Polypeptide for activating Treg cells to play anti-inflammatory effect

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide for activating Treg cells to play an anti-inflammatory role. According to the present invention, the new active small molecule polypeptide UMR2-705: DLLISIY is found in the invention; the UMR2-705 polypeptide is used for promoting proliferation and amplification of mouse Treg cells and proliferation of Treg cells in human PBMC (peripheral blood mononuclear cells) in an in-vitro test. The compound has good anti-inflammatory activity, and can obviously relieve the increase of levels of cell factors such as IL-6, TNF-alpha and IL-17A. In an LPS mouse inflammation model and a psoriasis mouse model, increase of levels of cell factors such as IL-6, TNF-alpha and IL-17A can be effectively inhibited, and a novel therapy is provided for treatment of inflammatory diseases. The polypeptide used in the invention has the advantages of low cost, stable and reliable effect and the like, and provides a new idea and theoretical basis for research and development of inflammation drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a polypeptide that activates Treg cells to exert anti-inflammatory effects. Background Art

[0002] Autoimmune diseases are mainly caused by the hyperactivity of the autoimmune system and the targeting of self-antigens, which leads to damage to the body's own tissues. The stability, function and number of Treg cells, which play an important role, have changed due to the presence of persistent inflammation. Treg cells are a type of T cell subset that controls the body's autoimmune response and participates in the regulation of multiple immune processes. They play an important role in maintaining the body's immune balance and preventing autoimmune diseases and transplant rejection. The abnormal decrease in the number of Treg cells or the decline in their function, resulting in immune tolerance defects, is the root cause of immune dysfunction. Glucocorticoids, immunosuppressants, biologics and other drugs commonly used in autoimmune diseases, as well as stem cell transplantation therapy, etc. Regulating inflammatory responses by regulating Treg homeostasis is a safe and effective strategy for autoimmune diseases.

[0003] There are many types of autoimmune diseases and their pathogenesis is still unclear. For those who are intolerant to or respond poorly to conventional therapies, other effective candidate treatment options need to be considered. Stem cell transplantation, biological agents, herbal preparations, and some immunomodulatory substances have shown good therapeutic prospects in anti-rheumatic treatment. For those who respond poorly or cannot tolerate first-line treatment alone or in combination, other potentially effective treatment options need to be considered, such as stem cell transplantation, biological agents or new herbal preparations, as well as some anti-inflammatory drugs that may regulate or suppress immunity, in order to alleviate the condition of patients with such refractory diseases and improve their quality of life.

[0004] Although glucocorticoids, immunosuppressants, biologics and other drugs have advantages in autoimmune diseases, there are still no reports on peptide drugs that regulate Treg cells to treat inflammation. The use of peptide drugs as drugs for the treatment of autoimmune diseases has good application prospects and safety, and peptide drugs have the advantages of low immunogenicity, easy synthesis and modification, and not easy to accumulate in the body. At the same time, the efficacy can also be provided by coupling ligands or by carrying nano-delivery systems. However, in the prior art, there is still a lack of better peptide drugs for the treatment of autoimmune diseases. Summary of the invention

[0005] The first aspect of the present invention aims to provide a small molecule polypeptide.

[0006] The purpose of the second aspect of the present invention is to provide a method for preparing the small molecule polypeptide of the first aspect of the present invention.

[0007] The purpose of the third aspect of the present invention is to provide the application of the small molecule polypeptide or its derivatives according to the first aspect of the present invention.

[0008] The fourth aspect of the present invention aims to provide a fusion protein.

[0009] The fifth aspect of the present invention aims to provide a biological agent.

[0010] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a small molecule polypeptide, the amino acid sequence of which is DLLISIY. The polypeptide is named UMR2-705 in the present invention, abbreviated as 705.

[0012] The second aspect of the present invention provides a method for preparing the small molecule polypeptide of the first aspect of the present invention, comprising at least one of a1) to a2):

[0013] a1) Chemical synthesis;

[0014] a2) Biosynthesis method.

[0015] In some embodiments of the present invention, the chemical synthesis method is Fomc solid phase peptide synthesis method.

[0016] Preferably, the DLLISIY polypeptide can also be synthesized by biosynthesis, specifically, by constructing an expression vector for expressing the DLLISIY polypeptide, and introducing the expression vector into a corresponding host cell for expression, and purifying and isolating the polypeptide.

[0017] The third aspect of the present invention aims to provide the use of the small molecule polypeptide or its derivatives according to the first aspect of the present invention in at least one of b1) to b2).

[0018] b1) preparing products for activating the anti-inflammatory function of Treg cells;

[0019] b2) Preparation of products for the treatment of autoimmune diseases.

[0020] Preferably, the derivative includes at least one of a pharmaceutically acceptable salt and a pharmaceutically acceptable modification.

[0021] Preferably, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0022] Preferably, the metal salt comprises an alkali metal salt or an alkaline earth metal salt.

[0023] Preferably, the alkali metal salt includes at least one of a sodium salt and a potassium salt.

[0024] Preferably, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt and aluminum salt.

[0025] Preferably, the salt formed with the organic base includes a salt formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and N,N'-dibenzylethylenediamine.

[0026] Preferably, the salt formed with an inorganic acid includes a salt formed with the following inorganic acids: at least one of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0027] Preferably, the salt formed with the organic acid includes a salt formed with the following organic acids: at least one of formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0028] Preferably, the salt formed with a basic amino acid includes a salt formed with the following basic amino acids: at least one of arginine, lysine and ornithine.

[0029] Preferably, the salt formed with the acidic amino acid includes a salt formed with the following acidic amino acids: at least one of aspartic acid and glutamic acid.

[0030] The pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, halogenation, polyethylene glycol modification, polysialic acid modification, and fatty acid modification. For example, PEG modification can be used to shield and hinder the surface of the protein, so that the enzymatic hydrolysis rate of the modified protein is significantly reduced, the stability is improved, and the half-life in vivo is prolonged; or fatty acid modification is used to react with the protein to undergo acylation modification, and the affinity of the protein to serum albumin can be increased by exposing carboxyl residues on the surface of the protein, thereby increasing its circulation time in the blood, thereby prolonging the half-life of the protein drug. Those skilled in the art can select the corresponding modification method according to the actual use of the drug.

[0031] Preferably, the product includes at least one of medicines and medical devices.

[0032] Preferably, the dosage form of the product includes at least one of an injection dosage form, a respiratory tract dosage form, a skin dosage form, a mucosal dosage form, and a cavity dosage form.

[0033] Preferably, the product includes an injectable dosage form and a dermal dosage form.

[0034] Furthermore, the injectable dosage forms include but are not limited to injection solutions, injection solutions, intravenous injection solutions, injection suspensions, sterile powders for injection, intravenous injections, water injections, injection emulsions, powder injections, injections, sterile powder injections, freeze-dried powder injections, etc.

[0035] Furthermore, the skin administration dosage forms include but are not limited to gels, dressings, transdermal patches, etc.

[0036] Preferably, the product further comprises pharmaceutically acceptable excipients.

[0037] Preferably, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0038] Furthermore, for the convenience of medication, the active ingredient UMR2-705 polypeptide or its derivatives can be processed into a specific dosage form with any one or more pharmaceutically acceptable excipients. These excipients can be solvents (such as water for injection, ethanol, propylene glycol and glycerol), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid and sodium tartrate), antioxidants (such as sodium sulfite, sodium pyrosulfite and sodium thiosulfate), antibacterial agents (such as phenol, benzyl alcohol and thimerosal), and isotonic regulators (such as sodium chloride and glucose).

[0039] Preferably, the autoimmune disease includes at least one of psoriasis, systemic lupus erythematosus, type I diabetes, inflammatory bowel disease, rheumatoid arthritis, and vitiligo.

[0040] The fourth aspect of the present invention provides a fusion protein, comprising the small molecule polypeptide described in the first aspect of the present invention and a coupling portion; the coupling portion comprises at least one of a detectable marker, a cytokine, and an enzyme.

[0041] Preferably, the detectable marker is selected from a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a label protein, a colored substance, or any combination thereof.

[0042] Preferably, the tag protein includes but is not limited to at least one of His, Flag, Myc, GST, GFP, and HA.

[0043] The fifth aspect of the present invention provides a biological preparation, comprising the small molecule polypeptide described in the first aspect of the present invention and / or the fusion protein described in the fourth aspect of the present invention; and a delivery vector.

[0044] Preferably, the delivery carrier comprises at least one of a hydrogel, a liposome, and a surfactant nanocarrier.

[0045] In some embodiments of the invention, the biologic is a nanoemulsion.

[0046] Preferably, the nanoemulsion is prepared by mixing A and B gels.

[0047] Among them, gel A is a gel system, and other common hydrogel systems in the art such as carbomer, sodium alginate, polyvinyl alcohol, polyacrylic acid, chitosan, etc. can be selected.

[0048] B gel is a liposome system, and other common nanoliposomes in the art such as polyethylene glycol (15)-hydroxystearate (Solutol HS15), polyethylene glycol (PEG400), oleic acid macrogol glyceride (Labrafil 1994cs) can be selected.

[0049] The small molecule polypeptide described in the first aspect of the present invention is mixed in the B-gel system, and the mass volume ratio of the small molecule polypeptide to the liposome is (1-50) mg / mL.

[0050] The beneficial effects of the present invention are:

[0051] The present invention discovered a new active small molecule polypeptide UMR2-705: DLLISIY. The UMR2-705 polypeptide promoted the proliferation and expansion of mouse Treg cells and the proliferation of Treg cells in human PBMC in in vitro experiments. It has good anti-inflammatory activity and can significantly alleviate the increase in the levels of cytokines such as IL-6, TNF-α and IL-17A. It can effectively inhibit the increase in the levels of cytokines such as IL-6, TNF-α and IL-17A in both the LPS mouse inflammation model and the psoriasis mouse model, providing a new therapy for the treatment of inflammatory diseases. The polypeptide used in the present invention has the advantages of low cost, stable and reliable effect, etc. The invention provides a new idea and theoretical basis for the research and development of inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0053] Figure 1 This is the result of UMR2-705 expanding the proportion of mouse Treg cells in vitro in Example 2 of the present invention.

[0054] Figure 2This is the result of UMR2-705 increasing the proportion of Treg cells in human PBMC in vitro in Example 3 of the present invention.

[0055] Figure 3 This is the result of UMR2-705 increasing the proportion of mouse Treg cells in vivo in Example 4 of the present invention.

[0056] Figure 4 This is the result of UMR2-705 in Example 5 of the present invention reducing the inflammation level in the LPS-induced mouse inflammation model in vivo, wherein A is IL6, B is TNF, and C is IL17.

[0057] Figure 5 This is the result of UMR2-705 reducing the inflammation level in the psoriasis mouse model in vivo in Example 5 of the present invention, wherein A is IL6, B is TNF, and C is IL17. DETAILED DESCRIPTION

[0058] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0059] Example 1 Synthesis of small molecule peptides

[0060] The first Fmoc-amino acid carboxyl group at the C-terminus of the candidate peptide is connected in the form of a covalent bond, and then the N-terminus of this amino acid is used as the starting point of the candidate multiple synthesis to dehydrate and condense with the carboxyl group of the second amino acid in the sequence to form a peptide bond. Then the Fmoc-amino acid protecting group at the N-terminus is deprotected, and then the N-terminus of the second amino acid is reacted with the carboxyl group of the following amino acid, and this process is repeated until the synthesis of the small molecule peptide DLLISIY is completed. Finally, the peptide is precipitated and washed. The molecular weight of the peptide identified by mass spectrometry is consistent with the theoretical molecular weight, and it is stored at -20°C.

[0061] Based on the same method, a control peptide WSLGYTG (a randomly screened control peptide without regulatory function) was synthesized.

[0062] The above polypeptides were obtained by the inventors in the early screening, among which the small molecule polypeptide DLLISIY (SEQ ID NO: 1) was named UMR2-705, and the control polypeptide WSLGYTG (SEQ ID NO: 2) was named UMR2-701.

[0063] Example 2 UMR2-705 polypeptide promotes proliferation and expansion of mouse Treg cells in vitro

[0064] 1. Experimental Materials

[0065] The experimental mice were wild-type C57BL / 6, 8 weeks old, 3 mice / group, from the Experimental Animal Center of the University of Macau. The relevant experiments were approved by the Ethics Review Committee of the University of Macau.

[0066] 2. Experimental Methods

[0067] Purification of CD4 T cells from lymph nodes and spleen of wild-type C57BL / 6 mice by magnetic bead sorting + T cells, using Cell Trace TM Voilet (Invitrogen TM , C34557) were used to stain the primary cells and add IL-2 10 ng / mL to culture CD4 + T cells. The cells were collected after treatment with different concentrations of UMR2-705 peptide (0, 5, 10, 20, 50, 100 μg / mL) for 72 hours and then used Dye (Invitrogen TM , L34975) to distinguish between live and dead cells, and use flow cytometry analysis to stain CD4, FOXP3 and other markers using flow cytometry antibodies. Analyze the effect of peptides on FOXP3 expression in vitro.

[0068] 3. Experimental results

[0069] The experimental results are shown in Figure 1 The proportion of mouse Treg cells increased with the increase of UMR2-705 peptide concentration ( Figure 1 A), and the Treg proliferation ability also increased with the increase of UMR2-705 peptide concentration ( Figure 1 (B), it can be seen that the UMR2-705 polypeptide can promote the proliferation and expansion of mouse Treg cells in in vitro experiments.

[0070] Example 3 UMR2-705 polypeptide promotes proliferation and expansion of human Treg cells in vitro

[0071] 1. Experimental Materials

[0072] Fresh human peripheral blood was collected from the Macau Blood Center, and related experiments were approved / permitted by the Ethics Committee of the University of Macau.

[0073] 2. Experimental Methods

[0074] PBMCs were obtained by gradient centrifugation of fresh human blood, IL-2 10 ng / mL was added to culture the cells in vitro, and different concentrations of UMR2-705 and UMR2-701 peptides (0, 25, 50, 100 μg / mL) were used to treat the cells for 72 hours before collection. Dye (Invitrogen TM , L34975) were stained to distinguish between live and dead cells, analyzed by flow cytometry, and FACS antibodies were used to stain CD4, FOXP3 and other markers to analyze the effect of the peptide on Treg cells under in vitro culture.

[0075] 3. Experimental results

[0076] The experimental results are shown in Figure 2 . CD4 + The proportion of cells increased with the increase of the concentration of UMR2-705 polypeptide, while the control polypeptide UMR2-701 had no such effect. It can be seen that UMR2-705 polypeptide can promote the proliferation of Treg cells in human PBMC.

[0077] Example 4 UMR2-705 polypeptide promotes proliferation and expansion of mouse Treg cells in vivo

[0078] 1. Experimental Materials

[0079] Same as Example 2.

[0080] 2. Experimental Methods

[0081] At 8 weeks of age, female mice were divided into 3 groups, with 3 mice in each group. The UMR2-705 and UMR2-705 groups were injected with the corresponding peptides (25 mg / kg), and the PBS group was injected with the same volume of PBS buffer. The injection was continued for three days until the mice were killed. Tissues were collected to obtain single-cell suspensions. The cells were resuspended in PBS and washed twice. After fixing the cells with the fixative for 12-18 hours, the cells were resuspended with a membrane permeabilizer and washed twice. After blocking for 30 minutes, CD45, CD3, CD4, and FOXP3 antibodies were added for staining for 30 minutes, and then washed twice with a membrane permeabilizer-containing washing solution and the proportion of Treg cells was detected on the machine.

[0082] 3. Experimental results

[0083] The results are as follows Figure 3 As shown, the UMR2-705 polypeptide can increase the proportion of Treg cells in the spleen and lymph nodes of mice in vivo, which is significant compared with the control group polypeptide UMR2-701 and PBS group.

[0084] Example 5 UMR2-705 polypeptide significantly alleviates inflammation levels in an inflammatory mouse model

[0085] 1. Experimental Materials

[0086] Same as Example 2.

[0087] 2. Experimental Methods

[0088] The mice were divided into four groups (PBS, LPS, LPS+705, LPS+701), with 3 mice in each group. The dosage of peptides in the peptide group was unified at 25 mg / kg. The LPS and PBS groups were injected with PBS only for the first two days. LPS+705 and LPS+701 were intraperitoneally injected with 705 and 701 peptides, respectively. The injection volume was unified for each group and injected for 3 consecutive days. On the third day, 0.2 mL of saline or 200 μg of LPS in 0.2 mL of saline were injected at the same time. After 24 hours, the mice were killed. According to the manufacturer's instructions, the BD Cytometric Bead Array (CBA) Mouse Th1 / Th2 / Th17 Cytokine Kit (Catalog No.: BD 560485) was used to measure mouse IL-6, TNF, and IL-17A.

[0089] 3. Experimental results

[0090] The results are as follows Figure 4 As shown, UMR2-705 polypeptide can effectively inhibit IL-6 ( Figure 4 A), TNF-α( Figure 4 Middle B) and IL-17A ( Figure 4 Middle C) Increased cytokine levels.

[0091] Example 6 UMR2-705 polypeptide preparation significantly improves inflammation levels in psoriasis mouse model

[0092] 1. Experimental Materials

[0093] The mice were the same as in Example 2.

[0094] UMR2-705, UMR2-701 nanoemulsion preparation method:

[0095] 1. Preparation of blank gel: Dissolve 400 mg of Carbomer 974N in 20 g of water and stir to swell (at least 2 hours, Carbomer ~2%).

[0096] 2. Preparation of nanoemulsion: First mix Labrafil 1994cs (1 mL), Solutol HS15 (1 mL) and PEG400 (0.5 mL), stir for five to ten minutes, then add UMR2-701 and UMR2-705 polypeptide 15 mg respectively, stir in a water bath at 50°C for about 1 hour, with a medium to slightly faster speed (the speed can keep the liquid surface uniform), add 7.5 mL of ultrapure water dropwise, and observe that the liquid surface changes from relatively clear to turbid and then to clear, with a slightly blue color, stop heating in the water bath, and stir for another 10-15 minutes until it reaches room temperature.

[0097] 3. Mix the two: nanoemulsion (mL): carbomer gel (g) = 3:2, stir and mix well, add triethanolamine dropwise, and measure the pH value.

[0098] 2. Experimental Methods

[0099] Imiquimod IMQ-induced mouse psoriasis model:

[0100] The experiment was divided into 5 groups, including normal mice (no treatment), psoriasis mice (only modeling without treatment, positive drug tacrolimus treatment, UMR2-705 peptide treatment, UMR2-701 peptide treatment), 3 mice in each group.

[0101] The treated mice were smeared with 0.2 g of nanoemulsion containing 701 and 705 polypeptides (10 mg / kg) or 0.3 mg of tacrolimus ointment (0.1% tacrolimus, Astellas Pharmaceuticals Co., Ltd., Japan). Treatment was performed 4 hours after IMQ modeling. Mice were killed after five consecutive days of treatment (IMQ modeling + treatment once a day), blood was collected, and plasma was separated after centrifugation. Mouse IL-6, TNF and IL-17A were measured using the BD Cytometric Bead Array (CBA) mouse Th1 / Th2 / Th17 cytokine kit (catalog number: BD 560485) according to the manufacturer's instructions.

[0102] 3. Experimental results

[0103] The results are as follows Figure 5 As shown, both UMR2-705 and UMR2-701 peptides can alleviate the inflammation level in the psoriasis mouse model and have a certain therapeutic effect on psoriasis.

[0104] In summary, the UMR2-705 polypeptide provided by the present invention has good anti-inflammatory activity and can significantly alleviate the increase in the levels of cytokines such as IL-6, TNF-α and IL-17A. In both the LPS mouse inflammation model and the psoriasis mouse model, the increase in the levels of cytokines such as IL-6, TNF-α and IL-17A can be effectively inhibited, providing a new therapy for the treatment of inflammatory diseases.

Claims

1. A small molecule polypeptide, characterized in that: The amino acid sequence of the small molecule polypeptide is DLLISIY.

2. The method for preparing the small molecule polypeptide according to claim 1, comprising at least one of a1) to a2): a1) Chemical synthesis; a2) Biosynthesis method.

3. Use of the small molecule polypeptide or its derivatives according to claim 1 in at least one of b1) to b2); b1) preparing products for activating the anti-inflammatory function of Treg cells; b2) Preparation of products for the treatment of autoimmune diseases.

4. The use according to claim 3, characterized in that: The derivative includes at least one of a pharmaceutically acceptable salt and a pharmaceutically acceptable modification.

5. The use according to claim 4, characterized in that: The pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid; The pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

6. The product according to claim 3, characterized in that: The products include pharmaceutical products; The dosage form of the product includes at least one of an injection dosage form, a respiratory tract dosage form, a skin dosage form, a mucosal dosage form, and a cavity dosage form.

7. The product according to claim 3, characterized in that: The autoimmune disease includes at least one of psoriasis, systemic lupus erythematosus, type I diabetes, inflammatory bowel disease, rheumatoid arthritis, and vitiligo.

8. A fusion protein, characterized in that: Comprising the small molecule polypeptide and a coupling part according to claim 1; The coupling part includes at least one of a detectable marker, a cytokine, and an enzyme.

9. A biological agent, characterized in that: It comprises the small molecule polypeptide according to claim 1 and / or the fusion protein according to claim 8; and a delivery vector.

10. The biological agent according to claim 9, characterized in that: The delivery carrier includes at least one of a hydrogel, a liposome, and a surfactant nanocarrier.

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