSGWYDA and YGRKKRRQRRRAAKALESPERPF). Figure 2 shown.

[0082] 2.1 Resin pretreatment (1) Swelling resin: a) Weigh 1.0 g of carboxyl resin (substitution value: 0.5 mmol / g) and place it in a synthesis column.

[0083] b) Add 10 mL of DMF and stir at room temperature for 30 minutes to allow the resin to fully swell.

[0084] c) Remove DMF by filtration and wash with fresh DMF three times (10 mL each time).

[0085] (2) The first amino acid is fixed: a) Dissolve the first amino acid at the C-terminus (carboxyl terminus) of the selected peptide in 5 mL of DMF. Add HOBt (3.0 eq) and DIC (3.0 eq) and activate for 5 minutes.

[0086] b) Add the activation solution to the resin and allow to react by nitrogen bubbling for 2 hours (at room temperature).

[0087] c) Filter and wash the resin with DMF (3 × 10 mL) and DCM (3 × 10 mL) in sequence.

[0088] d) Confirm the coupling is complete using the ninhydrin assay (a colorless resin bead indicates a complete reaction).

[0089] 2.2. Polypeptide chain extension (Fmoc cycle) (1) Fmoc deprotection: a) Add 20% piperidine / DMF solution (10 mL), bubble nitrogen through the mixture for 5 minutes, and filter.

[0090] b) Repeat once to ensure complete removal of the Fmoc group.

[0091] c) Wash with DMF six times (10 mL each time) to remove residual piperidine.

[0092] (2) Amino acid activation and coupling: a) Dissolve Fmoc-AA-OH (4.0 eq), HOBt (4.0 eq), and DIC (4.0 eq) in 5 mL DMF and activate for 5 minutes.

[0093] b) Add to the resin and bubble nitrogen through for 1 hour (standard coupling) or up to 2 hours (difficult sequences).

[0094] c) Filter and wash with DMF three times.

[0095] (3) Coupling monitoring: Take a small amount of resin and perform ninhydrin test: If the resin beads turn blue (free amino groups are present), repeat the coupling step.

[0096] 2.3 Special sequence processing (1) Arginine-rich regions: For consecutive Arg residues (e.g., RRRR), activation was performed using PyBOP (4.0 eq) / DIPEA (8.0 eq) at 50°C for 1 hour. After coupling, the residue was detected and a third coupling was performed if necessary.

[0097] (2) Aggregation-prone sequences: Add 5% DMSO / DMF to improve solubility.

[0098] 2.4. Peptide cleavage and deprotection (1) Preparation of lysis reagent: TFA / water / TIS / EDT = 94:2.5:2.5:1 (v / v / v / v), pre-cool in an ice bath.

[0099] (2) Cracking step: a) Transfer the dried resin to a 50 mL centrifuge tube and add 10 mL of lysis buffer.

[0100] b) Stir at room temperature for 2.5 hours (nitrogen protection).

[0101] c) Filter, wash the resin with a small amount of TFA (2 × 2 mL), and combine the filtrates.

[0102] (3) Peptide precipitation: a) Add the filtrate dropwise to 40 mL of cold ether (-20°C) and place in an ice bath for 30 minutes.

[0103] b) Centrifuge (4000 rpm, 10 minutes, 4°C) and discard the supernatant.

[0104] c) The precipitate was washed three times with cold ether and dried under vacuum for 24 hours.

[0105] 2.5. Peptide purification and identification The quality of the synthesized peptide was detected by HPLC and mass spectrometry (MS). Figure 3 )、Polypeptide P1( Figure 4 ) and peptide P2 ( Figure 5 ) have correct amino acid sequences and their purities are greater than 95%.

[0106] Example 3: Peptides P1 and P2 target and inhibit the expression of phosphorylated PGAM1 and PGK1 respectively According to step 1 of Example 1, mouse spleen naive CD4+ T cells were isolated and inoculated into culture plates pre-coated with anti-CD3 and anti-CD28 antibodies. Penetrating peptide (as a negative control group, NC), peptide P1, and peptide P2 were added, respectively, and cultured for 48 hours. The treated cells were collected, and proteins were extracted for immunoblotting analysis. Figure 6 As shown in Figures A and 6B, compared to the control group, the amount of phosphorylated PGAM1 protein was significantly reduced in the peptide P1-treated group, and the amount of phosphorylated PGK1 protein was significantly reduced in the peptide P2-treated group. These results indicate that the peptides can competitively inhibit the activation of the key glycolytic enzymes PGAM1 and PGK1.

[0107] Example 4: Peptides P1 and P2 reduce CD4+ T cell glycolysis 4.1 Experimental Methods (1) Isolate mouse spleen naive CD4+ T cells according to step 1 of Example 1.

[0108] (2) Cells were seeded into culture plates pre-coated with anti-CD3 and anti-CD28 antibodies.

[0109] (3) 5 μM of the membrane-penetrating peptide (as a negative control group NC), peptide P1, and peptide P2 were added to the culture medium and cultured for 48 hours.

[0110] (4) The glycolytic rate of the treated cells was detected using the Seahorse XF Glycolytic Rate Assay Kit (Agilent, 103344-100) according to the manufacturer's instructions.

[0111] a) Hydrate the probe cartridge with calibration solution at 37°C in the absence of CO2 24 hours in advance.

[0112] b) Prepare glycolysis stress test medium (GPS medium): Take XF basal medium, add 2 mM glutamine and 10 mM glucose, adjust the pH to 7.4, and preheat at 37°C.

[0113] c) Prepare 10 μM rotenone / antimycin A (Rot / AA) and 500 mM 2-DG in GPS medium. Add Rot / AA and 2-DG to the corresponding wells of the probe plate to a final concentration of 1 μM and 50 mM, respectively. Add the corresponding amount of GPS medium to the remaining two wells.

[0114] d) Transfer the treated T cells to a Seahorse cell plate, with A1, B4, C3, and D6 serving as blank controls with no cells added.

[0115] e) After centrifugation, remove the culture medium from the cell culture plate and add 1 mL of pre-warmed GPS medium to each well. Gently wash twice.

[0116] f) Finally, add 500 μL of GPS medium to each well and incubate at 37°C in a CO2-free incubator for 1 hour.

[0117] g) The test program is set up as follows 4.2 Experimental Results Figure 7 A: Compared with the control, the extracellular acidification rate (ECAR) of CD4+ T cells was inhibited after treatment with peptides P1 and P2.

[0118] Figure 7 B: After treatment with peptides P1 and P2, the proton efflux rate (PER) of CD4+ T cells decreased significantly.

[0119] Figure 7 C: After treatment with peptides P1 and P2, the basal glycolysis level of CD4+ T cells was significantly reduced.

[0120] Figure 7 D: After treatment with peptides P1 and P2, the basal proton efflux rate of CD4+ T cells decreased.

[0121] Figure 7 E: After treatment with peptides P1 and P2, the compensatory glycolysis rate of CD4+ T cells decreased.

[0122] Example 5: Peptides and their compositions inhibit CD4+ T cell activation, proliferation, and Th1 / Th17 subset differentiation Naive CD4+ T cells were isolated from mouse spleens according to step 1 of Example 1. The cells were seeded into culture plates pre-coated with anti-CD3 and anti-CD28 antibodies. A membrane-penetrating peptide and a peptide combination (P1 and P2, mixed at a ratio of 1:1, i.e., 2.5 μM each) were added to the culture medium.

[0123] 5.1. Flow cytometry detection of CD4+ T cell activation (1) After 16 hours of culture, collect the cells into a centrifuge tube and wash twice.

[0124] (2) Resuspend the cells in flow cytometry buffer and adjust the cell density to 1×10 6 / mL, and 100 μL of cells were transferred to a flow cytometry tube for staining.

[0125] (3) Dilute the antibodies using flow cytometry buffer: Add CD69 and CD44 antibodies at 1 μL / tube of antibody stock solution.

[0126] (4) Add 100 μL of diluted antibody to each staining tube and stain on ice for 20 minutes in the dark.

[0127] (5) Add 3 volumes of flow cytometry buffer to terminate staining, centrifuge at 300 g for 5 minutes, and discard the supernatant.

[0128] (6) Add 200 μL of cell staining fixative and test on the instrument.

[0129] 5.2. Cell Proliferation Detected by Live Tracking Dye (CellTrace™ Violet, CTV) Staining (1) Before cell inoculation, place the initial CD4+ T cells used in this experiment in a 15 mL centrifuge tube and add CTV dye (total concentration of 5 μM).

[0130] (2) Incubate in a 37°C water bath for 10 minutes.

[0131] (3) Add 3 volumes of complete medium to terminate staining, centrifuge at 300 g for 5 minutes, and discard the supernatant.

[0132] (4) The cells were seeded on culture plates coated with anti-CD3 antibodies and anti-CD28 antibodies, and treated with the cell-penetrating peptide and peptide combination, respectively.

[0133] (5) After 3 days of culture, the cells were collected into flow cytometry tubes, washed twice, and tested on the flow cytometer.

[0134] 5.3. Flow cytometry of Ki67 staining to reflect cell proliferation (1) After 48 hours of treatment with the cell-penetrating peptide and peptide combination, the cells were collected into a flow cytometry tube and washed twice with PBS.

[0135] (2) Centrifuge at 300 g for 5 minutes and discard the supernatant.

[0136] (3) Use the TF diluent in the transcription factor buffer kit to dilute the 4× Fix / Perm buffer to the required volume of 1× Fix / Perm working solution.

[0137] (4) Add 150 μL of 1× Fix / Perm working solution to each tube and fix at 4°C for 60 minutes.

[0138] (5) Dilute 5× Perm / Wash buffer to 1× Perm / Wash working solution using ddH2O.

[0139] (6) After fixation, add 1 mL of Perm / Wash working solution to each tube and centrifuge at 500 g for 5 minutes.

[0140] (7) Discard the supernatant and add 100 µL of Perm / Wash working solution to each tube to resuspend the cells.

[0141] (8) No antibody was added to the blank tubes and single-labeled surface marker tubes. 1 µL of Ki67 antibody was added to each single-labeled and stained tube.

[0142] (9) Shake to mix and incubate at 4°C in the dark for 1 hour.

[0143] (10) Add 1 mL of flow cytometry buffer to terminate staining and centrifuge at 500 g for 5 minutes.

[0144] (11) Tilt the flow tube, discard the supernatant, and gently wipe the remaining liquid at the tube mouth with absorbent paper.

[0145] (12) Add 200 μL of staining fixative to each flow cytometer tube and test on the flow cytometer.

[0146] 5.4 Effects of Peptide Combinations on CD4+ T Cell Subpopulation Differentiation Initial CD4+ T cells were inoculated into culture wells coated with anti-mouse CD3 / CD28 antibodies. Appropriate cytokines were added to the culture medium to induce differentiation into the corresponding T cell subsets: (1) Th1 cells, add IL-2 (10 ng / mL) and IL-12 (10 ng / mL).

[0147] (2) Th17 cells, IL-6 (10 ng / mL) and TGF-β (5 ng / mL) were added.

[0148] (3) Treg cells, add IL-2 (10 ng / mL) and TGF-β (10 ng / mL).

[0149] Under each differentiation condition, the cells were treated with a cell-penetrating peptide and a peptide combination, respectively. After 3-5 days of culture, the cells were harvested and subjected to flow cytometry.

[0150] 5.5 Experimental Results Figure 8 A: The peptide composition significantly reduced the expression of CD69 and CD44 on CD4+ T cells, indicating that the peptide composition inhibited CD4+ T cell activation.

[0151] Figure 8 B, C: The fluorescence decay of CTV markers in the peptide combination-treated group was slower than that in the control group, and the expression of Ki67 was significantly reduced. This result indicates that the peptide combination can inhibit the proliferation of CD4+ T cells.

[0152] Figure 8 D: Compared with the control, Treg differentiation of cells in the polypeptide-treated group was significantly enhanced.

[0153] Figure 8 E, F: Peptides can inhibit the differentiation of Th1 and Th17 cells.

[0154] Example 6: Peptide composition can reduce the incidence and improve the condition of type 1 diabetic (NOD) mice Thirty six-week-old wild-type spontaneously diabetic NOD mice were divided into two groups: a control group and an experimental group, with 15 mice in each group. Both the control and experimental groups were given intraperitoneal injections (1 mg / mL) of a cell-penetrating peptide and peptide combination (P1 and P2, mixed at a ratio of 1:1) twice weekly for two consecutive weeks. Blood glucose levels were measured twice weekly at the tail tip using a glucometer. Onset was defined as a blood glucose concentration ≥13.8 mmol / L on two consecutive occasions. The time of onset was recorded, and the incidence rate of the two groups was calculated. The experiment was terminated when the incidence rate reached 80% in either group or after 32 weeks of monitoring. T1D-related markers were compared between the two groups using HE, pancreatic immunofluorescence, ELISA, and flow cytometry.

[0155] Figure 9 A: Statistical graph of the incidence of two groups of NOD mice.

[0156] Figure 9 B: ELISA was used to detect the levels of C-peptide and insulin in mouse serum.

[0157] Figure 9 C: HE staining of pancreas and statistical diagram of inflammation grade.

[0158] Figure 9 D: Insulin immunofluorescence image of pancreatic islets.

[0159] Figure 9 EJ: The proportion (E), activation status (F), proliferation (G) and subset distribution (HJ) of CD4+ T cells in the pancreatic draining lymph nodes of mice.

[0160] The results showed that the onset of disease in the peptide composition-treated group of mice was delayed and the incidence rate was reduced ( Figure 9 A). The serum insulin and C-peptide levels in this group of mice were also significantly higher than those in the control group ( Figure 9 B). Figure 9 As shown in C, the pancreatic islet inflammation in the mice treated with the peptide composition was milder than that in the control group. Immunofluorescence results showed that the pancreatic islets of mice treated with the peptide composition produced more insulin ( Figure 9 D). Flow cytometry results showed that the proportion of CD4+ T cells in the peptide-treated mice decreased, and their proliferation and activation were significantly lower than those in the control group ( Figure 9 EG). In addition, the number of Treg cells in the peptide-treated mice increased significantly, the number of Th1 cells decreased significantly, and the proportion of Th17 cells also showed a downward trend ( Figure 9 HJ).

[0161] The above results show that the polypeptide combination can inhibit the activation of CD4+ T cells, reduce the proportion of effector CD4+ T cells, alleviate pancreatic β-cell damage, improve β-cell function, and thus reduce the incidence of T1D and delay disease progression.

[0162] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A polypeptide or polypeptide composition, characterized in that: The amino acid sequence of the polypeptide or polypeptide composition is shown as SEQ ID NO.2 and / or SEQ ID NO.

4.

2. The polypeptide or polypeptide composition according to claim 1, characterized in that: The first amino acid of the polypeptide or polypeptide composition is connected to a cell-penetrating peptide amino acid sequence.

3. The polypeptide or polypeptide composition according to claim 2, characterized in that: The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO.7, and the amino acid sequence of the polypeptide or polypeptide combination after connection is shown in SEQ ID NO.5 and / or SEQ ID NO.

6.

4. Use of the polypeptide or polypeptide combination according to any one of claims 1 to 3 in the preparation of a medicament for treating autoimmune diseases.

5. The application according to claim 4, characterized in that: The autoimmune disease is at least one of type 1 diabetes, inflammatory bowel disease, multiple sclerosis, and rheumatoid arthritis mediated by abnormal activation of CD4+ T cells.

6. The application according to claim 4, characterized in that: The polypeptide or polypeptide combination achieves the treatment of autoimmune diseases by inhibiting effector CD4+ T cells.

7. A drug for treating autoimmune diseases, characterized in that: The drug comprises the polypeptide or polypeptide composition according to any one of claims 1 to 3.

8. The drug according to claim 7, characterized in that: The drug also includes pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that: The auxiliary materials include fillers, diluents, binders, disintegrants and emulsifiers.

Images