Use of blocker and biological material with lck / fyn-mediated STAT3 phosphorylation as target

Abstract

The present invention provides use of an Lck / Fyn-mediated STAT3 activation pathway as a target in preparing or screening a product. The product blocks the Lck / Fyn-mediated STAT3 activation pathway, inhibits the differentiation of Th17 cells, and promotes the differentiation of Th17 cells into Treg cells without affecting the differentiation of Th1 and Th2 cells, thereby preventing or treating Th17 cell-mediated immune diseases. According to the target, the present invention provides use of a blocker against Lck / Fyn-mediated STAT3 activation in preparing a product, and the blocker is selected from an Lck / Fyn kinase inhibitor or a substance that specifically inhibits the correct binding of Lck / Fyn to STAT3. The present invention provides use of one or more biological materials in preparing a product for preventing or treating a Th17 cell-mediated immune disease. The present invention further provides a medicament for preventing or treating a Th17 cell-mediated immune disease and a therapeutic method thereof.

Description

Use of inhibitors and biomaterials targeting Lck / Fyn-mediated STAT3 phosphorylation Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to uses of a blocker and a biomaterial targeting Lck / Fyn-mediated STAT3 phosphorylation. Background Art

[0002] Th17 cells are a type of CD4 that secretes IL-17, IL-17F, and IL-22. + Th17 cells are a subset of T cells that play a very important pathogenic role in a series of autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, psoriasis, ankylosing spondylitis and inflammatory bowel disease. Th17 cells are differentiated from naive CD4 T cells stimulated by antigens, and their differentiation requires cytokines such as TGFβ, IL-1β, IL-6 and IL23. As found in "STAT3 regulates cytokine-mediated generation of inflammatory helper T cells", cytokines IL-6 / IL-23 are the main transcription factors that regulate the transcription of Th17 cells through the JAK-STAT3 pathway. This mediates the differentiation of Th17 cells. Antibodies that block cytokines that induce Th17 cell differentiation, such as anti-IL6 / IL6R antibodies like tocilizumab, salicizumab, satelizumab, and siltuximab, anti-IL23 antibodies like risankizumab, and antibodies that block IL-17, a cytokine secreted by Th17 cells, like secukinumab, have all been approved by the FDA for marketing. However, these antibody drugs are large in size, making them difficult to reach certain specific sites, such as the central nervous system, and are relatively expensive. JAK inhibitors such as fedratinib, upadacitinib, filgotinib, and abrocitinib, inhibit the signaling of many cytokines and therefore have relatively significant side effects, resulting in FDA blacklist warnings. Clinical studies have shown that patients treated with JAK inhibitors are more likely to experience recurrences of viral diseases they carry, such as hepatitis. Therefore, safer and more effective treatments for autoimmune diseases are needed.

[0003] Summary of the Invention

[0004] In view of the shortcomings of the above-mentioned prior art, this study found that during the differentiation of Th17 cells, the SH2 domain of Lck / Fyn can bind to the 328-335 polypeptide on STAT3, causing the conformation of Lck / Fyn to change, further enhancing the kinase activity of Lck / Fyn, and the kinase active site of Lck / Fyn directly phosphorylates STAT3. Phosphorylated STAT3 mediates the differentiation of antigen-stimulated CD4 T cells into Th17 cells. Blocking the phosphorylation of STAT3 mediated by Lck / Fyn selectively inhibits the in vitro and in vivo differentiation of Th17 cells and Th17 cell-mediated immune diseases, but Lck / Fyn does not directly affect the phosphorylation of STAT4, STAT5, and STAT6. Therefore, inhibiting the activity of Lck / Fyn does not affect the differentiation of Th1 and Th2. Therefore, the purpose of the present invention is to provide a blocker that blocks the phosphorylation of STAT3 mediated by Lck / Fyn, thereby selectively inhibiting the in vitro and in vivo differentiation of Th17 cells and Th17 cell-mediated immune diseases.

[0005] In a first aspect, the present invention provides the use of the Lck / Fyn-mediated STAT3 activation pathway as a target in the preparation or screening of a product, wherein the product blocks the Lck / Fyn-mediated STAT3 activation pathway and has any one or more of the following effects:

[0006] (1) Inhibits the differentiation of Th17 cells without affecting the differentiation of Th1 and Th2 cells;

[0007] (2) Promote the transdifferentiation of Th17 cells into Treg cells;

[0008] (3) Prevent or treat Th17 cell-mediated immune diseases.

[0009] A second aspect of the present invention provides the use of a blocker of Lck / Fyn-mediated STAT3 activation in the preparation of a product, wherein the product has one or more of the following effects:

[0010] (1) Inhibits the differentiation of Th17 cells without affecting the differentiation of Th1 and Th2 cells;

[0011] (2) Promote the transdifferentiation of Th17 cells into Treg cells;

[0012] (3) Prevent or treat Th17 cell-mediated immune diseases.

[0013] The third aspect of the present invention provides the use of any of the following biomaterials in the preparation of a product for preventing or treating Th17 cell-mediated immune diseases:

[0014] a. A polypeptide comprising a STAT3 mutant or a fragment thereof, wherein the STAT3 mutant or the fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 1; preferably, the STAT3 mutant or the fragment thereof is further fused with a cell-penetrating peptide; further preferably, the cell-penetrating peptide is located at the N-terminus of the STAT3 mutant or the fragment thereof; further preferably, the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO: 5;

[0015] b. a polynucleotide encoding the polypeptide;

[0016] c. a construct comprising the polynucleotide described in b;

[0017] d. A host cell comprising the construct described in c or a host cell whose genome has been integrated with the polynucleotide described in b.

[0018] The fourth aspect of the present invention provides a drug for preventing or treating Th17 cell-mediated immune diseases, comprising any one or more of the above-mentioned biomaterials for preparing products for preventing or treating Th17 cell-mediated immune diseases.

[0019] The fifth aspect of the present invention provides a method for treating an immune disease, comprising administering to a subject an effective amount of the blocker used in the above-mentioned use for preparing a product for preventing or treating a Th17 cell-mediated immune disease, and / or any one or more of the biomaterials used in the above-mentioned use for preparing a product for preventing or treating a Th17 cell-mediated immune disease.

[0020] The beneficial effects of the present invention are:

[0021] (1) Based on the discovery in the present invention that Lck / Fyn binds to STAT3 and activates Lck / Fyn, thereby causing the activated Lck / Fyn to phosphorylate STAT3, it was found that blocking Lck / Fyn-mediated STAT3 phosphorylation with a blocker selectively inhibited the in vitro and in vivo differentiation of Th17 cells and Th17 cell-mediated immune diseases, thus opening up a new therapeutic approach for the treatment of Th17 cell-mediated immune diseases.

[0022] (2) The present invention provides a blocker of Lck / Fyn-mediated STAT3 activation for the treatment of Th17-mediated immune diseases. By binding the Lck / Fyn kinase inhibitor product to the Lck / Fyn kinase active site, the activity of Lck / Fyn is inhibited, thereby inhibiting the phosphorylation of STAT3. Blocking the phosphorylation of STAT3 mediated by Lck / Fyn selectively inhibits the in vitro and in vivo differentiation of Th17 cells and Th17 cell-mediated immune diseases. However, Lck / Fyn does not directly affect the phosphorylation of STAT4, STAT5, and STAT6. Therefore, inhibiting the activity of Lck / Fyn does not affect the differentiation of Th1 and Th2. Therefore, Lck / Fyn kinase inhibitors are highly selective. Lck / Fyn kinase inhibitors have a small molecular weight and can easily reach special parts such as the central nervous system. They also have low toxicity and side effects and are safe and highly effective.

[0023] (3) The present invention also provides a substance that specifically inhibits the correct binding of Lck / Fyn to STAT3 for use in treating Th17-mediated immune diseases. By blocking the binding of the 328-335 polypeptide on STAT3 to the SH2 domain of Lck / Fyn, the conformational change of Lck / Fyn is blocked, and Lck / Fyn cannot expose the active site, thereby inhibiting the activity of Lck / Fyn and thus inhibiting the phosphorylation of STAT3. Blocking the phosphorylation of STAT3 mediated by Lck / Fyn selectively inhibits the differentiation of Th17 cells in vivo and in vitro and Th17 cell-mediated immune diseases, thereby achieving the effect of treating Th17 cell-mediated immune diseases. A sequence rich in basic amino acids is added to the polypeptide sequence of the mutant STAT3 MUT to help the polypeptide enter the cell.

[0024] (4) The present invention provides new clinical application value for preparations targeting Lck / Fyn kinase in the field of treatment of Th17 cell-mediated immune diseases, opening up broad prospects for its further market application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1. Principle of the Lck / Fyn-mediated STAT3 phosphorylation activation pathway. During TH17 cell differentiation, TCR-Lck / Fyn directly phosphorylates STAT3 at Y705 and, together with cytokine-JAK kinase, activates STAT3.

[0026] Figure 2. Inhibition of Lck / Fyn kinase activity inhibits TH17 cell differentiation and promotes Treg differentiation.

[0027] A. Under the optimal Th17 differentiation conditions containing cytokines TGFβ, IL-1β, IL-6 and IL23, the effect of different concentrations of Srci1 on the differentiation of IL-17+ Cells and FOXP3 + Scri1 inhibited the differentiation of Th17 cells and promoted the transformation of Th17 cells into Treg cells.

[0028] B. Effects of different concentrations of Srci1 on the mRNA expression levels of Th17-related gene loci under optimal Th17 differentiation conditions containing cytokines TGFβ, IL-1β, IL-6, and IL23.

[0029] C. Effect of different concentrations of Srci1 on the differentiation of IL-17 cells under standard Th17 differentiation conditions containing cytokines TGFβ and IL-6. + Cells and FOXP3 + Scri1 inhibits the differentiation of Th17 cells and promotes the transformation of Th17 cells into Treg cells.

[0030] D. Effects of different concentrations of Srci1 on the mRNA expression levels of Th17-related gene loci under standard Th17 differentiation conditions containing cytokines TGFβ and IL-6.

[0031] E. In the presence of cytokines TGFβ and IL-2, the effect of different concentrations of Srci1 on the Treg differentiation of IL-17 + Cells and FOXP3 + Srci1 promotes Treg cell differentiation.

[0032] F. Effect of Srci1 administration on the mRNA expression levels of Treg-related genes under Treg differentiation conditions containing cytokines TGFβ and IL-2.

[0033] G. Effect of different concentrations of Srci1 on IFNγ expression under Th1 differentiation conditions + Scri1 did not affect Th1 differentiation.

[0034] H. Effect of different concentrations of Srci1 on IL-4 in Th2 differentiation conditions + Scri1 did not affect the differentiation of Th2 cells.

[0035] IK. Using point mutation STAT3C to mimic activated STAT3, STAT3C reversed the effect of Srci1 on TH17 cell differentiation.

[0036] Figure 3. Administration of Srci1 to inhibit the kinase activity of Lck / Fyn or expression of STAT3 mutant (Dup 328-330and R335W) on the in vivo differentiation of Th17 cells and EAE disease.

[0037] A. When EAE disease symptoms begin, the effect of daily administration of 50 mg / kg of Scri1 or DMSO on EAE disease scores. Scri1 alleviates EAE symptoms.

[0038] BD. Flow cytometric data of infiltrating T cells from the CNS, including IL-17, after administration of Srci1 and DMSO. + Cells and FOXP3 + Scri1 inhibits the differentiation of Th17 cells and promotes the differentiation of Treg cells in the central nervous system.

[0039] E. IFNγ expression in CNS-infiltrating T cells after administration of Srci1 and DMSO. + Scri1 does not affect the differentiation of Th1 cells.

[0040] F. IL-17+IFNγ expression of infiltrating T cells from the CNS after administration of Srci1 and DMSO. + Percentage of cells.

[0041] G. FOXP3 expression in CNS-derived T cells after administration of Srci1 and DMSO. + Cells and IL-17 + Scri1 inhibits the differentiation of Th17 cells and promotes the differentiation of Treg cells in the central nervous system.

[0042] H.Stat3 fl / fl CD4 cre Schematic diagram of adoptive transfer of EAE in 2D2 mice.

[0043] IL. Empty vector, STAT3 WT, Dup 328-330 After adoptive transfer of WT STAT3-overexpressing T cells into Rag1- / - mice and then immunization with MOG, the EAE disease scores at different days, the maximum disease scores during the entire course of the disease, the weight loss at different days, and the incidence rate at different days were significantly higher in mice transplanted with WT STAT3-overexpressing T cells than in mice transplanted with empty vector, Dup 328-330 Mice transplanted with R335W-overexpressing T cells rarely develop the disease or the symptoms are very mild.

[0044] MO received empty vector, WT STAT3, Dup 328-330 In mice transplanted with T cells overexpressing R335W, after MOG immunization, the number of CD3+ CD4 + The percentage of CD3 in CNS + CD4 + The percentage of CD3 in CNS + CD4 + The number of Dup 328-330 and R335W had no significant effect on inhibiting Th17 differentiation in vivo.

[0045] PS. received empty vector, WT STAT3, Dup 328-330 After MOG immunization, CD4 + Flow cytometry data of cells, including IL-17 + The percentage of cells, FOXP3 + The percentage of cells, IFNγ + The percentage of cells, T cells overexpressing WT STAT3 can be well induced to differentiate into Th17 cells in mice, while overexpressing empty vector, Dup 328-330 R335W and R335W could not induce differentiation into Th17.

[0046] Figure 4 Effects of STAT3 MUT on Th17 cell differentiation.

[0047] AB.STAT3 MUT can inhibit TH17 cell differentiation and promote Treg differentiation.

[0048] Figure 5. Interaction between Lck / Fyn and STAT3 and TCR stimulation induces STAT3 phosphorylation through Lck / Fyn.

[0049] AC.TCR stimulation can induce STAT3 phosphorylation through Lck / Fyn.

[0050] IJ.STAT3 binds to Lck / Fyn.

[0051] K. Inhibition of Lck / Fyn kinase activity will inhibit the recruitment of STAT3 to gene sites specifically expressed in Th17 cells. DETAILED DESCRIPTION

[0052] In the present invention, the immune disease is mainly a Th17 cell-mediated immune disease, selected from autoimmune diseases or non-autoimmune diseases; preferably, the autoimmune disease is selected from autoimmune encephalomyelitis, multiple sclerosis, rheumatoid arthritis, psoriasis, ankylosing spondylitis and inflammatory bowel disease, and / or other autoimmune diseases mediated by Th17 cells, and the non-autoimmune disease is a cytokine storm generated during infection or treatment mediated by Th17 cells.

[0053] Lck / Fyn-mediated STAT3 activation pathway

[0054] In the present study, Lck / Fyn plays an important role in the differentiation process of Th17 cells. The SH2 domain of Lck / Fyn can bind to the polypeptide at positions 328-335 on STAT3, causing a conformational change in Lck / Fyn, thereby further exposing the kinase active site of Lck / Fyn and optimizing the kinase activity of Lck / Fyn. Lck / Fyn then directly phosphorylates STAT3 through the kinase active site, and then cooperates with the JAK kinase-induced STAT3 pathway to transcriptionally regulate the master transcription factor of Th17 cells. This mediates the differentiation of Th17 cells, as shown in Figure 1.

[0055] Therefore, inhibiting Lck / Fyn activity or blocking the correct binding of Lck / Fyn to STAT3 can block STAT3 activation, thereby inhibiting Th17 cell-mediated immune diseases; but it does not affect the phosphorylation of other STAT transcription factors (STAT4, STAT5, STAT6) or the differentiation of other T cell subsets. Therefore, it is believed that Lck / Fyn kinase inhibitors can inhibit Th17 differentiation by inhibiting STAT3 activation, and thus can be used to treat Th17 cell-mediated immune diseases or provide preventive / therapeutic capabilities for Th17 cell-mediated immune diseases.

[0056] The differentiation of Th17 cells refers to the differentiation of CD4 naive T cells into Th17 cells in the presence of relevant cytokines.

[0057] The autoimmune diseases include autoimmune encephalomyelitis, multiple sclerosis, rheumatoid arthritis, psoriasis, ankylosing spondylitis and inflammatory bowel disease.

[0058] STAT phosphorylation regulator Lck / Fyn

[0059] Lck and Fyn are members of the Src family of kinases. Activation of Lck / Fyn is a key factor in initiating the TCR signaling pathway. When the TCR is stimulated, Lck / Fyn is activated and can directly phosphorylate STAT3, thereby inducing the differentiation of Th17 cells. During the differentiation of Th17 cells, the SH2 domain of Lck / Fyn can bind to the polymorphic sequence at positions 328-335 on STAT3, maximizing the kinase activity of Lck / Fyn. The kinase active site of Lck / Fyn can directly phosphorylate STAT3, but Lck / Fyn does not directly affect the phosphorylation of STAT4, STAT5, or STAT6.

[0060] Blockers of Lck / Fyn-mediated STAT3 activation

[0061] The Lck / Fyn-mediated STAT3 activation inhibitor is suitable for weakening, blocking or changing the interaction between Lck / Fyn and STAT3, and / or inhibiting the activity of Lck / Fyn kinase to inhibit or block the phosphorylation or activation of STAT3.

[0062] The Lck / Fyn-mediated STAT3 activation blocker is selected from: Lck / Fyn kinase inhibitors; substances that specifically inhibit the correct binding of Lck / Fyn to STAT3.

[0063] Lck / Fyn kinase inhibitors

[0064] Compounds that inhibit the activity of the STAT3 phosphorylation regulator Lck / Fyn kinase include, but are not limited to, inhibiting Lck / Fyn kinase gene transcription or expression, or Lck / Fyn kinase inhibitors binding to the kinase active site, thereby inhibiting its activity.

[0065] The Lck / Fyn kinase inhibitor can be selected from siRNA, shRNA, antibodies and small molecule compounds.

[0066] The small molecule compound can be Srci1, Dasatinib, Bosutinib, Saracatinib, 1-Naphthyl PP1 hydrochloride, TL02-59, RK 24466, eCF506, Ginkgolic acid C17:1, Nintedanib, XL228, Pelitinib (EKB-569), AMG-47a, PP121, 1-Naphthyl PP1 (1-NA-PP1), Tirbanibulin, Dehydroabietic acid, MNS (3,4-Methylenedioxy-β-nitrostyrene), 7-Hydroxy-4-chromone, Myristic Acid, HPK1-IN-2, 1-NM-PP1, KX1-004, PP2, PP1, WH-4-023, CCT196969, SU6656, Dasatinib Monohydrate, ON123300, AD80, Repotrectinib (TPX-0005), UM-164, Elzovantinib (TPX-0022), DGY-06-116, TPX-0046, MLR-1023 and other small molecule compounds.

[0067] Small molecule compounds

[0068] The small molecule compound in the present invention refers to a compound with a molecular weight of less than 1000 Daltons.

[0069] Srci1

[0070] Chemical name: 6,7-Dimethoxy-N-(4-phenoxyphenyl)-4-quinazolinamine

[0071] Molecular formula: C 22 H 19 N3O3

[0072] IC50: Src, 44nM; Lck, 88nM

[0073] Cas No.179248-59-0

[0074] The structural formula is as follows:

[0075] Dasatinib

[0076] Chemical Name:

[0077] N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-yl]amino]-1,3-thiazole-5-carboxamide

[0078] Molecular formula: C 22 H 26 C l N7O2S

[0079] IC50: Bcr-Abl, 1.0nM; Src, 0.5nM; Lck, 0.4nM; Yes, 0.5nM; c-Kit, 5.0nM; PDGFRβ, 28nM; p38, 100nM; Her1, 180nM; Her2, 710nM; FGFR-1, 880nM; MEK, 1700nM

[0080] Cas No.302962-49-8

[0081] The structural formula is as follows:

[0082] Bosutinib

[0083] Chemical Name:

[0084] 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methyl-1-piperazinyl)propox y]-3-quinolinecarbonitrile

[0085] Molecular formula: C 26 H 29 C l2 N5O3

[0086] IC50: Src, 1.2nM; Abl, 1nM

[0087] Cas No.380843-75-4

[0088] The structural formula is as follows:

[0089] Saracatinib

[0090] Chemical Name:

[0091] N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine

[0092] Molecular formula: C 27 H 32 C l N5O5

[0093] IC50: Src, 2.7nM; v-Abl, 30nM; EGFR, 66nM; c-Kit, 200nM

[0094] Cas No.379231-04-6

[0095] The structural formula is as follows:

[0096] 1-Naphthyl PP1hydrochloride

[0097] Chemical Name:

[0098] 1-tert-butyl-3-(naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyriMidin-4-aMine hydrochloride

[0099] Molecular formula: C 19 H 19 N5ClH

[0100] IC50: c-Fyn, 0.6μM; c-Abl, 0.6μM; v-Src, 1.0μM; CDK2, 18μM; CAMKII, 22μM

[0101] Cas No.956025-47-1

[0102] The structural formula is as follows:

[0103] TL02-59

[0104] Chemical Name:

[0105] 3-[(6,7-Dimethoxy-4-quinazolinyl)oxy]-N-{4-[(4-ethyl-1-piperazinyl)methyl]-3-(trifluorom ethyl)phenyl}-4-methylbenzamide

[0106] Molecular formula: C 32 H 34 F3N5O4

[0107] IC50: Fgr, 0.03nM

[0108] CAS No.1315330-17-6

[0109] The structural formula is as follows:

[0110] RK 24466

[0111] Chemical name: 7-Cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

[0112] Molecular formula: C 23 H 22 N4O

[0113] IC50: Lck, 0.001μM; Lckcd, 0.002μM

[0114] CAS No.213743-31-8

[0115] The structural formula is as follows:

[0116] Correct binding of Lck / Fyn to STAT3

[0117] The correct binding is that the polypeptide at positions 328-335 on STAT3 binds to the SH2 domain of Lck / Fyn, causing a conformational change in Lck / Fyn, thereby further exposing the kinase active site of Lck / Fyn, thereby allowing the kinase activity of Lck / Fyn to reach an optimal state; then Lck / Fyn phosphorylates STAT3 through the kinase active site.

[0118] Substances that specifically inhibit the correct binding of Lck / Fyn to STAT3

[0119] The substance that specifically inhibits the correct binding of Lck / Fyn to STAT3 inhibits the interaction between Lck / Fyn and STAT3, or blocks the conformational change of Lck / Fyn; preferably, the substance that specifically inhibits the correct binding of Lck / Fyn to STAT3 inhibits the binding of the Lck / Fyn domain to the 328-335 peptide segment on STAT3, inhibits the conformational change of Lck / Fyn kinase, and inhibits the exposure of the active site of Lck / Fyn kinase; more preferably, the substance that specifically inhibits the binding of Lck / Fyn to STAT3 is selected from antibodies, small molecule compounds or polypeptides.

[0120] The substance that specifically inhibits the binding of Lck / Fyn to STAT3 includes a STAT3 mutant or a fragment thereof. Preferably, the STAT3 mutant or the fragment thereof includes a mutation in the 328-335 peptide segment compared to the wild-type STAT3.

[0121] The mutation is two point mutations occurring simultaneously in the amino acid sequence at positions 328-335 compared to the wild-type STAT3, including duplication of cysteine, methionine, and proline (CMP) in the amino acid sequence at positions 328-330 of the wild-type STAT3, and simultaneous mutation of the amino acid at position 335 of the wild-type STAT3 from arginine (R) to tryptophan (W).

[0122] Blocker preparation product of the present invention

[0123] The product must contain a blocker of Lck / Fyn-mediated STAT3 phosphorylation, and the blocker of Lck / Fyn-mediated STAT3 phosphorylation serves as an active ingredient for the aforementioned function.

[0124] In the product, the active ingredient that exerts the aforementioned function may be only the inhibitor of Lck / Fyn-mediated STAT3 phosphorylation, or may contain other chemicals that can exert similar functions.

[0125] The product can be a single-component product containing only a blocker for Lck / Fyn-mediated STAT3 phosphorylation, or a multi-component product containing a blocker for Lck / Fyn-mediated STAT3 phosphorylation.

[0126] The form of the product is not particularly limited and can be in the form of solid, liquid, gel, semi-fluid, aerosol or other substances.

[0127] The product may be used in the pharmaceutical field, health care product field, food field, etc.

[0128] The product is used for mammals or T cells of the mammals.

[0129] Biomaterials for preparing products for preventing or treating Th17 cell-mediated immune diseases

[0130] The biological material may be one or more of the following:

[0131] a. A polypeptide comprising a STAT3 mutant or a fragment thereof, wherein the STAT3 mutant or the fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 1; preferably, the STAT3 mutant or the fragment thereof is further fused with a cell-penetrating peptide; further preferably, the cell-penetrating peptide is located at the N-terminus of the STAT3 mutant or the fragment thereof; further preferably, the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO: 5;

[0132] b. a polynucleotide encoding the polypeptide;

[0133] c. a construct comprising the polynucleotide described in b;

[0134] d. A host cell comprising the construct described in c or a host cell whose genome has been integrated with the polynucleotide described in b.

[0135] The polypeptide is highly homologous to wild-type STAT3. Compared with the amino acid sequence of STAT3, two point mutations occur simultaneously in the amino acid sequence at positions 328-335, including duplication of cysteine, methionine, and proline (CMP) in the amino acid sequence at positions 328-330 of wild-type STAT3, and mutation of the amino acid at position 335 of wild-type STAT3 from arginine (R) to tryptophan (W).

[0136] The construct is a recombinant vector comprising a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 1. Preferably, the construct is a recombinant expression vector comprising a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 1.

[0137] In the present invention, the vector refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 1 can be inserted. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector may contain multiple elements for controlling expression.

[0138] The host cell refers to a cell into which the above construct is introduced or into which a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 1 is integrated into the genome.

[0139] Biomaterial preparation product of the present invention

[0140] The product must contain at least one of the above-mentioned biological materials, including a polypeptide having a sequence as shown in SEQ ID NO: 1, a polynucleotide encoding the polypeptide, a construct containing the polynucleotide, a host cell containing the construct, or a host cell with the polynucleotide integrated into its genome; and the biological material serves as the active ingredient for the aforementioned function.

[0141] In the product, the active ingredient that performs the aforementioned functions may be only one or more of the aforementioned biological materials, or may include other biological materials that can perform similar functions.

[0142] The product may be only one or more of the above-mentioned biomaterials, or may be a multi-component product containing one or more of the above-mentioned biomaterials.

[0143] The product is used for mammals or T cells of the mammals.

[0144] Preparation of medicines from biological materials in the present invention

[0145] It contains at least one of the above-mentioned biological materials, the sequence of which includes a polypeptide shown in SEQ ID NO: 1, a polynucleotide encoding the polypeptide, a construct containing the polynucleotide, a host cell containing the construct, or a host cell whose genome is integrated with the polynucleotide; optionally, it also contains one or more pharmaceutically acceptable excipients.

[0146] Lck / Fyn kinase inhibitors for drug preparation

[0147] The drug is prepared using the Lck / Fyn kinase inhibitor as the main active ingredient or one of the main active ingredients. Generally, in addition to the active ingredient, the drug also includes one or more pharmaceutically acceptable carriers or excipients according to the requirements of different dosage forms.

[0148] The above-mentioned "pharmaceutically acceptable" means that when the molecular entities and compositions are appropriately administered to animals or humans, they will not produce adverse, allergic or other untoward reactions.

[0149] "Pharmaceutically acceptable carriers or excipients" should be compatible with the Lck / Fyn kinase inhibitor, meaning they can be co-mingled with it without significantly reducing the efficacy of the pharmaceutical composition under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer. These materials are used as needed to aid in the stability of the formulation or to help increase the activity or its bioavailability or to produce an acceptable taste or flavor in the case of oral administration.

[0150] In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited and can be prepared into injections, oral liquids, tablets, capsules, dripping pills, sprays, etc., and can be prepared by conventional methods. The choice of pharmaceutical dosage form should match the mode of administration.

[0151] The drug is used for mammals or T cells of the mammals.

[0152] Methods for treating Th17 cell-mediated immune diseases

[0153] In the present invention, the method for treating Th17 cell-mediated immune diseases comprises administering to a subject an effective amount of the above-mentioned Lck / Fyn-mediated STAT3 activation blocker, and / or any one or more of the above-mentioned biomaterials.

[0154] The effective amount refers to a dose that can achieve the treatment, prevention, alleviation and / or relief of the Th17 cell-mediated immune disease or disorder described in the present invention in the subject.

[0155] The treatment method can be carried out by conventional administration methods such as oral feeding, sublingual administration, rectal administration, intraperitoneal injection, intravenous injection, intramuscular injection, etc.

[0156] The treatment method can be to administer the above-mentioned product for treating Th17 cell-mediated immune diseases to the subject during the prevention period of Th17 cell-mediated immune diseases, or during the early, middle, or late stages of treatment.

[0157] In addition to using the above-mentioned products, drugs or biological materials for treating Th17 cell-mediated immune diseases, the treatment method may also simultaneously or sequentially administer to the subject an effective amount of other drugs for treating Th17 cell-mediated immune diseases and / or implement other means of treating Th17 cell-mediated immune diseases on the subject.

[0158] The other drugs for treating Th17 cell-mediated immune diseases refer to drugs for treating Th17 cell-mediated immune diseases other than the blockers, biomaterials, products, and drugs of the present invention. They can be selected from one or more of Tocilizumab, Sarituzumab, Satlizumab, Siltuximab, Risankizumab, Secukinumab, Fedratinib, Upadacitinib, Filgotinib, Abrocitinib, etc.

[0159] The other means of treating Th17 cell-mediated immune diseases can be selected from one or more of the following: administration of anti-inflammatory drugs, administration of corticosteroids, administration of analgesics, administration of immunosuppressive drugs, surgical treatment, etc.

[0160] The subject of the treatment method is a mammal or T cells of the mammal.

[0161] The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, or the like.

[0162] CD4 naive T cells

[0163] Naive CD4 T cells are activated after interacting with antigen-MHC complexes and differentiate into specific subsets based on the cytokine environment of the microenvironment. Following activation, they differentiate into different subsets under the regulation of different cytokines, including Th1, Th2, Th9, Th17, Th22, Tfh, and regulatory T cells (Tregs).

[0164] Th17 cells

[0165] Th17 cells participate in inflammatory responses and defense against extracellular pathogens, particularly in the intestinal and respiratory mucosa. The characteristic transcription factor of Th17 cells is RORγt, and they secrete IL-17A, IL-17F, IL-21, IL-22, and IL-26. Th17 cells are associated with various autoimmune diseases, such as autoimmune encephalomyelitis, multiple sclerosis, rheumatoid arthritis, psoriasis, and inflammatory bowel disease.

[0166] Treg cells

[0167] Treg cells are a type of cell that negatively regulate immune responses. They express CD25 and Foxp3. Abnormal Treg function can lead to various autoimmune and inflammatory diseases.

[0168] Th1 cells

[0169] Th1 cells participate in cellular immune responses and defense against pathogenic microbial infection, including promoting the proliferation and differentiation of cytotoxic T cells and the phagocytic function of macrophages. The characteristic transcription factor of Th1 cells is T-bet, and they secrete IL-2, IFN-γ, TNF-α, and LT-α. The pro-inflammatory properties of Th1 cells enable pathogen clearance and anti-tumor immunity. However, Th1 and IFN-γ can contribute to inflammatory diseases (such as inflammatory bowel disease) and autoimmune diseases (such as type 1 diabetes and rheumatoid arthritis).

[0170] Th2 cells

[0171] Th2 cells participate in humoral immune responses and defense against parasitic infections, including promoting B cell proliferation and differentiation into plasma cells and antibody production. The characteristic transcription factor of Th2 cells is GATA3, and they secrete cytokines such as IL-4, IL-5, IL-6, IL-10, and IL-13. Excessive Th2 responses are associated with allergies and asthma.

[0172] IL-17 cytokine

[0173] Interleukin-17 is a key pro-inflammatory cytokine produced mainly by Th17 cells and is highly expressed in patients with a variety of autoimmune diseases, including psoriasis, psoriatic arthritis, rheumatoid arthritis and multiple sclerosis.

[0174] FOXP3 transcription factor

[0175] It is a Treg cell-specific transcription factor that plays an important role in regulating the development and function of Treg cells. It maintains immune system homeostasis by regulating the stability and suppressive function of Treg cells.

[0176] IFN-γ cytokine

[0177] Interferon γ is a Th1 cell and CD8 + The cytokines secreted by CTL cells can act on dendritic cells and macrophages to induce the secretion of more IL-12. Once Th1 cells mature, they secrete IFN-γ, which promotes further Th1 differentiation through autocrine pathways.

[0178] IL-4 cytokine

[0179] That is, interleukin-4. The differentiation of Th2 cells depends on IL-4. IL-4 is necessary for Th2 differentiation and maturation. It is also an autocrine product during the maturation of Th2 cells, that is, IL-4 can be produced by Th2 cells themselves.

[0180] STAT3

[0181] STAT3 is a protein composed of 770 amino acids, and its complete amino acid sequence is:

[0182] Dup 328-330

[0183] The STAT3 mutant fragment, compared with the STAT3323-340 peptide segment, has a duplication of the amino acid sequence at positions 328-330 of the STAT3 wild type.

[0184] R335W

[0185] The STAT3 mutant fragment has a polypeptide sequence that is different from the STAT3323-340 peptide fragment, in which the arginine at position 335 of the wild-type STAT3 is mutated to tryptophan.

[0186] STAT3 WT

[0187] The wild-type fragment of STAT3 is the 323-340 peptide segment of STAT3.

[0188] STAT3 MUT

[0189] The STAT3 mutant fragment, the polypeptide sequence is compared with the STAT3323-340 peptide segment, and the amino acids 328-330 of STAT3 are repeated and the arginine at position 335 is mutated to tryptophan.

[0190] Cell-penetrating peptides

[0191] A short peptide that can effectively transport small molecule drugs, proteins, peptides, nucleic acid fragments and nanocarriers (such as liposomes, polymer micelles, inorganic nanoparticles, etc.) across the cell membrane. In the embodiment of the present invention, Dup 328-330 , R335W, STAT3 WT, and STAT3 MUT further include a cell-penetrating peptide, the amino acid sequence of which is shown in SEQ ID NO:5.

[0192] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0193] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the examples, any prior art methods, devices, and materials similar or equivalent to those in the examples may be used to implement the present invention, based on the knowledge of the prior art by those skilled in the art and the disclosure of this invention.

[0194] Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein utilize conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These conventional techniques are all described in detail in the published literature.

[0195] 1. Materials and Reagents

[0196] RPMI 1640 culture medium, fetal bovine serum, and penicillin-streptomycin were purchased from Invitrogen; Srci1 was purchased from Sigma; and BMS509744 was purchased from MedChemExpress.

[0197] Anti-mouse CD3(clone 145-2C11,16-0031), anti-mouse CD28(clone 37.51,16-0281), anti-mouse IFNγ(clone XMG1.2,16-7311), anti-mouse IL-4(clone 11B11,16-7041), anti-mouse FOXP3(clone FJK-16s, 17-5773) was purchased from eBioscience;

[0198] Anti-mouse CD4 (clone RM4-5, 550954), anti-mouse CD25 (clone 7D4, 558642), anti-mouse CD44 (clone IM7, 559250), anti-mouse CD62L (clone MEL-14, 553150), anti-mouse IL-17 (clone TC11-18H10, 559502), anti-mouse IFNγ (clone XMG1.2, 561040, for staining), PE Anti-STAT3 (pY705) (clone 4 / P-STAT3) (612569), PerCP-Cy5.5 Anti-STAT4 (pY693) (clone 38 / p-STAT4) (561217), Alexa Fluor 647 Anti-STAT5 (pY694) (clone 47 / STAT5) (612599), Alexa Fluor 488 Anti-STAT6 (pY641) (clone J71-773.58.11) (558243) and anti-CD90.1 (Thy1.1) (clone OX-7, 561406), PE Mouse IgG2a, κ Isotype Control (clone MOPC-173) (558595), PerCP-Cy5.5 Mouse IgG2b, κ Isotype Control (clone 27-35) (565380), Alexa Fluor 647 Mouse IgG1κ Isotype control (clone MOPC-21) (557783), Alexa Fluor 488 Mouse IgG1κ Isotype Control (clone MOPC-21) (557782), BV421 Rat Anti-Mouse CD126 (IL-6RA) (clone D7715A7), PE Rat anti-Mouse CD360 (IL-21 Receptor) (clone 4A9) were purchased from BD Bioscience.

[0199] CD130 (IL-6ST) (clone KGP130) was purchased from Invitrogen; Anti-mTOR (clone 7C10, 2983), p-mTOR (clone D9C2, 5536), anti-AKT (clone C57E7, 4691), anti-phospho-AKT Ser473(clone#D9E,4060),anti-4E-BP1(236B4,2855),anti-p70S6K(clone 49D7,2708),anti-p-p70S6K(9208),anti-phospho-STAT3(clone D3A7,9145),anti-STAT3(clone 79D7,4904),anti-STAT3(clone124H6,4904,for endogenous Co-IP),anti-Lck(2752,for WB), anti-Flag (clone 9A3, 8146), anti-V5 (clone D3H8Q, 13202) were purchased from Cell Signaling Technology.

[0200] Anti-c-Myc (clone Y69) was purchased from Abcam; Mouse IL-6 antibody, mouse IL-23 antibody and mouse IL-21 antibody were purchased from R&D systems; Mouse IL3, mouse IL-6, mouse SCF were purchased from PeproTech; CD90.1 magnetic beads were purchased from Milltenyi Biotec; EasySep Mouse Hematopoietic Progenitor Cell Enrichment Kit was purchased from StemCell Technology.

[0201] 2. Experimental Animals

[0202] STAT3 fl / fl CD4 cre Mice were obtained from Chen Dong (Tsinghua University) with permission from Dr. Shizuo Akira (Osaka University, Japan).

[0203] C57BL / 6 and Rag1- / - mice were from The Jackson Laboratory.

[0204] 3. CD4 Naive T Cell Culture

[0205] CD4 naive T cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin-streptomycin, 55 μM β-mercaptoethanol, and 2 mM glutamine.

[0206] 4. Differentiation of CD4 Naive T Cells

[0207] Approximately 400,000 naive CD4 T cells were seeded into a 48-well plate and coated with 2 μg / ml anti-mouse CD3 and 1 μg / ml anti-mouse CD28. The conditions for inducing CD4 naive T cell differentiation are as follows:

[0208] Tregs: 0.5ng / ml TGFβ, 200U / ml mouse IL-2, 1μg / ml anti IFNγ and 1μg / ml anti-mouse IL-4.

[0209] Optimal conditions for Th17: 2.5 ng / ml TGFβ, 10 ng / ml mouse IL-1β, 10 ng / ml mouse IL-6, 10 ng / ml mouse IL-23, 1 μg / ml anti-IFNγ and 1 μg / ml anti-mouse IL-4.

[0210] Standard conditions for Th17 cells: 2.5 ng / ml TGFβ, 20 ng / ml mouse IL-6, 1 μg / ml anti-IFNγ, and 1 μg / ml anti-mouse IL-4.

[0211] Th1: 10ng / ml mouse IL-12, 1μg / ml anti-mouse IL-4.

[0212] Th2: 20ng / ml mouse IL-4, 1ug / ml anti-IFNγ.

[0213] 24 hours after inoculation, the indicated concentrations of Scri1 or BMS509744 were added. Fresh culture medium was replenished on the fourth day of inoculation. For experimental groups containing small molecule compounds, fresh culture medium containing the same concentration of small molecule compounds was replenished. On the fifth day of inoculation, cells were stimulated with phorbol myristate myristate (PMA), ionomycin, and Golgistop for 4-6 hours to activate T cells and secrete cytokines. Intracellular staining was performed in the absence of the small molecule compounds.

[0214] 5. Analysis of mRNA Expression by qRT-PCR

[0215] At the end of differentiation, T cells were restimulated with anti-CD3 and anti-CD28-coated T cells for 5 hours. mRNA expression of the corresponding genes was determined by qRT-PCR. β-actin (Actb) was used as an internal control for normalization.

[0216] 6. Preparation of Retroviral Vectors and T Cell Infection

[0217] Preparation of retroviral vector: encoding wild-type STAT3 WT or its mutant Dup 328-330 The gene of mutant R335W was cloned into pMIG-retroviral vector, Dup 328-330 , R335W, and MUT are mutants of STAT3, and the GFP gene is replaced by the Thy1.1 reporter gene. 328-330 293T cells (ATCC, CRL-3216) were transfected with the pMIG-retroviral vector encoding the R335W gene and the pCL-ECO retroviral packaging vector using the eukaryotic cell nucleic acid transfection reagent LipoMax. All cells were tested for mycoplasma contamination. After transfection, cells were cultured for 48–72 hours. The supernatant was collected after filtration through a 45 μm filter.

[0218] T cell infection: Add the virus-containing supernatant to pre-activated naive CD4 T cells (20 hours post-inoculation). Centrifuge the pre-activated CD4 T cells at 1000g for 2 hours, then incubate for another 2 hours. Wash and culture the naive CD4 T cells under Th17 differentiation conditions. On the second day, reinfect the naive CD4 T cells and culture them for 4 days. Add fresh medium if necessary and repeat the above steps for stimulating the cells for intracellular staining.

[0219] 7. Establishment of EAE Model in Mice

[0220] EAE model in C57BL / 6 mice: 8-week-old C57BL / 6 mice were divided into groups of approximately 10. Myeloid oligodendrocyte glycoprotein MOG35-55 (MEVGWYRSPFSROVHLYRNGK) peptide was emulsified in complete Freund's adjuvant (CFA). C57BL / 6 mice were immunized twice with 300 μg of MOG35-55 peptide, and then pertussis toxin was injected intraperitoneally to establish the EAE model.

[0221] 2D2 mouse transfer-induced EAE model: from STAT3 fl / fl CD4 cre CD4 naive T cells (CD4 + CD25 - CD62 high CD44low ) and cultured under TH17 differentiation conditions. After pre-activation of anti-CD3 and anti-CD28 for 20 h, Thy1.1 cells were infected with retroviral vectors encoding wild-type STAT3 WT or its mutants at 37°C and 2000 rpm. + CD4 naive T cells were incubated for 2 hours and then cultured for another 2 hours. The cells were then washed and replaced with fresh medium containing Th17 differentiation conditions. The next day, CD4 naive T cells were re-infected and cultured for another 3 days. After the above cells were sorted, they were re-cultured on 48-well plates that were pre-coated with 2μg / ml anti-CD3 (clone 145-2C11) and 1μg / ml anti-CD28 (clone 37.51) antibodies and cultured under TH17 conditions for 48 hours. Thy1.1 + The cells were injected intravenously into Rag1- / - mice, with 10 cells per mouse 6 cells. Mice were immunized with 200 μg of the MOG35-55 (MEVGWYRSPFSROVHLYRNGK) peptide emulsified in complete Freund's adjuvant (CFA). On days 0 and 2 after immunization, 250 ng of pertussis toxin were intraperitoneally injected. Disease scores range from 0 to 5: 0, no clinical symptoms; 1, tail weakness or staggering gait with a tight tail; 2, staggering gait; 3, hindlimb paralysis; 4, hindlimb and forelimb paralysis; and 5, death. Mice were randomly assigned to treatment and control groups, and the scores were scored blindly.

[0222] When clinical symptoms began to appear, DMSO or Scri1 were injected intraperitoneally daily at 50 mg / kg body weight. At the end of the experiment, all mice, including those with and without EAE symptoms, were euthanized and analyzed for T cell infiltration into the central nervous system. Statistically significant results were analyzed using a t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0223] All animal experiments were approved by the institutional IACUC committee.

[0224] Example

[0225] Example 1 Scri1 inhibits the differentiation of Th17 cells and promotes the differentiation of Treg cells by inhibiting the activity of Lck / Fyn

[0226] Experimental Methods: Naive CD4 T cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin-streptomycin, 55 μM β-mercaptoethanol, and 2 mM glutamine. Approximately 400,000 naive CD4 T cells were plated in 48-well plates and coated with 2 μg / ml anti-mouse CD3 and 1 μg / ml anti-mouse CD28, respectively. Optimal differentiation conditions were established by adding 2.5 ng / ml TGFβ, 10 ng / ml mouse IL-1β, 10 ng / ml mouse IL-6, 10 ng / ml mouse IL-23, 1 μg / ml anti-IFNγ, and 1 μg / ml anti-mouse IL-4 to induce Th17 differentiation of naive CD4 T cells. 2.5 ng / ml TGFβ, 20 ng / ml mouse IL-6, 1 μg / ml anti-IFNγ, and 1 μg / ml anti-mouse IL-4 were added as standard differentiation conditions to induce Th17 differentiation of naive CD4 T cells. 0.5 ng / ml TGFβ, 200 U / ml mouse IL-2, 1 μg / ml anti-IFNγ, and 1 μg / ml anti-mouse IL-4 were added to induce Treg differentiation of naive CD4 T cells. 10 ng / ml mouse IL-12 and 1 μg / ml anti-mouse IL-4 were added to induce Th1 differentiation of naive CD4 T cells. 20 ng / ml mouse IL-4 and 1 μg / ml anti-IFNγ were added to induce Th2 differentiation of naive CD4 T cells. 24 hours after inoculation, 2.5 μM, 5 μM, and 7.5 μM Srci1 were added as experimental groups, and DMSO was added as a control group. On day 4 after inoculation, the experimental group received fresh culture medium containing the corresponding concentrations of small molecule compounds, while the control group received fresh culture medium. On day 5 after inoculation, T cells were activated to secrete cytokines and intracellular staining was performed. Finally, the degree of T cell differentiation was assessed by flow cytometry analysis of cytokine secretion. At the end of differentiation, T cells were restimulated with coated anti-CD3 and anti-CD28 for 5 hours. mRNA expression of the corresponding genes was determined by qRT-PCR.

[0227] The purpose of this example is to study the effect of Scri1 on the differentiation of Th17, Treg, Th1 and Th2 cells. The experiment inhibited the activity of Lck / Fyn kinase by a series of Lck / Fyn kinase inhibitors, reduced the differentiation of Th17 cells, and transformed Th17 into Treg cells, but had no effect on the differentiation of Th1 and Th2 mediated by STAT4 and STAT6. The experimental results are shown in Figure 2A-H. Under the optimal Th17 differentiation conditions, the experimental group with Scri1 added had a higher IL-17 level than the control group without Scri1. + The percentage of cells decreased significantly, and with the increase of Srci1 concentration, IL-17 + The percentage of cells decreased in sequence, indicating that the differentiation of Th17 cells was inhibited. The experimental group with Scri1 had a higher FOXP3 + The percentage of cells increased significantly, and with the increase of Srci1 concentration, FOXP3 + The percentage of cells increased successively, indicating that the differentiation of Treg cells was promoted. The results of qRT-PCR showed that the mRNA expression level of Foxp3 was greatly increased, and the mRNA expression levels of IL-17a, IL-17f, IL23r, and IL21 were significantly reduced, which also showed that the differentiation of Th17 cells was inhibited and the differentiation of Treg cells was promoted. The same method was used under the standard differentiation conditions of Th17 to obtain results similar to those under the optimal differentiation conditions of Th17. Under the differentiation conditions of Th1, the experimental groups with 2.5μM and 5μM Scri1 added showed a significant increase in IFNγ compared with the control group without Scri1. + There was no significant change in the percentage of cells, indicating that Scri1 did not affect Th1 differentiation. Under Th2 differentiation conditions, the experimental groups with 2.5μM and 5μM Scri1 showed a significant increase in IL-4 + There was no significant change in the percentage of cells, indicating that Scri1 does not affect Th2 differentiation. This example demonstrates at the cellular level that Lck / Fyn kinase inhibitors can inhibit STAT3-mediated Th17 cell differentiation by reducing Lck / Fyn activity without affecting Th1 and Th2 cell differentiation. Therefore, Lck / Fyn kinase inhibitors can be used as products for treating autoimmune diseases caused by Th17 cells.

[0228] Example 2 The Lck / Fyn kinase inhibitor Scri1 significantly improves the symptoms of the EAE model

[0229] Experimental Methods: Eight-week-old C57BL / 6 mice were divided into groups of approximately 10. The myeloid oligodendritic cell glycoprotein MOG35-55 (MEVGWYRSPFSROVHLYRNGK) peptide, emulsified in complete Freund's adjuvant (CFA), was immunized twice with 300 μg of the MOG35-55 peptide. The EAE model was then established by intraperitoneal injection of pertussis toxin. When clinical symptoms began to appear, DMSO or Scri1 were administered intraperitoneally daily at 50 mg / kg body weight. DMSO served as a vehicle control. At the end of the experiment, all mice, including those with and without EAE symptoms, were euthanized, and T cells infiltrating the central nervous system were analyzed.

[0230] The purpose of this example is to study the therapeutic effect of Scri1 on autoimmune diseases (EAE). The experiment effectively slowed down the clinical symptoms of EAE mice by administering Scri1 to EAE model mice, reduced the differentiation of Th17 cells, and converted Th17 into Treg cells, but had no effect on Th1 cells. The experimental results are shown in Figure 3. In Figure 3A-G, after the mice developed clinical symptoms of EAE, the disease score curve of Scri1 was flatter and the disease score was lower than that of the solvent control group that did not administer Scri1, indicating that the clinical symptoms of EAE were effectively alleviated. And the experimental group that administered Scri1 had IL-17 + A lower percentage of cells had FOXP3 + The percentage of cells was higher in the control group, indicating that Scri1 inhibited the differentiation of Th17 cells in mice and promoted the transformation of Th17 cells into Treg cells, but IFNγ + There was no significant difference in the percentage of cells, indicating that Scri1 had no effect on Th1. This indicates that Scri1 has a significant therapeutic effect on autoimmune diseases and that the use of Lck / Fyn kinase inhibitors as a product for the treatment of autoimmune diseases can effectively treat autoimmune diseases.

[0231] Example 3 Single mutation STAT3 mutant Dup 328-330 Effects of R335W and R335W on Th17 differentiation and EAE disease

[0232] Experimental method: From STAT3 fl / fl CD4 cre CD4 naive T cells (CD4 + CD25 - CD62 high CD44 low) and cultured under TH17 differentiation conditions. After pre-activation of anti CD3 and anti CD28 for 20 hours, CD4 naive T cells were infected with retroviral vectors or empty vectors containing STAT3 WT or Dup328-330 and R335W encoding genes at 37°C and 2000rpm for 2 hours, and then cultured for another 2 hours. The cells were then washed and replaced with fresh culture medium containing Th17 differentiation conditions. The CD4 naive T cells were infected again the next day and cultured for another 3 days. After sorting the above cells, they were re-cultured on a 48-well plate that was pre-coated with 2μg / ml anti CD3 (clone 145-2C11) and 1μg / ml anti CD28 (clone 37.51) antibodies and cultured under TH17 conditions for 48 hours. Thy1.1 was collected + The cells were injected intravenously into Rag1- / - mice, with 10 cells per mouse 6 cells. Rag1- / - mice were immunized with 200 μg of the MOG35-55 (MEVGWYRSPFSROVHLYRNGK) peptide emulsified in complete Freund's adjuvant (CFA). On days 0 and 2 after immunization, 250 ng of pertussis toxin were injected intraperitoneally. At the end of the experiment, all mice, including those with and without EAE symptoms, were euthanized, and T cells infiltrating the central nervous system were analyzed.

[0233] The purpose of this example is to explore the single mutant STAT3 mutant Dup 328-330 Effects of Dup and R335W on Th17 differentiation and EAE disease. 328-330 , R335W are mutant STAT3, in which the amino acids at positions 328-330 of the polypeptide sequence are repeated and the arginine at position 335 is replaced by tryptophan. In Figure 3H-S, mice expressing STAT3 WT and Dup were injected into the body. 328-330 , R335W cells, caused the mice to develop clinical symptoms of EAE, but Dup 328-330 The disease scores of R335W and STAT3 WT were significantly lower than those of STAT3 WT, and Dup 328-330 There was no significant difference in the disease scores between R335W and the empty vector; further comparison of the maximum disease scores of the four, the empty vector and Dup 328-330 , R335W were also significantly lower than STAT3 WT; in addition, the comparison of weight loss and morbidity of mice also showed that Dup 328-330 The pathogenicity of STAT3 WT was higher than that of R335W. 328-330, R335W cells to make mice IL-17 + A lower percentage of cells had FOXP3 + The percentage of cells was higher, and the injection of cells expressing STAT3 WT made the mice IL-17 + A higher percentage of cells had FOXP3 + The percentage of cells was lower, indicating that STAT3 WT promoted the differentiation of Th17 cells, while Dup 328-330 , R335W had no significant effect on inhibiting the differentiation of Th17 cells. + There was no significant difference in the number of cells, indicating that it had no effect on the differentiation of Th1 cells.

[0234] Example 4 Effect of mutant STAT3 MUT on Th17 cell differentiation

[0235] Experimental Methods: The gene encoding STAT3 WT or STAT3 MUT was cloned into the pMIG retroviral vector, and the GFP gene was replaced with the Thy1.1 gene. The pMIG retroviral vectors encoding STAT3 WT or STAT3 MUT and the pCL-ECO retroviral packaging vector were transfected into 293T cells (ATCC, CRL-3216) using the eukaryotic cell nucleic acid transfection reagent LipoMax. All cells were tested for mycoplasma contamination. After transfection, the cells were cultured for 48–72 hours. The supernatant was filtered through a 45 μm filter and collected. The virus-containing supernatant was added to preactivated naive CD4 T cells (20 hours after plating). PBS was also added as a blank control. The preactivated CD4 T cells were centrifuged at 1000 g for 2 hours and then incubated for another 2 hours. The naive CD4 T cells were washed and cultured under Th17 differentiation conditions. The next day, the naive CD4 T cells were reinfected and cultured for 4 days. If necessary, add fresh culture medium and repeat the above steps to stimulate cells for intracellular staining.

[0236] The purpose of this example is to explore the effect of the mutant STAT3 MUT with duplication at 328-330 and mutation of R335 to W335 on Th17 cell differentiation. The experimental results are shown in Figure 4A-B. Compared with the blank control, the IL17 + The percentage of cells increased significantly, FOXP3 + The percentage of IL17 cells in the STAT3 MUT group was significantly decreased. + The percentage of cells with FOXP3 +The percentage of cells increased significantly, indicating that STAT3 MUT has a significant inhibitory effect on Th17 cells and promotes the differentiation of Treg cells, which may have a therapeutic effect on autoimmune diseases induced by Th17 cells.

[0237] Example 5 Interaction between Lck / Fyn and STAT3

[0238] The interaction between Lck / Fyn and STAT3 was demonstrated by co-immunoprecipitation assay. As shown in Figure 5I-J , in the co-immunoprecipitation, obvious bands appeared when STAT3 and Lck and STAT3 and Fyn were co-expressed, indicating that STAT3 interacted with Lck / Fyn.

[0239] Example 6 Lck / Fyn kinase inhibitors inhibit STAT3 phosphorylation

[0240] Experimental Methods: Naive CD4+ T cells were cultured under the conditions indicated in Figure 5A. After 48 hours, the cells were harvested, fixed, permeabilized, and stained with a fluorescently labeled antibody that recognizes STAT3 phosphorylated at Y705, and analyzed by flow cytometry. T cells treated as described above were harvested, lysed, and STAT3 phosphorylation was detected using an anti-STAT3 Y705 phosphorylated antibody and Western blotting.

[0241] Experimental results: As shown in Figures 5A-B, TCR stimulation induced STAT3 phosphorylation, which could be inhibited by the Lck / Fyn inhibitor Srci1. In the presence of proinflammatory cytokines IL-6 and IL23, STAT3 reached the optimal phosphorylation level. Under these conditions, the STAT3 phosphorylation level after Scri1 administration was significantly lower than that after DMSO administration. Figure 5C shows that TCR stimulation alone can induce STAT3 phosphorylation, while maintaining the same total amount of STAT3 in each sample, and that phosphorylation can be inhibited by the Lck / Fyn inhibitor Srci1.

[0242] Example 7 Recruitment of STAT3 to Th17 cell-specifically expressed gene sites

[0243] Experimental method: Differentiating Th17 cells were fixed with paraformaldehyde, then treated with cell lysis buffer, centrifuged to enrich the nuclei, and then churned with nuclease (MNase) and sonicated to break the chromatin into 150p-900bp fragments. The fragments were then incubated with antibodies containing STAT3 (because STAT can bind to the gene sites of Th17-related proteins). STAT3 was then precipitated using beads coupled with protein A / G. The STAT3-bound DNA was then eluted and recovered. The primers marked in the figure were then used to quantify the DNA content at each site by qPCR, that is, the ability of STAT3 to recruit / bind to the indicated gene sites.

[0244] Experimental results: As shown in Figure 5K, administration of the Lck / Fyn kinase inhibitor Scri1 inhibited the activity of Lck / Fyn and suppressed the recruitment of STAT3 to gene sites specifically expressed in Th17 cells.

[0245] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Use of the Lck / Fyn-mediated STAT3 activation pathway as a target in the preparation or screening of a product, wherein the product blocks the Lck / Fyn-mediated STAT3 activation pathway and has any one or more of the following effects: (1) Inhibiting the differentiation of Th17 cells without affecting the differentiation of Th1 and Th2 cells; (2) Promoting the transdifferentiation of differentiating Th17 cells into Treg cells; (3) Preventing or treating Th17 cell-mediated immune diseases.

2. Use of an agent that blocks Lck / Fyn-mediated STAT3 activation in the preparation of a product, characterized in that the product has one or more of the following effects: (1) Inhibiting the differentiation of Th17 cells without affecting the differentiation of Th1 and Th2 cells; (2) Promoting the transdifferentiation of differentiating Th17 cells into Treg cells; (3) Preventing or treating Th17 cell-mediated immune diseases.

3. The use according to claim 2, characterized in that the agent that blocks Lck / Fyn-mediated STAT3 activation is selected from: Lck / Fyn kinase inhibitors; substances that specifically inhibit the correct binding of Lck / Fyn to STAT3.

4. The use according to claim 3, characterized in that the Lck / Fyn kinase inhibitor inhibits the activity of Lck / Fyn kinase or inhibits the transcription or expression of the Lck / Fyn kinase gene; preferably, the Lck / Fyn kinase inhibitor is selected from siRNA, shRNA, antibodies or small molecule compounds; the substance that specifically inhibits the correct binding of Lck / Fyn to STAT3 inhibits the interaction between Lck / Fyn and STAT3, or blocks the conformational change of Lck / Fyn; preferably, the substance that specifically inhibits the correct binding of Lck / Fyn to STAT3 inhibits the binding of the domain of Lck / Fyn to the peptide segment 328-335 on STAT3, or inhibits the conformational change of Lck / Fyn kinase, or inhibits the exposure of the binding site of Lck / Fyn kinase to STAT3; more preferably, the substance that specifically inhibits the correct binding of Lck / Fyn to STAT3 is selected from antibodies, small molecule compounds or polypeptides.

5. The use according to claim 3, characterized in that The Lck / Fyn kinase inhibitor is selected from one or more of Srci1, Dasatinib, Bosutinib, Saracatinib, 1-Naphthyl PP1 hydrochloride, TL02-59, RK 24466, eCF506, Ginkgolic acid C17:1, Nintedanib, XL228, Pelitinib, AMG-47a, PP121, 1-Naphthyl PP1, Tirbanibulin, Dehydroabietic acid, MNS, 7-Hydroxy-4-chromone, Myristic Acid, HPK1-IN-2, 1-NM-PP1, KX1-004, PP2, PP1, WH-4-023, CCT196969, SU6656, Dasatinib Monohydrate, ON123300, AD80, Repotrectinib, UM-164, Elzovantinib, DGY-06-116, TPX-0046, MLR-1023; The substance that specifically inhibits the correct binding of Lck / Fyn to STAT3 includes a STAT3 mutant or a fragment thereof. Preferably, compared with wild-type STAT3, the STAT3 mutant or the fragment thereof includes a mutation in the peptide segment of 328-335. Preferably, the STAT3 mutant or the fragment thereof includes the amino acid sequence shown in SEQ ID NO:

1. Preferably, the STAT3 mutant or the fragment thereof is further fused with a transmembrane peptide; more preferably, the transmembrane peptide is located at the N-terminus of the STAT3 mutant or the fragment thereof; more preferably, the amino acid sequence of the transmembrane peptide is as shown in SEQ ID NO:

5.

6. The use according to claim 2, wherein, the object of use of the product is a mammal or T cells of the mammal.

7. The use according to claim 2, wherein, the immune disease is selected from autoimmune diseases or non-autoimmune diseases; preferably, the autoimmune disease is selected from autoimmune encephalomyelitis, multiple sclerosis, rheumatoid arthritis, psoriasis, ankylosing spondylitis and inflammatory bowel disease, and / or other autoimmune diseases mediated by Th17 cells, and the non-autoimmune disease is cytokine storm generated during Th17 cell-mediated infection or treatment.

8. The use of any of the following biomaterials in the preparation of a product for preventing or treating Th17 cell-mediated immune diseases: a. A polypeptide comprising a STAT3 mutant or a fragment thereof, wherein the STAT3 mutant or the fragment thereof comprises the amino acid sequence shown in SEQ ID NO:1; preferably, the STAT3 mutant or the fragment thereof is further fused with a cell-penetrating peptide; more preferably, the cell-penetrating peptide is located at the N-terminus of the STAT3 mutant or the fragment thereof; more preferably, the amino acid sequence of the cell-penetrating peptide is as shown in SEQ ID NO:5; b. A polynucleotide encoding the polypeptide described in a; c. A construct comprising the polynucleotide described in b; d. A host cell comprising the construct described in c or a host cell whose genome is integrated with the polynucleotide described in b.

9. A drug for preventing or treating Th17 cell-mediated immune diseases, comprising any one or more of the biomaterials in the use described in claim 8.

10. A method for treating Th17 cell-mediated immune diseases, comprising administering an effective amount of the blocker in any one of the uses described in claims 2 to 7, and / or any one or more of the biomaterials in the use described in claim 8 to a subject.

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