Polypeptides targeting phosphorylated pgam1 and compositions and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a polypeptide targeting phosphorylated PGAM1 and a composition and application thereof, and an amino acid sequence thereof is shown as SEQ ID NO. 5 and / or SEQ ID NO. 6. The application discloses, for the first time, a positive correlation between a PGAM1 S23 phosphorylation level and a CD4+ T cell activation degree, and develops a polypeptide drug capable of specifically and competitively inhibiting phosphorylated PGAM1 and PGK1, which can effectively block CD4+ T cell overactivation, and provides a new treatment strategy for type 1 diabetes. In view of the universality of the CD4+ T cell metabolic mechanism, the technology can also be applied to the treatment of multiple sclerosis, rheumatoid arthritis and other autoimmune diseases mediated by abnormal activation of CD4+ T cells, and has a wide clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a polypeptide targeting phosphorylation of PGAM1 and a composition and application thereof. BACKGROUND

[0002] The core pathological feature of autoimmune diseases is chronic inflammatory response driven by abnormal activation of CD4+ T cells. In Type 1 diabetes (T1D), autoreactive CD4+ T cells recognize pancreatic beta cell antigens and secrete pro-inflammatory cytokines (such as IFN-γ, IL-17), leading to beta cell destruction and insulin secretion deficiency. Similarly, in other autoimmune diseases mediated by over-activation of CD4+ T cells (such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, etc.), the same immune activation mechanism repeatedly occurs, indicating that a universal treatment strategy targeting CD4+ T cells has important clinical value.

[0003] Current treatment methods for such diseases mainly include: 1. Immunosuppressive therapy (such as glucocorticoids, anti-CD3 mAb): although it can alleviate symptoms, it has systemic side effects (such as increased risk of infection). 2. Antigen-specific immunomodulation (such as insulin peptide treatment of T1D): only effective for specific diseases, and the efficacy is limited. 3. Metabolic intervention (such as rapamycin inhibition of mTOR): lack of targeting, which may interfere with normal immune function. The existing methods do not solve the fundamental problem of "metabolic dependence of CD4+ T cell over-activation", and it is urgent to develop new targeted therapies.

[0004] Activated CD4+ T cells exhibit significant metabolic reprogramming, which depends on glycolysis rather than oxidative phosphorylation to provide energy and biosynthetic precursors. Studies have shown that inhibition of glycolysis can selectively inhibit the differentiation of pro-inflammatory Th1 / Th17 cells, while retaining the function of regulatory T cells (Treg). The present application proves by experiments that in activated CD4+ T cells, the phosphorylation levels of PGAM1 S23 and PGK1 S203 are significantly increased. In the peripheral blood of T1D patients, the phosphorylation levels of PGAM1 and PGK1 in autoreactive CD4+ T cells are also significantly higher than those in healthy controls.

[0005] However, existing small molecule glycolysis inhibitors (such as 2-deoxyglucose) are difficult to be clinically applied due to non-specific targeting and toxicity problems. Polypeptide drugs have become a new tool for the treatment of autoimmune diseases due to their high specificity and good safety. However, existing polypeptide therapies mainly target antigen presentation or T cell receptor signals, and there is no research on regulating T cell metabolism by polypeptides to competitively inhibit the phosphorylation sites of key enzymes of glycolysis.

[0006] Therefore, the application develops a glycolysis inhibition strategy based on polypeptides, which selectively blocks the metabolic reprogramming of CD4+ T cells by targeting the PGAM1 S23 or / and PGK1 S203 phosphorylation site. The sequence of the polypeptide is derived from the phosphorylation regulation region of PGAM1 (amino acids 18-28) and PGK1 (amino acids 198-208) respectively, and can be connected with a cell-penetrating peptide (such as TAT) to enhance the delivery efficiency. Experiments have proved that this strategy can significantly inhibit the activation of CD4+ T cells and the differentiation of pro-inflammatory subsets, while retaining the function of regulatory T cells, providing a safer and more precise treatment for autoimmune diseases. SUMMARY

[0007] To solve the above problems, the application provides a therapeutic polypeptide and polypeptide composition targeting metabolic reprogramming of CD4+ T cells and applications thereof. The polypeptide and polypeptide composition effectively block the abnormal activation of CD4+ T cells by specifically inhibiting the phosphorylation modification of the PGAM1 S23 and / or PGK1 S203 sites of the key glycolytic enzyme. The therapeutic polypeptide provided by the application has the following outstanding advantages: 1) high targeting specificity, specifically binds to the PGAM1 / PGK1 phosphorylation site; 2) excellent penetration, achieves efficient intracellular delivery through TAT cell-penetrating peptides; 3) extremely low immunogenicity, avoiding adverse reactions; 4) wide applicability, can treat various CD4+ T cell-mediated autoimmune diseases; 5) industrialization advantage, mature synthesis process, controllable cost. This innovative therapy breaks through the existing treatment limitations, combining efficacy and safety, and has significant clinical value and application prospect.

[0008] To achieve the above-mentioned purpose, the application adopts the following technical solutions:

[0009] In a first aspect, the application provides a polypeptide or polypeptide composition, wherein the amino acid sequence of the polypeptide or polypeptide composition is shown in SEQ ID NO. 2 and / or SEQ ID NO. 4.

[0010] In the above technical solution, a cell-penetrating peptide is connected before the first amino acid of the polypeptide or polypeptide composition to improve its delivery efficiency.

[0011] In the above technical solution, 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 composition after connection is shown in SEQ ID NO. 5 and / or SEQ ID NO. 6.

[0012] In a second aspect, the application provides the use of the above-mentioned polypeptide or polypeptide composition in the preparation of a drug for treating autoimmune diseases.

[0013] In the above technical solution, the autoimmune disease is at least one of type 1 diabetes, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis mediated by abnormal activation of CD4+ T cells.

[0014] In the above technical solution, the polypeptide or polypeptide composition achieves the treatment of autoimmune diseases by inhibiting effector CD4+ T cells.

[0015] The exogenous polypeptide sequence provided by the present application is consistent with the phosphorylation target sequence of the endogenous protein PGAM and PGK1, can be used as a "decoy" to competitively bind protein kinases, thereby reducing the interaction of endogenous proteins with kinases and inhibiting their phosphorylation.

[0016] In a third aspect, the present application provides a medicine for treating autoimmune diseases, which comprises the above-mentioned polypeptide or polypeptide composition.

[0017] In the above technical solution, the medicine further comprises a pharmaceutically acceptable excipient.

[0018] In the above technical solution, the excipient comprises a filler, a diluent, a binder, a disintegrant, an emulsifier.

[0019] The present application has the following beneficial effects:

[0020] (1) The polypeptide P1 of the present application can inhibit phosphorylated PGAM1, and the polypeptide P2 can inhibit phosphorylated PGK1, both of which alone or in combination can reduce the glycolysis level of cells and significantly inhibit effector CD4+ T cells. The polypeptide group was verified in a type 1 diabetes model to significantly inhibit the activation of autoreactive CD4+ T cells, delay the onset time and reduce the incidence.

[0021] (2) Compared with the full-length protein, the polypeptide of the present application has small molecular weight, is easy to process and synthesize, has weak immunogenicity, and has relatively weak side effects; and has a cell membrane penetrating peptide for better absorption, providing a reference for the clinical treatment of autoimmune diseases.

[0022] (3) Based on the universality of the CD4+ T cell metabolism mechanism, this strategy can be expanded to other autoimmune diseases mediated by abnormal activation of CD4+ T cells (such as multiple sclerosis, rheumatoid arthritis, etc.).

[0023] The embodiments of the present application will be further illustrated by specific examples, but the scope of protection is not limited to these examples. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 : Expression of PGAM1 S23 and PGK1 S203 phosphorylated proteins in CD4+ T cells.

[0025] Figure 2 Synthesis of cell-penetrating peptide, polypeptides P1 and P2.

[0026] Figure 3 Mass spectrometry and high performance liquid chromatography detection results of cell-penetrating peptide. Figure 3 A is the mass spectrometry detection results of phosphorylated polypeptide, Figure 3 B is the high performance liquid chromatography detection results of cell-penetrating peptide.

[0027] Figure 4 Mass spectrometry and high performance liquid chromatography detection results of polypeptide P1. Figure 4 A is the mass spectrometry detection results of polypeptide P1, Figure 4 B is the high performance liquid chromatography detection results of polypeptide P1.

[0028] Figure 5 Mass spectrometry and high performance liquid chromatography detection results of polypeptide P2. Figure 5 A is the mass spectrometry detection results of polypeptide P2, Figure 5 B is the high performance liquid chromatography detection results of polypeptide P2.

[0029] Figure 6 Targeted inhibitory effect of polypeptides P1 and P2 on phosphorylated PGAM1 and PGK1 expression, respectively.

[0030] Figure 7 Inhibitory effect of polypeptides on CD4+ T cell glycolysis.

[0031] Figure 8 Inhibitory effect of polypeptide composition on effector CD4+ T cells.

[0032] Figure 9 Polypeptide composition reduces the incidence of type 1 diabetes (NOD) mice and improves their condition. DETAILED DESCRIPTION

[0033] In order to better illustrate the purposes, technical solutions and advantages of the present application, the present application will be further described below in combination with specific examples. The present application can be implemented in many different forms, and should not be understood as being limited to the examples set forth herein. On the contrary, these examples are provided so that the present disclosure will be thorough and complete, and will fully convey the idea of the present application to those skilled in the art, and the present application will be limited only by the claims.

[0034] Unless otherwise specifically indicated, various raw materials, reagents, instruments and equipment, etc. used in the present application can be purchased from the market or can be prepared by existing methods.

[0035] The application provides a group of polypeptides or polypeptide compositions capable of targeting inhibition of glycolysis, including polypeptide 1 and / or polypeptide 2. The polypeptide 1 at least includes amino acid residues 18-28 at the N-terminal of PGAM1 protein. The polypeptide 2 at least includes amino acid residues 198-208 at the N-terminal of PGK1 protein.

[0036] PGAM1 protein amino acid sequence SEQ ID NO. 1: MAAYKLVLIRHGESAWNLENRFSGWYDADLSPAGHEEAKRGGQALRDAGYEFDICFTSVQKRAIRTLWTVLDAIDQMWLPVVRTWRLNERHYGGLTGLNKAETAAKHGEAQVKIWRRSYDVPPPPMEPDHPFYSNISKDRRYADLTEDQLPSCESLKDTIARALPFWNEEIVPQIKEGKRVLIAAHGNSLRGIVKHLEGLSEEAIMELNLPTGIPIVYELDKNLKPIKPMQFLGDEETVRKAMEAVAAQGKVKK.

[0037] Polypeptide 1 amino acid sequence SEQ ID NO. 2: LENRFSGWYDA.

[0038] PGK1 protein amino acid sequence SEQ ID NO. 3: MSLSNKLTLDKLDVKGKRVVMRVDFNVPMKNNQITNNQRIKAAVPSIKFCLDNGAKSVVLMSHLGRPDGVPMPDKYSLEPVAAELKSLLGKDVLFLKDCVGPEVENACANPAAGTVILLENLRFHVEEEGKGKDASGNKVKAEPAKIDAFRASLSKLGDVYVNDAFGTAHRAHSSMVGVNLPQKAGGFLMKKELNYFAKALESPERPFLAILGGAKVADKIQLINNMLDKVNEMIIGGGMAFTFLKVLNNMEIGTSLYDEEGAKIVKDLMSKAEKNGVKITLPVDFVTADKFDENAKTGQATVASGIPAGWMGLDCGTESSKKYAEAVGRAKQIVWNGPVGVFEWEAFARGTKSLMDEVVKATSRGCITIIGGGDTATCCAKWNTEDKVSHVSTGGGASLELLEGKVLPGVDALSNV.

[0039] Polypeptide 1 amino acid sequence SEQ ID NO. 4: AKALESPERPF.

[0040] As preferred, the first amino acid of the polypeptide 1 and 2 is connected with a transmembrane peptide sequence, and the synthetic polypeptide P1 (sequence shown in SEQ ID NO. 5) and polypeptide P2 (sequence shown in SEQ ID NO. 6) are obtained.

[0041] SEQ ID NO. 5: YGRKKRRQRRRALENRFSGWYDA

[0042] SEQ ID NO. 6: YGRKKRRQRRRAAKALESPERPF.

[0043] The transmembrane peptide is a kind of short peptide that can carry macromolecular substances into cells, including naturally occurring cell transmembrane peptides and artificially synthesized cell transmembrane peptides. The 47-57 amino acid residues (YGRKKRRQRRR) derived from human immunodeficiency virus (HIV)-1 are the most commonly used cell transmembrane peptides. In the specific embodiments of the present application, in order to enhance the binding of the transmembrane sequence to polypeptides 1 and 2, an alanine (A) is added at the C-terminal of the YGRKKRRQRRR sequence as a cell transmembrane peptide sequence, i.e. the cell transmembrane peptide sequence used is YGRKKRRQRRRA (SEQ ID NO. 7).

[0044] The present application also provides the use of the polypeptide or polypeptide composition in the preparation of a drug for treating autoimmune diseases.

[0045] As preferred, the autoimmune disease is at least one of CD4+ T cell abnormal activation mediated type 1 diabetes, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis.

[0046] That is, the polypeptide 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.

[0047] The present application also provides a drug for treating autoimmune diseases, which contains at least one or two polypeptides described above.

[0048] As preferred, the drug further comprises a pharmaceutically acceptable excipient. The excipient includes a filler, a diluent, a binder, a disintegrant, an emulsifier.

[0049] In the specific embodiments of the present application, the polypeptides are all polypeptides connected with transmembrane peptides. Preferably, they are synthesized by GenScript Biotech (Shanghai) Co., Ltd., and are identified by MS and have a purity of more than 95% detected by high performance liquid chromatography (HPLC).

[0050] Example 1: Expression of PGAM1 S23 and PGK1 S203 phosphorylated proteins in CD4+ T cells

[0051] 1.1. Detection of changes in PGAM1 and PGK1 phosphorylated proteins before and after activation of mouse naive CD4+ T cells

[0052] (1) 8-week-old C57BL / 6 wild-type mice were sacrificed by cervical dislocation and soaked in 75% alcohol for 5 minutes, and their spleens were removed.

[0053] (2) The spleen was gently ground at the end of a 1 mL syringe, and 1640 basal medium was added to rinse the cells through a 70 μm filter, and the spleen cell suspension was collected in a 50 mL centrifuge tube.

[0054] (3) Centrifugation at 300 g for 5 minutes, and the spleen cells were deposited at the bottom of the tube.

[0055] (4) Discard the supernatant, add 2 mL of red blood cell lysis solution, gently mix the cells, and lyse for 5 minutes on ice.

[0056] (5) Add about 8 mL of D-PBS to terminate lysis.

[0057] (6) Centrifugation at 400 g for 5 minutes at room temperature.

[0058] (7) The cells were deposited at the bottom of the tube, and the supernatant was discarded.

[0059] (8) Use the mouse naive CD4 + T cell sorting kit (BioLegend, 480040) for subsequent sorting work:

[0060] a) Add 100 μL of D-PBS to resuspend 1×10 7 cells.

[0061] b) Add 10 μL of mixed antibodies, mix well, and incubate on ice for 15 minutes.

[0062] c) Add an equal amount of streptavidin nanomagnetic beads to the antibodies, and continue to incubate on ice for 15 minutes.

[0063] d) Add D-PBS to expand the cell suspension volume to about 3.5 mL, and transfer to a sterile flow tube.

[0064] e) Place the flow tube containing the cell suspension in the magnetic adsorption rack, and stand for 1 minute.

[0065] f) Tilt the magnetic rack and flow tube as a whole, and transfer the cell suspension that is not combined with antibodies and magnetic beads to a sterile 15 mL centrifuge tube.

[0066] g) 300 g centrifuge for 5 minutes, the cell pellet at the bottom of the tube is the sorted naive CD4+ T cells.

[0067] (9) The naive CD4+ T cells were divided into two parts, one part was directly extracted for protein; the other part was suspended in 200 μL / well in the culture well coated with anti-mouse CD3 / CD28 antibody, and the total protein was extracted after the cells were collected after 48 hours of culture.

[0068] (10) The protein expression of the two groups of cells was analyzed by Western blotting.

[0069] 1.2, Analysis of PGAM1 and PGK1 phosphorylated protein expression levels in CD4+ T cells of healthy controls and T1D patients

[0070] (1) Collect 3-5 mL of venous blood from T1D patients and healthy volunteers using heparin anticoagulant tubes.

[0071] (2) Take an equal volume of lymphocyte separation medium into the centrifuge tube.

[0072] (3) Tilt the centrifuge tube at 45° and slowly spread the blood cells on the upper layer of the lymphocyte solution.

[0073] (4) 600 g gradient (3 speed up and 1 speed down) centrifuge for 25 minutes.

[0074] (5) After centrifugation, the liquid is divided into 4 layers, and the white mist layer cells are carefully aspirated.

[0075] (6) Add 10 mL of PBS to wash the cells.

[0076] (7) 300 g centrifuge for 5 minutes, the cell pellet at the bottom of the tube is the peripheral blood mononuclear cells (PBMCs).

[0077] (8) Add D-PBS and wash twice.

[0078] (9) Resuspend the cells with D-PBS, adjust the cell density to about 1 x 10 8 / mL. Use the human CD4 + T cell sorting kit for subsequent sorting.

[0079] a) Add 10 μL of antibody to every 1 x 10 7 cells, mix well and incubate on ice for 15 minutes.

[0080] b) Fix the LS column on the magnetic adsorption rack, and rinse the column with 2 mL of D-PBS.

[0081] c) Add 1 mL D-PBS to dilute the cell antibody mixture and transfer to the LS column, and let it flow out naturally.

[0082] d) After the cell suspension is completely added to the column, continue to add 1.5-2 mL D-PBS for rinsing.

[0083] e) After the last drop of liquid flows out naturally, remove the LS column and place it on a 15 mL centrifuge tube.

[0084] f) Add 2 mL 1640 base medium to the LS column, and quickly flush out the cells in the column with a pusher. The cells are CD4+ T cells.

[0085] (10) Extract the total protein of the obtained cells and perform immunoblotting analysis.

[0086] 3. Experimental results

[0087] Figure 1 A: The expression of PGAM1 S23 and PGK1 S203 phosphorylated proteins in the initial CD4+ T cells significantly increased after stimulation by α-CD3 / CD28 activation.

[0088] Figure 1 B: The levels of PGAM1 S23 and PGK1 S203 phosphorylated proteins in CD4+ T cells in T1D patients were significantly higher than those in healthy controls.

[0089] Example 2: Synthesis of cell-penetrating peptides, polypeptides P1 and P2

[0090] The base sequences encoding the cell-penetrating peptide (YGRKKRRQRRRA), polypeptide P1 (YGRKKRRQRRRALENRFSGWYDA), and polypeptide P2 (YGRKKRRQRRRAAKALESPERPF) were designed. Among them, an alanine (A) variant was added to the carboxy terminus of the 47-57 amino acid residues (YGRKKRRQRRR) of human immunodeficiency virus (HIV)-1, forming the cell-penetrating peptide sequence (YGRKKRRQRRRA); the sequences of polypeptides P1 and P2 are derived from the phosphorylation regulation region of PGAM1 (18-28 amino acids) and PGK1 (198-208 amino acids), respectively, and the amino terminus is connected with the cell-penetrating peptide, forming a cell-penetrating peptide-polypeptide fusion sequence (YGRKKRRQRRRALENRFSGWYDA and YGRKKRRQRRRAAKALESPERPF), as shown in Figure 2 .

[0091] 2.1, Resin pretreatment

[0092] (1) Swelling resin:

[0093] a) Weigh 1.0 g of carboxyl resin (substitution: 0.5 mmol / g) and place it in a synthesis column.

[0094] b) Add 10 mL of DMF and stir at room temperature for 30 minutes to allow the resin to swell completely.

[0095] c) Remove the DMF by suction filtration and wash the resin with fresh DMF three times (10 mL each time).

[0096] (2) First amino acid attachment:

[0097] a) Dissolve the first amino acid of the polypeptide C-terminus (carboxyl end) in 5 mL of DMF, add HOBt (3.0 eq) and DIC (3.0 eq), and activate for 5 minutes.

[0098] b) Add the activated solution to the resin and bubble nitrogen for 2 hours (room temperature).

[0099] c) Suction filter and wash the resin with DMF (3 x 10 mL) and DCM (3 x 10 mL) in sequence.

[0100] d) Confirm complete coupling by ninhydrin detection (resin beads are colorless, indicating complete reaction).

[0101] 2.2, Polypeptide chain extension (Fmoc cycle)

[0102] (1) Fmoc deprotection:

[0103] a) Add 20% piperidine / DMF solution (10 mL) and bubble nitrogen for 5 minutes, then suction filter.

[0104] b) Repeat once to ensure complete removal of the Fmoc group.

[0105] c) Wash with DMF six times (10 mL each time) to remove residual piperidine.

[0106] (2) Amino acid activation and coupling:

[0107] a) Dissolve the next Fmoc-AA-OH (4.0 eq), HOBt (4.0 eq), and DIC (4.0 eq) in 5 mL of DMF and activate for 5 minutes.

[0108] b) Add to the resin and bubble nitrogen for 1 hour (standard coupling) or extend to 2 hours (difficult sequence).

[0109] c) Suction filter and wash with DMF three times.

[0110] (3) Coupling monitoring: take a small amount of resin for ninhydrin detection: if the resin beads are blue (free amino group exists), repeat the coupling step.

[0111] 2.3, Special sequence processing

[0112] (1) Arg-rich region: for consecutive Arg (such as RRRR), use PyBOP (4.0 eq) / DIPEA (8.0 eq) activation, 50°C reaction for 1 hour. After coupling detection, if necessary, the third coupling is carried out.

[0113] (2) Aggregation-prone sequence: add 5% DMSO / DMF to improve solubility.

[0114] 2.4, Polypeptide cleavage and deprotection

[0115] (1) Cleavage reagent preparation: TFA / water / TIS / EDT = 94:2.5:2.5:1 (v / v / v / v), pre-cooled in ice bath.

[0116] (2) Cleavage step:

[0117] a) Transfer the dried resin to a 50 mL centrifuge tube, add 10 mL cleavage solution.

[0118] b) Stir at room temperature for 2.5 hours (under nitrogen protection).

[0119] c) Filter, wash the resin with a small amount of TFA (2x2 mL), and combine the filtrate.

[0120] (3) Polypeptide precipitation:

[0121] a) Add the filtrate dropwise to 40 mL of cold ether (-20°C), and stand in ice bath for 30 minutes.

[0122] b) Centrifuge (4000 rpm, 10 minutes, 4°C), discard the supernatant.

[0123] c) Wash the precipitate with cold ether 3 times, and dry in vacuum for 24 hours.

[0124] 2.5, Polypeptide purification and identification

[0125] Use HPLC and mass spectrometry (MS) to detect the quality of the synthesized peptide segment. The detection results show that the amino acid sequences of the synthesized cell-penetrating peptide NC ( Figure 3 ), polypeptide P1 ( Figure 4 ) and polypeptide P2 ( Figure 5 ) are correct, and the purity of each is greater than 95%.

[0126] Example 3: Polypeptides P1 and P2 respectively target and inhibit the expression of phosphorylated PGAM1 and PGK1

[0127] Mouse splenic naive CD4+ T cells were isolated according to the procedure of Step 1 in Example 1 and inoculated in culture plates precoated with anti-CD3 antibody and anti-CD28 antibody, and 5 μM of transmembrane peptide (as negative control group NC), polypeptide P1 and polypeptide P2 were added respectively, and cultured for 48 hours. The treated cells were collected and the protein was extracted for immunoblotting analysis. As shown in Figs. 6A and 6B, the amount of phosphorylated PGAM1 protein in the polypeptide P1 treatment group was significantly reduced, and the amount of phosphorylated PGK1 protein in the polypeptide P2 treatment group was significantly reduced, as compared with the control group. This result indicates that the polypeptides can competitively inhibit the activation of key glycolytic enzymes PGAM1 and PGK1. Figure 6 A and 6B, the amount of phosphorylated PGAM1 protein in the polypeptide P1 treatment group was significantly reduced, and the amount of phosphorylated PGK1 protein in the polypeptide P2 treatment group was significantly reduced, as compared with the control group. This result indicates that the polypeptides can competitively inhibit the activation of key glycolytic enzymes PGAM1 and PGK1.

[0128] Example 4: Polypeptides P1 and P2 reduce glycolysis of CD4+ T cells

[0129] 4.1, Experimental method

[0130] (1) Mouse splenic naive CD4+ T cells were isolated according to the procedure of Step 1 in Example 1.

[0131] (2) The cells were inoculated in culture plates precoated with anti-CD3 antibody and anti-CD28 antibody.

[0132] (3) 5 μM of transmembrane peptide (as negative control group NC), polypeptide P1 and polypeptide P2 were added respectively in the culture medium, and cultured for 48 hours.

[0133] (4) The glycolytic rate of the aforementioned treated cells was detected using Seahorse XF Glycolytic Rate Assay Kit (Agilent, 103344-100) according to the instructions.

[0134] a) The probe plate was hydrated with calibration solution at 37°C without CO2 for 24 hours in advance.

[0135] b) The glycolysis pressure test medium (GPS medium) was configured: XF base medium was taken, 2 mM glutamine and 10 mM glucose were added, the pH was adjusted to 7.4, and it was preheated at 37°C for standby.

[0136] c) 10 μM of Rotenone / Antimycin A (Rot / AA) and 500 mM of 2-DG were configured using GPS medium. Rot / AA and 2-DG were added to the corresponding drug wells of the probe plate to make the final concentration 1 μM and 50 mM, and the corresponding amount of GPS medium was added to the remaining two wells.

[0137] d) Transfer the treated T cells to Seahorse cell plates, where A1, B4, C3, D6 are left blank without cells.

[0138] e) After centrifugation of the cell culture plates, the original culture medium in the cell plates was aspirated, and 1 mL of pre-warmed GPS medium was added to each well, and washed twice gently.

[0139] f) Finally, 500 μL of GPS medium was added to each well, and incubated in a 37°C CO2-free incubator for 1 hour.

[0140] g) The detection program was set as follows

[0141]

[0142] 4.2, Experimental results

[0143] Figure 7 A: The extracellular acidification rate (ECAR) of CD4+ T cells was inhibited after treatment with polypeptides P1 and P2 compared with the control.

[0144] Figure 7 B: The proton efflux rate (PER) of CD4+ T cells was significantly reduced after treatment with polypeptides P1 and P2.

[0145] Figure 7 C: The basal glycolysis level of CD4+ T cells was significantly reduced after treatment with polypeptides P1 and P2.

[0146] Figure 7 D: The basal proton efflux rate of CD4+ T cells was reduced after treatment with polypeptides P1 and P2.

[0147] Figure 7 E: The compensatory glycolysis rate of CD4+ T cells was reduced after treatment with polypeptides P1 and P2.

[0148] Example 5: Polypeptides and their compositions inhibit the activation, proliferation and Th1 / Th17 subset differentiation of CD4+ T cells

[0149] Mouse spleen naive CD4+ T cells were isolated according to the operation of step 1 in Example 1. The cells were inoculated in culture plates coated with anti-CD3 antibody and anti-CD28 antibody in advance, and the transmembrane peptide and polypeptide composition (P1 and P2 were mixed at 1:1, i.e. 2.5 μM each) were added to the culture medium.

[0150] 5.1, Flow detection of CD4+ T cell activation

[0151] (1) After 16 hours of culture, the cells were collected into centrifuge tubes and washed twice.

[0152] (2) Resuspend cells with flow buffer and adjust cell density to 1 x 10 6 / mL, and take 100 μL cells into flow tube for staining.

[0153] (3) Dilute antibodies with flow buffer: add CD69, CD44 antibodies at 1 μL / tube of antibody stock solution.

[0154] (4) Add 100 μL of diluted antibodies per staining tube, stain for 20 minutes on ice in the dark.

[0155] (5) Stop staining by adding 3 times volume of flow buffer, centrifuge at 300 g for 5 minutes, discard supernatant.

[0156] (6) Add 200 μL of cell staining fixative, and detect on machine.

[0157] 5.2, Cell proliferation detected by CellTrace™ Violet (CTV) staining

[0158] (1) Before cell seeding, place the initial CD4+ T cells used in this experiment into a 15 mL centrifuge tube, and add CTV dye (total concentration of 5 μM).

[0159] (2) Incubate in a 37 °C water bath for 10 minutes.

[0160] (3) Stop staining by adding 3 times volume of complete medium, centrifuge at 300 g for 5 minutes, discard supernatant.

[0161] (4) Seed the cells in culture plates coated with anti-CD3 antibody and anti-CD28 antibody, and add transmembrane peptide and polypeptide composition, respectively.

[0162] (5) After 3 days of culture, collect the cells into flow tubes, wash twice, and detect on machine.

[0163] 5.3, Flow detection of Ki67 staining to reflect cell proliferation

[0164] (1) After 48 hours of transmembrane peptide and polypeptide composition treatment, collect the cells into flow tubes, and wash twice with PBS.

[0165] (2) Centrifuge at 300 g for 5 minutes, discard supernatant.

[0166] (3) Dilute 4x Fix / Perm buffer to 1x Fix / Perm working solution to the required volume using the TF diluent in the transcription factor buffer kit.

[0167] (4) Add 150 μΐ, of 1x Fix / Perm working solution to each tube, fix at 4 °C for 60 min.

[0168] (5) Dilute 5x Perm / Wash buffer to 1x Perm / Wash working solution with ddH2O.

[0169] (6) After fixation, add 1 mL of Perm / Wash working solution to each tube, centrifuge at 500 g for 5 min.

[0170] (7) Discard the supernatant, resuspend the cells with 100 μΐ, of Perm / Wash working solution per tube.

[0171] (8) No antibody is added to the blank tube and the single marker tube of surface molecules. Add 1 μΐ, of Ki67 antibody to each of the single marker tube and the staining tube.

[0172] (9) Shake well, incubate at 4 °C for 1 h in the dark.

[0173] (10) Add 1 mL of flow buffer to terminate the staining, centrifuge at 500 g for 5 min.

[0174] (11) Incline the flow tube, discard the supernatant, and use a water-absorbing paper to gently dab the residual liquid in the tube.

[0175] (12) Add 200 μΐ, of staining fixative to each flow staining tube, and detect on the machine.

[0176] 5.4, Effect of polypeptide composition on differentiation of CD4+ T cell subgroups

[0177] Initial CD4+ T cells were inoculated into culture wells coated with anti-mouse CD3 / CD28 antibody. The corresponding cytokines were added in the culture medium to induce differentiation into corresponding T cell subgroups:

[0178] (1) Th1 cells, add IL-2 (10 ng / mL) and IL-12 (10 ng / mL).

[0179] (2) Th17 cells, add IL-6 (10 ng / mL) and TGF-β (5 ng / mL).

[0180] (3) Treg cells, add IL-2 (10 ng / mL) and TGF-β (10 ng / mL).

[0181] Under each differentiation condition, transmembrane peptide and polypeptide composition were added for treatment. After 3-5 days of culture, the above-mentioned cells were harvested and subjected to cell line flow detection.

[0182] 5.5, Experimental results

[0183] Figure 8 A: The polypeptide composition significantly reduces the expression of CD69 and CD44 of CD4+ T cells. This indicates that the polypeptide composition inhibits the activation of CD4+ T cells.

[0184] Figure 8 B, C: The fluorescence decay of CTV labeling of the polypeptide composition-treated group is slower than that of the control group, and the expression of Ki67 is significantly reduced. This result indicates that the polypeptide composition can inhibit the proliferation of CD4+ T cells.

[0185] Figure 8 D: Compared with the control, the differentiation of Tregs in the polypeptide-treated group is significantly enhanced.

[0186] Figure 8 E, F: The polypeptide can inhibit the differentiation of Th1 and Th17 cells.

[0187] Example 6: The polypeptide composition can reduce the incidence of type 1 diabetes (NOD) mice and improve their state

[0188] Thirty 6-week-old wild-type spontaneous diabetic NOD mice were divided into two groups, i.e., a control group and an experimental group, with 15 mice in each group. The control group and the experimental group of mice were given transmembrane peptides and a polypeptide composition (P1 and P2 mixed at a ratio of 1:1) intraperitoneally (1 mg / mL), twice a week, for two consecutive weeks. The blood glucose meter was used to detect the blood glucose of the mice twice a week by taking the tail end of the mice, and the incidence was recorded when the blood glucose concentration was ≥13.8 mmol / L for two consecutive times. The incidence of mice in the two groups was calculated, and the experiment was terminated when the incidence of any group reached 80% or when the monitoring was completed for 32 weeks. The T1D-related indicators of the mice in the two groups were compared by using HE and immunofluorescence pancreatic pathological detection, ELISA, and flow cytometry.

[0189] Figure 9 A: A graph of the incidence of NOD mice in the two groups.

[0190] Figure 9 B: ELISA detection of C-peptide and insulin levels in mouse serum.

[0191] Figure 9 C: Pancreatic HE staining graph and inflammation grading statistical graph.

[0192] Figure 9 D: Insulin immunofluorescence graph of islets.

[0193] Figure 9 E-J: The proportion (E), activation state (F), proliferation (G), and subgroup distribution (H-J) of CD4+ T cells in the mouse pancreatic lymph nodes.

[0194] The results show that the onset time of the mice in the polypeptide composition treatment group is delayed and the incidence rate is decreased Figure 9 A). The insulin and C-peptide levels in the serum of this group of mice are also significantly higher than those of the control group Figure 9 B). As shown in Figure 9 C, the islet inflammation of the mice in the polypeptide composition treatment group is lighter than that of the control group. The immunofluorescence results show that the mice in the polypeptide composition treatment group produce more insulin Figure 9 D). The flow cytometry results show that the proportion of CD4+ T cells in the mice in the polypeptide treatment group is decreased, and the proliferation and activation of the CD4+ T cells are significantly lower than those of the control group Figure 9 E-G). In addition, the Treg cells in the mice in the polypeptide treatment group are significantly increased, the Th1 cells are significantly decreased, and the proportion of Th17 cells also shows a decreasing trend ​ H-J).

[0195] The above results show that the polypeptide composition can inhibit the activation of CD4+ T cells, reduce the proportion of effector CD4+ T cells, alleviate the damage to islet β cells, improve the function of β cells, and thus reduce the incidence of T1D and delay the progression of the disease.

[0196] Obviously, the above examples are only examples for clearly illustrating but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. All the embodiments do not need to be exhausted here. The changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A polypeptide composition, characterized by: The polypeptide composition comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO: 5 and a polypeptide with an amino acid sequence as shown in SEQ ID NO:

6.

2. Use of the polypeptide composition of claim 1 in the preparation of a medicament for treating an autoimmune disease, the autoimmune disease being at least one of CD4+ T cell abnormal activation mediated type 1 diabetes, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis.

3. Use according to claim 2, characterized in that: The polypeptide composition achieves the treatment of autoimmune diseases by inhibiting the activity of phosphorylated PGAM1 and phosphorylated PGK1.

4. A medicament for treating an autoimmune disease, characterized by: The medicament comprises the polypeptide composition of claim 1.

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