Use of blocker targeting lck / fyn-mediated STAT3 phosphorylation, and use of biomaterial

Abstract

The present invention provides a use of an Lck / Fyn-mediated STAT3 activation pathway as a target in the preparation or screening of a product. The product blocks the Lck / Fyn-mediated STAT3 activation pathway, inhibits differentiation of Th17 cells, and promotes transdifferentiation of the Th17 cells into Treg cells, thereby preventing or treating a Th17 cell-related immune disease. On the basis of the target, the present invention provides a use of an Lck / Fyn-mediated STAT3 activation blocker in the preparation of a product. The blocker is selected from an Lck / Fyn kinase inhibitor or a substance that specifically inhibits correct binding of Lck / Fyn and STAT3. The present invention provides a use of one or more biomaterials in the preparation of a product for preventing or treating the Th17 cell-related immune disease. The present invention also provides a drug for preventing or treating the Th17 cell-related immune disease and a treatment method thereof. Blocking Lck / Fyn-mediated STAT3 phosphorylation can be applied to the treatment of the Th17 cell-related immune disease or improvement of the capability to prevent an immune disease caused by the Th17 cells.

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 CD4+ T cell subset that secrete IL-17, IL-17F, and IL-22. They play a crucial pathogenic role in a range of autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, and inflammatory bowel disease. Th17 cells differentiate from antigen-stimulated naive CD4+ T cells and require cytokines such as TGFβ, IL-1β, IL-6, and IL23. As shown in the study "STAT3 Regulates Cytokine-Mediated Generation of Inflammatory Helper T Cells," IL-6 and IL-23 both regulate the master transcription factor RORγt and other characteristic genes of Th17 cells through the JAK-STAT3 pathway, thereby mediating Th17 cell differentiation. 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. However, these antibodies are bulky, 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, which inhibit the signaling of many cytokines, have relatively significant side effects and have received 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 and herpes viruses. 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 a conformational change in Lck / Fyn, further enhancing the kinase activity of Lck / Fyn. The kinase active site of Lck / Fyn directly phosphorylates STAT3, and synergistically with the STAT3 phosphorylated by the cytokine-JAK signaling pathway 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-related 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-related immune diseases.

[0005] The first aspect of the present invention provides a method for inhibiting Th17 cell differentiation and promoting Treg cell differentiation, which is used to treat Th17 cell-related diseases. The method inhibits or blocks the mutual binding of Lck / Fyn and STAT3, thereby inhibiting the phosphorylation of STAT3 by Lck / Fyn.

[0006] The second aspect of the present invention provides a drug for preventing or treating Th17 cell-related immune diseases, which includes a small molecule inhibitor of Lck / Fyn and a polypeptide shown in SEQ ID NO: 5; preferably, the small molecule inhibitor of Lck / Fyn includes Srci1, Dasatinib, Bosutinib, and Saracatinib.

[0007] A third aspect of 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:

[0008] (1) Inhibits the differentiation of Th17 cells without affecting the differentiation of Th1 and Th2 cells;

[0009] (2) Promote the transdifferentiation of Th17 cells into Treg cells;

[0010] (3) Prevent or treat Th17 cell-related immune diseases.

[0011] A fourth aspect of the present invention provides 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:

[0012] (1) Inhibits the differentiation of Th17 cells without affecting the differentiation of Th1 and Th2 cells;

[0013] (2) Promote the transdifferentiation of Th17 cells into Treg cells;

[0014] (3) Prevent or treat Th17 cell-related immune diseases.

[0015] A fifth aspect of the present invention provides use of any of the following biomaterials in the preparation of a product for preventing or treating Th17 cell-related immune diseases:

[0016] 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 or C-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;

[0017] b. a polynucleotide encoding the polypeptide;

[0018] c. a construct comprising the polynucleotide described in b;

[0019] 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.

[0020] In a sixth aspect, the present invention provides a drug for preventing or treating Th17 cell-related immune diseases, comprising any one or more of the biomaterials described above for preparing products for preventing or treating Th17 cell-related immune diseases.

[0021] The seventh aspect of the present invention provides a method for treating Th17 cell-related immune diseases, 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 Th17 cell-related immune diseases, and / or any one or more of the biomaterials used in the above-mentioned use for preparing a product for preventing or treating Th17 cell-related immune diseases.

[0022] The beneficial effects of the present invention are:

[0023] (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-related immune diseases, which opened up a new therapeutic idea for the treatment of Th17 cell-related immune diseases.

[0024] (2) The present invention provides a blocker of Lck / Fyn-mediated STAT3 activation for the treatment of Th17 cell-related 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-related 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 effective.

[0025] (3) The present invention also provides a substance that specifically inhibits the correct binding of Lck / Fyn to STAT3 for use in treating Th17 cell-related 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 vitro and in vivo and Th17 cell-related immune diseases, thereby achieving the effect of treating Th17 cell-related 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.

[0026] (4) The present invention provides new clinical application value for preparations targeting Lck / Fyn kinase in the field of treatment of Th17 cell-related immune diseases, opening up broad prospects for its further market application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1-Figure 3. TCR stimulation can directly induce STAT3 phosphorylation.

[0028] Figure 1A-C. CD4+ naive T cells were cultured in 48-well culture dishes coated with (or without, or under conditions of cytokine stimulation alone) anti-CD3 / CD28 antibodies according to the culture conditions indicated in the figure. After 24 hours, Srci1 or DMSO was added. After 48 hours, cells were collected for intracellular staining or lysed for Western blotting.

[0029] Figure 1D. CD4+ naive T cells were cultured in 48-well culture dishes coated with anti-CD3 / CD28 antibodies (or without antibody coating, under conditions of cytokine stimulation alone) according to the culture conditions indicated in the figure. Cells were collected at the designated time and stained with STAT3 phosphorylation antibodies.

[0030] Figure 2A. Supernatants from CD4+ T cells after TCR stimulation alone were collected and then used to culture T cells. It was found that these supernatants did not activate STAT3, thus ruling out that TCR stimulation activates STAT3 through paracrine or autocrine mechanisms.

[0031] FIG2B . Blocking antibodies against IL-6 / IL-21 / IL-23 and gp130 do not affect TCR-induced STAT3 phosphorylation.

[0032] Figures 2C-2F. Cross-linked anti-CD3 / CD28 antibodies induce rapid STAT3 phosphorylation, and this TCR-stimulated STAT3 phosphorylation can be inhibited by Lck / Fyn inhibitors but not by JAK inhibitors or blocking antibodies to IL-6 / IL-21 / IL-23 and gp130, further indicating that TCR directly phosphorylates STAT3 through Lck / Fyn rather than affecting STAT3 phosphorylation through autocrine or paracrine mechanisms.

[0033] Figure 3A. Co-immunoprecipitation experiments showed that Lck / Fyn can directly bind to STAT3.

[0034] Figure 3B. CHIP-qPCR experiments showed that Srci1 treatment can significantly inhibit the recruitment of STAT3 to Th17-related gene sites.

[0035] Figure 4-6. Inhibition of Lck / Fyn kinase activity inhibits Th17 cell differentiation and promotes Treg differentiation.

[0036] Figure 4A. Effect of different concentrations of Srci1 on the differentiation of IL-17 cells under optimal Th17 differentiation conditions containing cytokines TGFβ, IL-1β, IL-6, and IL23. + Cells and FOXP3 +Scri1 inhibited the differentiation of Th17 cells and promoted the transformation of Th17 cells into Treg cells.

[0037] Figure 4B. Effects of administration of different concentrations of Srci1 on the mRNA expression levels of Th17-related genes under optimal Th17 differentiation conditions containing cytokines TGFβ, IL-1β, IL-6, and IL23.

[0038] Figure 4C. Effect of different concentrations of Srci1 on the expression of IL-17 in 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.

[0039] Figure 4D. Effects of administration of different concentrations of Srci1 on the mRNA expression levels of Th17-related genes under standard Th17 differentiation conditions containing cytokines TGFβ and IL-6.

[0040] Figure 5A. Effect of different concentrations of Srci1 on the expression of IL-17 in Treg differentiation cells under conditions containing cytokines TGFβ and IL-2. + Cells and FOXP3 + Srci1 promotes Treg cell differentiation.

[0041] FIG5B . Effects of Srci1 administration on the mRNA expression levels of Treg-related genes under Treg differentiation conditions containing cytokines TGFβ and IL-2.

[0042] Figure 5C. Effect of different concentrations of Srci1 on IFNγ expression under Th1 differentiation conditions. + Scri1 did not affect the differentiation of Th1 cells.

[0043] Figure 5D. Effect of different concentrations of Srci1 on IL-4 expression under Th2 differentiation conditions. + Scri1 did not affect Th2 differentiation.

[0044] Figure 6A-C. Using point mutant STAT3C to mimic activated STAT3, STAT3C reversed the effect of Srci1 on Th17 cell differentiation.

[0045] Figure 7-9. TCR stimulation induces phosphorylation of STAT3, but not STAT4, STAT5, and STAT6.

[0046] Figure 7A-D. Increasing the stimulation intensity of TCR increased the phosphorylation of STAT3 and promoted the differentiation of Th17 cells.

[0047] Figure 8A-C. Effects of TCR stimulation on STAT4, STAT5 and STAT6 phosphorylation.

[0048] FIG9A-F . ITK inhibitors do not affect TCR stimulation-induced STAT3 phosphorylation, although ITK inhibitors can inhibit Th17 cell differentiation.

[0049] Figure 10. TCR stimulation-induced STAT3 phosphorylation is independent of the cytokine-JAK pathway.

[0050] 10A-G . Effects of TCR stimulation on Th17 cell-associated cytokines or cytokine receptors.

[0051] Figure 10H. STAT3 phosphorylation induced by TCR stimulation can be inhibited by Srci1 but not by a broad-spectrum inhibitor of JAK kinases, indicating that TCR stimulation directly activates STAT3.

[0052] Figures 11-12. Effects of Srci1 inhibition of Lck / Fyn activity on gene expression during Th17 cell differentiation.

[0053] Figure 11A. T cells were differentiated under Th17 or Treg conditions, and Srci1 was added 24 hours later. After differentiation, cells were harvested for mRNA sequencing. The results show that Srci1 treatment can convert Th17 cells into Treg cells.

[0054] Figure 11B-C. Approximately 50% of the genes affected by Srci1 treatment are also affected by STAT3 deficiency.

[0055] Figure 11D. GSEA analysis showed that Srci1 treatment comprehensively downregulated the expression of Th17-related genes and upregulated the expression of Treg-related genes.

[0056] Figure 11E. Pathway enrichment analysis showed that Srci1 treatment mainly affected the expression of cytokines and immune-related genes.

[0057] 12A-B . Srci treatment inhibited many direct target genes of STAT3 related to Th17 cell differentiation; and upregulated many direct target genes of FOXP3.

[0058] Figure 13-15. Srci1 does not affect T cell proliferation and survival.

[0059] 13A-D. Srcl treatment does not affect T cell proliferation and survival.

[0060] 14A-B . Srci1 treatment does not affect c-Myc expression or the mTOR signaling pathway.

[0061] Figure 15A-B. Constitutively activated AKT cannot reverse the inhibitory effect of Srci1 on Th17 cell differentiation, indicating that Srci treatment does not affect Th17 cell differentiation by affecting the mTOR signaling pathway.

[0062] Figure 16-Figure 17. Effects of Srci1 treatment on Th17 cell differentiation.

[0063] FIG16A . Determination of STAT3 direct target genes during Th17 cell differentiation.

[0064] FIG16B . KEGG pathway analysis of gene expression changes induced by Srci1 treatment.

[0065] FIG16C . Process for determining direct target genes of FOXP3.

[0066] 17A-B . Effects of Srci1 treatment on the expression of genes related to the TCR downstream signaling pathway and the mTOR signaling pathway.

[0067] Figure 18-19. Disruption of the interaction between STAT3 and Lck / Fyn inhibits Th17 cell differentiation.

[0068] FIG18A . Sites on STAT3 that cause Th17 cell deficiency.

[0069] Figure 18B. AlphaFold 2 Multimer software was used to predict the binding between SH2 / SH3 domain and STAT3.

[0070] FIG18C . Amino acid residues on the SH2 domain of Lck / Fyn that bind to STAT3 at positions 328-335 are conserved among multiple Src family members.

[0071] Figure 18D-E. Mutation of amino acids 328-335 of STAT3 inhibits the interaction between STAT3 and Lck / Fyn.

[0072] Figure 19A-C. Mutations at 328-335 do not affect the ability of STAT3C to promote Th17 cell differentiation, indicating that these mutations do not affect the DNA binding ability of STAT3.

[0073] Figure 19D-F. Mutations at 328-335 do not affect the ability of STAT3C to reverse the differentiation of Th17 cells by Srci1, indicating that these mutations affect the differentiation of Th17 cells not by affecting the DNA binding ability of STAT3 but by affecting the phosphorylation of STAT3.

[0074] Figure 19G-J. Mutations at 328-335 significantly affected STAT3-mediated Th17 cell differentiation.

[0075] Figure 20-21. Mutations at positions 328-330 of STAT3 affect TCR-Lck / Fyn axis-mediated STAT3 phosphorylation but do not affect cytokine-induced STAT3 phosphorylation.

[0076] Figure 20A. Experimental flow shows that LSK cells (rich in blood stem cells) were isolated from STAT3 conditional knockout mice, then overexpressed empty vector, WT STAT3, or STAT3 mutants, and then transplanted into Rag1-deficient mice. CD4+ naive T cells were isolated from them 2 months later for subsequent experiments.

[0077] Figure 20B-E. The CD4+ naive T cells obtained above were cultured under standardized conditions, and the cells were collected for STAT3 phosphorylation staining (BC) after 48 hours or for intracellular cytokine staining on the fifth day.

[0078] 21A-E . Effects of polypeptide sequences containing STAT3 wild-type polypeptide and cell-penetrating peptide on STAT3 phosphorylation and Th17 cell differentiation.

[0079] Figure 21F-H. In vitro kinase activity assays showed that Lck phosphorylates STAT3. The polypeptide containing STAT3 328-330 can promote Lck-mediated STAT3 phosphorylation, while the mutant polypeptide loses this ability.

[0080] Figure 22-Figure 24. Inhibition of TCR stimulation-induced STAT3 phosphorylation can inhibit Th17 cell differentiation in vivo and improve EAE disease in mice.

[0081] FIG22A shows the effects of daily administration of 50 mg / kg of Scri1 or DMSO on EAE disease scores at the onset of EAE disease. Scri1 alleviates EAE symptoms.

[0082] Figure 22B-C. Flow cytometry data of infiltrating T cells from the CNS after administration of Srcil and DMSO, including IL-17+ cells and FOXP3 +Scri1 treatment significantly reduced the proportion of Th17 cells and increased the proportion of Treg cells in the central nervous system.

[0083] FIG22D . IFNγ expression in CNS-infiltrating T cells after administration of Srcil and DMSO. + Scri1 does not affect the differentiation of Th1 cells.

[0084] FIG22E . IL-17 expression in CNS-infiltrating T cells after administration of Srcl and DMSO. + and IFNγ + The percentage of cells.

[0085] FIG22F . FOXP3 expression in infiltrating T cells from the CNS after administration of Srcil 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.

[0086] FIG. 22G . Number of infiltrating T cells from the CNS after administration of Srcl and DMSO.

[0087] Figure 22H. Representative flow cytometry plots of infiltrating T cells from the CNS.

[0088] Figure 22I. Stat3 fl / fl CD4 cre Schematic diagram of adoptive transfer of EAE in 2D2 mice.

[0089] Figure 23A-D. 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 showed little or no disease-related symptoms.

[0090] Figure 24A-C. Receiving empty vector, WT STAT3, Dup 328-330 The percentage of CD3+CD4+ in the spleen, the percentage of CD3+CD4+ in the CNS, and the number of CD3+CD4+ in the CNS of mice transplanted with R335W-overexpressing T cells after MOG immunization.

[0091] Figure 24D-F. Receiving empty vector, WT STAT3, Dup 328-330 Flow cytometric data of CD4+ cells isolated from the CNS of mice transplanted with R335W-overexpressing T cells after MOG immunization, 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 and R335W T cells could not be induced to differentiate into Th17 cells.

[0092] Figure 24G. Receiving empty vector, WT STAT3, Dup 328-330 Figure 3. Flow cytometry of CD4+ T cells isolated from the CNS of mice transplanted with R335W-overexpressing T cells after MOG immunization.

[0093] Figure 25A-E. The conserved sequence (MAS motif) in STAT3 interacts with the SH2 domain of Lck / Fyn. AlphaFold2Multimer was used to predict the binding between the SH2 / SH3 domain on Lck / Fyn and STAT3. The results showed that the SH2 domain on Lck / Fyn can bind to amino acids 328-335 on STAT3; while the SH3 domain does not bind to STAT3.

[0094] 26A-B . Effects of STAT3 MUT polypeptides containing transmembrane sequences on Th17 cell differentiation. STAT3 MUT polypeptides containing transmembrane sequences can inhibit TH17 cell differentiation and promote Treg differentiation.

[0095] Figure 27. Principle of the Lck / Fyn-mediated STAT3 phosphorylation activation pathway. During TH17 cell differentiation, TCR-Lck / Fyn directly phosphorylates STAT3 at Y705 and synergizes with cytokine-JAK kinase to maximize STAT3 activation. DETAILED DESCRIPTION

[0096] In a method provided by the present invention for inhibiting Th17 cell differentiation and promoting Treg cell differentiation, the method inhibits or blocks the mutual binding of Lck / Fyn and STAT3, thereby inhibiting the phosphorylation of STAT3 by Lck / Fyn; thereby, it is used to treat Th17 cell-related diseases.

[0097] The method includes using antibodies, small molecule compounds, peptides or other substances to inhibit the activity of Lck / Fyn kinase; or directly inhibiting the mutual binding between the SH2 domain on Lck / Fyn and the 320-340 position on STAT3; or inhibiting the binding between the kinase domain of Lck / Fyn and the domain near Y705 on STAT3; or inhibiting the conformational change of Lck / Fyn kinase, thereby inhibiting the phosphorylation of STAT3 by Lck / Fyn; and also includes using siRNA, shRNA, antibodies or their derivatives to inhibit the expression of Lck / Fyn or STAT3, thereby inhibiting the mutual binding between Lck / Fyn and STAT3.

[0098] In the present invention, the immune disease is mainly a Th17 cell-related 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, inflammatory bowel disease, asthma and systemic lupus erythematosus, and / or other autoimmune diseases related to Th17 cells, and the non-autoimmune disease is a Th17 cell-related infection or cytokine storm generated during treatment.

[0099] Lck / Fyn-mediated STAT3 activation pathway

[0100] In the study of the present invention, 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, so that the kinase activity of Lck / Fyn reaches the optimal state; then Lck / Fyn directly phosphorylates STAT3 through the kinase active site, and then cooperates with the JAK kinase-induced STAT3 pathway to transcriptionally regulate the expression of the master transcription factor RORγt and other Th17-related genes of Th17 cells, thereby mediating the differentiation of Th17 cells, as shown in Figure 1.

[0101] Therefore, inhibiting Lck / Fyn activity or blocking the correct binding of Lck / Fyn to STAT3 can inhibit STAT3 activation, thereby suppressing Th17 cell-related immune diseases; but it does not affect the phosphorylation of other STAT transcription factor proteins (STAT4, STAT5, STAT6) or the differentiation of other T cell subsets. Therefore, it is believed that Lck / Fyn kinase inhibitors can inhibit the differentiation of Th17 cells by inhibiting STAT3 activation, and thus can be used to treat Th17 cell-related immune diseases or provide preventive / therapeutic capabilities for Th17 cell-related immune diseases.

[0102] The differentiation of Th17 cells refers to the differentiation of CD4+ naive T cells into Th17 cells after receiving antigen stimulation in the presence of relevant cytokines.

[0103] The autoimmune diseases include autoimmune encephalomyelitis, multiple sclerosis, rheumatoid arthritis, psoriasis, ankylosing spondylitis and inflammatory bowel disease.

[0104] Lck / Fyn

[0105] Lck / Fyn refers to Lck or Fyn, both of which 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, the 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 polypeptide sequence at positions 328-335 on STAT3, thereby 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.

[0106] Blockers of Lck / Fyn-mediated STAT3 activation

[0107] 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.

[0108] The inhibitor of Lck / Fyn-mediated STAT3 activation is selected from: Lck / Fyn kinase inhibitors or derivatives thereof; and substances that specifically inhibit the correct binding of Lck / Fyn to STAT3.

[0109] Lck / Fyn kinase inhibitors

[0110] 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.

[0111] The Lck / Fyn kinase inhibitor can be selected from siRNA, shRNA, antibodies or small molecule compounds and their derivatives.

[0112] The derivative refers to a compound formed by replacing atoms or atomic groups in the parent compound molecule with other atoms or atomic groups, but the compound formed after the atoms or atomic groups are replaced can still perform the same function as the parent compound, especially a compound that can still inhibit the phosphorylation of STAT3 by Lck / Fyn.

[0113] 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 or one or more thereof.

[0114] Small molecule compounds

[0115] The small molecule compound in the present invention refers to a compound with a molecular weight of less than 1000 Daltons.

[0116] Srci1

[0117] Chemical name: 6,7-Dimethoxy-N-(4-phenoxyphenyl)-4-quinazolinamine

[0118] Molecular formula: C 22 H 19 N3O3

[0119] IC50: Src, 44nM; Lck, 88nM

[0120] Cas No.179248-59-0

[0121] The structural formula is as follows:

[0122] Dasatinib

[0123] Chemical Name:

[0124] N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-yl]amino]-1,3-thiazole-5-carboxamide

[0125] Molecular formula: C 22 H 26 C l N7O2S

[0126] 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

[0127] Cas No.302962-49-8

[0128] The structural formula is as follows:

[0129] Bosutinib

[0130] Chemical Name:

[0131] 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methyl-1-piperazinyl)propoxy]-3-quinolinecarbonitrile

[0132] Molecular formula: C 26 H 29 C l2 N5O3

[0133] IC50: Src, 1.2nM; Abl, 1nM

[0134] Cas No.380843-75-4

[0135] The structural formula is as follows:

[0136] Saracatinib

[0137] Chemical Name:

[0138] N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine

[0139] Molecular formula: C 27 H 32 C l N5O5

[0140] IC50: Src, 2.7nM; v-Abl, 30nM; EGFR, 66nM; c-Kit, 200nM

[0141] Cas No.379231-04-6

[0142] The structural formula is as follows:

[0143] 1-Naphthyl PP1 hydrochloride

[0144] Chemical Name:

[0145] 1-tert-butyl-3-(naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyriMidin-4-aMine hydrochloride

[0146] Molecular formula: C 19 H 19 N5ClH

[0147] IC50: c-Fyn, 0.6μM; c-Abl, 0.6μM; v-Src, 1.0μM; CDK2, 18μM; CAMKII, 22μM

[0148] Cas No.956025-47-1

[0149] The structural formula is as follows:

[0150] TL02-59

[0151] Chemical Name:

[0152] 3-[(6,7-Dimethoxy-4-quinazolinyl)oxy]-N-{4-[(4-ethyl-1-piperazinyl)methyl]-3-(trifluoromethyl)phenyl}-4-methylbenzamide

[0153] Molecular formula: C 32 H 34 F3N5O4

[0154] IC50: Fgr, 0.03nM

[0155] CAS No.1315330-17-6

[0156] The structural formula is as follows:

[0157] RK 24466

[0158] Chemical name: 7-Cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

[0159] Molecular formula: C 23 H 22 N4O

[0160] IC50: Lck, 0.001μM; Lckcd, 0.002μM

[0161] CAS No.213743-31-8

[0162] The structural formula is as follows:

[0163] Correct binding of Lck / Fyn to STAT3

[0164] 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.

[0165] Substances that specifically inhibit the correct binding of Lck / Fyn to STAT3

[0166] 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.

[0167] 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.

[0168] 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).

[0169] Blocker preparation product of the present invention

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] The product may be used in the pharmaceutical field, health care product field, food field, etc.

[0175] The product is used for mammals or T cells of the mammals.

[0176] Biomaterials for preparing products for preventing or treating Th17 cell-related immune diseases

[0177] The biological material may be one or more of the following:

[0178] 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 or C-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;

[0179] b. a polynucleotide encoding the polypeptide;

[0180] c. a construct comprising the polynucleotide described in b;

[0181] 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.

[0182] 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).

[0183] 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.

[0184] 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.

[0185] 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.

[0186] Biomaterial preparation product of the present invention

[0187] 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.

[0188] 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.

[0189] 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.

[0190] The product is used for mammals or T cells of the mammals.

[0191] Preparation of medicines from biological materials in the present invention

[0192] 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.

[0193] Lck / Fyn kinase inhibitors for drug preparation

[0194] 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.

[0195] 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.

[0196] "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.

[0197] 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.

[0198] The drug is used for mammals or T cells of the mammals.

[0199] Method for treating Th17 cell-related immune diseases

[0200] In the present invention, the method for treating Th17 cell-related 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.

[0201] The effective amount refers to a dose that can achieve the treatment, prevention, alleviation and / or relief of the Th17 cell-related immune diseases or disorders described in the present invention in the subject.

[0202] 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.

[0203] The treatment method can be to administer the above-mentioned product for treating Th17 cell-related immune diseases to the subject during the prevention period of Th17 cell-related immune diseases, or during the early, middle, or late stages of treatment.

[0204] In addition to using the above-mentioned products, drugs or biomaterials for treating Th17 cell-related immune diseases, the treatment method may also simultaneously or sequentially administer to the subject an effective amount of other drugs for treating Th17 cell-related immune diseases and / or implement other means of treating Th17 cell-related immune diseases on the subject.

[0205] The other drugs for treating Th17 cell-related immune diseases refer to drugs for treating Th17 cell-related 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, and Abrocitinib.

[0206] The other means of treating Th17 cell-related 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.

[0207] The subject of the treatment method is a mammal or T cells of the mammal.

[0208] The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, or the like.

[0209] CD4+ naive T cells

[0210] Naive CD4+ T cells are activated after interacting with antigen-MHC complexes and differentiate into specific subsets based on the cytokine milieu of the microenvironment. Following activation, they differentiate into different subsets under the regulation of various cytokines, including Th1, Th2, Th17, Tfh, and regulatory T cells (Tregs).

[0211] Th17 cells

[0212] 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 a variety of autoimmune diseases, such as autoimmune encephalomyelitis, multiple sclerosis, rheumatoid arthritis, psoriasis, and inflammatory bowel disease.

[0213] Treg cells

[0214] 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.

[0215] Th1 cells

[0216] 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).

[0217] Th2 cells

[0218] 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.

[0219] IL-17 cytokine

[0220] 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.

[0221] FOXP3 transcription factor

[0222] 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.

[0223] IFN-γ cytokine

[0224] 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.

[0225] IL-4 cytokine

[0226] 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.

[0227] STAT3

[0228] STAT3 is a protein composed of 770 amino acids, and its complete amino acid sequence is:

[0229] Dup 328-330

[0230] The STAT3 mutant fragment has a polypeptide sequence that is repeated at amino acid positions 328-330 compared to the STAT3 323-340 peptide segment of the STAT3 wild type.

[0231] R335W

[0232] The STAT3 mutant fragment has a polypeptide sequence that is comparable to the STAT3 323-340 peptide fragment, in which the arginine at position 335 of the wild-type STAT3 is mutated to tryptophan.

[0233] STAT3 WT

[0234] The wild-type fragment of STAT3 is the 323-340 peptide segment of STAT3.

[0235] STAT3 MUT

[0236] The STAT3 mutant fragment, whose polypeptide sequence is compared with the STAT3 323-340 peptide segment, has duplication of amino acids 328-330 and mutation of arginine at position 335 to tryptophan.

[0237] Cell-penetrating peptides

[0238] 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 also include a cell-penetrating peptide. Such cell-penetrating peptides are known in the art and include any amino acid sequence that can help a polypeptide or protein enter a cell. Those skilled in the art can select a conventional one based on actual circumstances. For example, the amino acid sequence of the cell-penetrating peptide can be as shown in SEQ ID NO: 5.

[0239] Furthermore, in a specific embodiment of the present invention, the Dup containing the cell-penetrating peptide 328-330The complete sequence is: SYGRKKRRQPRRR VERQPCMPCMPMHPDRPLVIK (SEQ ID NO: 7); the complete sequence of 335W containing a cell-penetrating peptide is: SYGRKKRRQPRRR VERQPCMPMHPDWPLVIK (SEQ ID NO: 8); the complete sequence of STAT3 WT containing a cell-penetrating peptide is: SYGRKKRRQPRRR VERQPCMPMHPDRPLVIK (SEQ ID NO: 9); the complete sequence of STAT3 MUT containing a cell-penetrating peptide is SYGRKKRRQPRRRVERQPCMPCMPMHPDWPLVIK (SEQ ID NO: 10). In a specific embodiment of the present invention, the STAT3 MUT, Dup 328-330、 Mutants such as R335W all contain cell-penetrating peptides.

[0240] STAT3C

[0241] In the embodiments of the present invention, STAT3C refers to STAT3 in which amino acids 662 and 664 of STAT3 are mutated to cysteine, which makes STAT3 no longer dependent on phosphorylation at position Y705 to enter the cell nucleus and exert transcriptional activity. It is also called constitutively active STAT3 (STAT3C).

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 1. Materials and Reagents

[0246] 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.

[0247] 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;

[0248] 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.

[0249] 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), and anti-V5 (clone D3H8Q,13202) were purchased from Cell Signaling Technology.

[0250] 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.

[0251] 2. Experimental Animals

[0252] STAT3 fl / fl CD4 cre Mice were obtained from Chen Dong (Tsinghua University) with permission from Dr. Shizuo Akira (Osaka University, Japan).

[0253] C57BL / 6 and Rag1- / - mice were from The Jackson Laboratory.

[0254] 3. CD4 Naive T Cell Culture

[0255] 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.

[0256] 4.CD4+ naive T cell differentiation

[0257] Approximately 400,000 CD4+ naive 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, respectively. The conditions for inducing CD4+ naive T cell differentiation are as follows:

[0258] Tregs: 0.5ng / ml TGFβ, 200U / ml mouse IL-2, 1μg / ml anti-IFNγ and 1μg / ml anti-mouse IL-4.

[0259] 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.

[0260] 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.

[0261] Th1: 10ng / ml mouse IL-12, 1μg / ml anti-mouse IL-4.

[0262] Th2: 20ng / ml mouse IL-4, 1ug / ml anti-IFNγ.

[0263] Specified concentrations of ScRI1, Ruxolitinib, Baricitinib, or BMS509744 were added 24 hours after inoculation. 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.

[0264] 5. Analysis of mRNA Expression by qRT-PCR

[0265] 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.

[0266] 6. Preparation of Retroviral Vectors and T Cell Infection

[0267] 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.

[0268] T cell infection: Add the virus-containing supernatant to pre-activated CD4+ naive 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 CD4+ T cells under Th17 differentiation conditions. On the second day, reinfect the 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.

[0269] 7. Establishment of EAE Model in C57BL / 6 Mice

[0270] 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.

[0271] 8.2D2 mouse transfer-induced EAE model: from STAT3 fl / fl CD4 cre CD4+ naive T cells (CD4 + CD25 - CD62high CD44 low ) 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 by centrifugation at 37°C and 2000 rpm. + CD4+ T cells were cultured 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+ T cells were infected again 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.

[0272] 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).

[0273] All animal experiments were approved by the institutional IACUC committee.

[0274] 9. Obtaining bone marrow chimeric reconstructed mice

[0275] Bone marrow cells were obtained by flushing the tibia and femur of STAT3fl / flCD4cre mice. Lineage-positive cells were enriched using the EasySep Mouse Hematopoietic Progenitor Cell Enrichment Kit (StemCell Technology, 19856), and lineage-negative cells were collected and stained for c-Kit, Sca-1, and Lin. LSK cells (LinSca1 + cKit + ). LSK cells were cultured overnight in RPMI1640 containing 10% FBS with mouse IL-3 (20 ng / ml), mouse IL-6 (50 ng / ml) and mouse SCF (50 ng / ml) (all from PeproTech). The next day, LSK cells were infected with retrovirus expressing empty vector or WT STAT3 or its mutants by centrifugation at 2000 rpm for 2 h at 36 ° C. After centrifugation, the cells were incubated for another 3 h, and the supernatant was replaced with fresh medium containing cytokines. After 48 h of culture, Thy1.1 was evaluated by flow cytometry (FACS). + The infection efficiency was determined by quantification, and Thy1.1 was collected using CD90.1 MicroBeads (Milltenyi Biotec, 130-121-273). + LSK cells were injected with Thy1.1 + LSK cells were adoptively transferred into lethally irradiated Rag1- / - recipient mice (at least 10 per mouse). 5 LSK cells).

[0276] Eight weeks after reconstitution, CD4+ T cells were isolated from lymph nodes and spleen for further experimental analysis.

[0277] 10. ChIP-qPCR

[0278] Ten million differentiating Th17 cells were cross-linked with 1% formaldehyde (Sigma-Aldrich) for 10 minutes, quenched with 125 mM glycine for 5 minutes, and then washed with cold PBS. The cell pellet was resuspended in chilled lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40 plus protease inhibitors) for 30 minutes with rotation at 4°C. The nuclear pellet was resuspended in chilled MNase reaction buffer (50 mM Tris-HCl, 5 mM CaCl2, 100 μg / ml BSA, 1 mM DTT, pH 8.0, 100 U MNase, NEB, M0247S) and incubated at 37°C for 20 minutes to digest DNA to approximately 150-900 bp. Enzymatic digestion was blocked by adding 10 μL of 0.5 M EDTA. The nuclear pellet was resuspended in 500 μL of ice-cold ChIP dilution buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% BSA, and protease inhibitors) and incubated on ice for 10 min. Chromatin was fragmented by pulsed sonication (on / off for 20 s, 30% output, 3 cycles). 2% of the sample was injected. The remaining sample was incubated with rabbit anti-STAT3 (CST, clone 79D7, 4904) and normal rabbit IgG (Abcam, ab172730) at 4°C with rotation overnight. Dynabeads Protein A+G (Thermo Fisher Scientific) were added and incubated at 4°C with rotation for 2 hours. The immune complexes were washed twice using RIPA buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% na-deoxycholate), high salt buffer (RIPA buffer + 350 mM NaCl), LiCl buffer (10 mM Tris-HCl, 250 mM LiCl, 0.5% NP-40, 0.5% na-deoxycholate), and TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The collected immunomagnetic beads were resuspended in 200 μL of elution buffer (1% SDS and 0.1 M NaHCO3) and incubated at 65°C for 45 min. The supernatant was collected into a new tube containing 8 μL of 5 M NaCl and incubated overnight to reverse the DNA-protein linkage. Add 1 μL of RNase to the supernatant and incubate at 37°C for 30 min. Then, add 4 μL of 0.5 M EDTA, 8 μL of 1 M Tris-HCl, and 4 μL of Proteinase K to the supernatant and incubate at 45°C for 2 h. Purify the decrosslinked DNA using Zymo spin columns (Zymo, D5205). Quantify the immunoprecipitated DNA and the injected DNA by RT-PCR.ChIP data were analyzed using the percentage input method of 100*2 (adjusted input - Ct(IP)). The primers used for ChIP-qPCR are shown in Table 1.

[0279] Table 1 Primers used in ChIP-qPCR

[0280] 11. Co-immunoprecipitation

[0281] Collect cells and wash with cold PBS. Add cold cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, mixed protease inhibitors) and incubate on ice for 30 minutes. Centrifuge (12,000 g, 4°C, 10 minutes) and collect the supernatant.

[0282] For endogenous Co-IP, Th17 cell lysates were immunoprecipitated with mouse anti-STAT3 (CST, 124H6, 9139S) and normal mouse IgG (Abcam, ab18415) overnight at 4°C with rotation. 20 μl of Protein A / G agarose (Beyotime, P2055) was added and incubated for an additional 2 hours at 4°C. For exogenous Co-IP, Flag-mCherry-Lck / Fyn was first stably transfected using retroviral vectors, followed by transient transfection with V5-STAT3 or its mutants using LipoMAX. Cell lysates were harvested 48 hours after transfection and immunoprecipitated with anti-Flag (specifically, a short amino acid sequence, DYKDDDDK) magnetic agarose (Invitrogen, A36797) overnight at 4°C with rotation. The agarose gel was then washed four times with wash buffer (20 mM Tris-HCl, pH 7.5, 0.1% Triton X-100, 150 mM NaCl plus protease inhibitors). The resulting samples were analyzed by SDS-PAGE. Western blotting was performed using anti-STAT3 (CST, 4904), anti-Lck (Thermo Fisher Scientific, PA5-79587), anti-Fyn (Affinity, AF6102), anti-V5 (CST, 13202), anti-Flag (CST, 8146), and anti-GAPDH (CST, 5174) antibodies.

[0283] 12.mRNA-Seq

[0284] The designated cells were collected for mRNA sequencing. RNA sequences were aligned to mouse Gencode (GRCm38, version M23) using STAR (version 2.7.9a). Sequence and genomic annotation data were obtained from Ensembl release 98 (https: / / www.gencodegenes.org). Read counts and transcripts per kilobase million (TPM) were determined using RSEM (version 1.3.3). Stat3 (WT) Th0 and Th17 (48h) read data from the GSE40918 dataset were processed using the same method as in the article (Ciofani et al, 2012). Count output was used to identify differentially expressed genes (TH17_DMSO vs TH17_Srci1, TH17_DMSO vs TH17_STAT3_KO) using DESeq2 (version 1.34.0). The resulting p-values ​​were used to create false discovery rate (FDR) estimates using the Benjamini-Hochberg (BH) method. Gene expression differences were considered significant if the FDR estimate was less than 0.05 and the log2FC was greater than 0.58. Microarray expression data for TH17, iTreg, and nTreg genes from GSE14308 were preprocessed using the MAS5 algorithm and quantile normalized. For each gene in the two categories (TH17 vs. iTreg), differential gene expression was then calculated using limma (version 3.42.2) based on the log2(MAS5+1) values ​​of the expression values ​​in the geo-provided microarray dataset. Genes were considered differentially expressed if the FDR for differential expression was less than 0.05 and the log2FC was greater than 2. STAT3 direct target genes were identified using two criteria: the presence of a STAT3 binding peak at the gene locus and significant suppression of gene expression by STAT3 knockout. FOXP3 direct target genes were defined using the same method, with binding peaks called using MACS2 software (settings: -q 0.05 -g mm). Gene ontology (GO) enrichment analysis was performed using metscape, using the widely adopted hypergeometric test. The calculated p-values ​​were corrected using the Benjamini-Hochberg algorithm according to the recommendations of the topGO website. Gene set enrichment analysis (GSEA, version 4.2.3) was performed on the Treg-associated gene set in GSE14308 to generate hypotheses and test the hypotheses for the TH17_DMSO and TH17_Srci1 gene sets. The gene expression dataset is available in the GEO database: GSE229377.

[0285] 13. Protein complex structure prediction and interface analysis

[0286] Since the AlphaFold2 modeling structure has been successfully used to predict protein binding sites (Yuan et al, 2022), this example modeled the complex structure of STAT3 using the LCK and FYN sequences from NCBI according to the tutorial at https: / / github.com / deepmind / alphafold (Sayers et al, 2021). In this example (Jumper et al., 2021; Evans et al., 2021), model parameters and genetic databases, including BFD (Steinegger et al., 2019a), Mgnify (Mitchell et al., 2020), UniProt (2021), UniRef90 (Suzek et al., 2007), Uniclust30 (Mirdita et al., 2017), PDB70 (Steinegger et al., 2019b), PDB seqres (Berman et al., 2000), and PDB (Berman et al., 2000), were downloaded to the Tianhe-2 supercomputer to implement AlphaFold-Multitimer. The "max_template_date" parameter was set to use protein templates before 2022-05-19, and the predicted structures were relaxed using the Amber relaxation program (Hornak et al., 2006) to resolve structural violations and conflicts. According to the coordinates of the predicted structure, the composite interface is defined in this embodiment as the Cα-Cα atom Euclidean distance less than The interchain residue pairs were further visualized using the PyMOL package.

[0287] Example

[0288] Example 1 TCR stimulation can directly induce STAT3 phosphorylation

[0289] CD4+ naive T cells were cultured in 48-well culture dishes coated with anti-CD3 / CD28 antibodies (under conditions of cytokine stimulation alone, cells were cultured in dishes not coated with anti-CD3 / CD28 antibodies) under the conditions indicated in Figure 1A. After 24 hours, Scri1 or DMSO was added. After 48 hours, cells were harvested and subjected to intracellular staining, lysed for Western blot analysis, or stained with an antibody against Y705-phosphorylated STAT3. The results, as shown in Figures 1A-C, show that TCR stimulation induced STAT3 phosphorylation in an Lck / Fyn-dependent manner and synergized with the proinflammatory cytokines IL-6 and IL23 to induce optimal STAT3 phosphorylation. Scri1, a selective inhibitor of Lck / Fyn, significantly inhibited STAT3 phosphorylation induced by TCR stimulation alone and by dual TCR and cytokine stimulation. In Figure 1C, under the premise that the total amount of STAT3 in each sample remains consistent, it shows that TCR stimulation alone can induce STAT3 phosphorylation, and phosphorylation can be inhibited by Srci1, an inhibitor of Lck / Fyn. As shown in Figure 1D, the dynamic changes of STAT3 phosphorylation induced by TCR stimulation and cytokines over time

[0290] The supernatants of CD4+ T cells after TCR stimulation alone were collected and then used to culture T cells. The T cells were stained with Y705 phospho-STAT3 antibody. As shown in Figure 2A, it was found that these supernatants could not activate STAT3, thus ruling out that TCR stimulation activates STAT3 in a paracrine or autocrine manner.

[0291] CD4+ naive T cells were cultured under the conditions indicated in Figure 2B, and blocking antibodies against IL-6 / IL-21 / IL-23 and gp130 were added. After 48 hours, the cells were collected and subjected to intracellular staining or lysed for Western blot analysis. The cells were then stained with antibodies against Y705-phosphorylated STAT3. As shown in Figure 2B, blocking antibodies against IL-6 / IL-21 / IL-23 and gp130 did not affect the phosphorylation of STAT3 induced by TCR stimulation.

[0292] CD4+ naive T cells were cultured under the conditions indicated in Figure 2B, and DSMO, Scri1, Ruxolitinib, Baricitinib, IL-6 / IL-21 / IL-23, and gp130 blocking antibodies were added. Subsequently, Y705-phosphorylated STAT3 antibody staining was performed. As shown in Figures 2C-2F, cross-linked anti-CD3 / CD28 antibodies induced rapid STAT3 phosphorylation, and this TCR-stimulated STAT3 phosphorylation could be inhibited by Lck / Fyn inhibitors but not by JAK inhibitors or IL-6 / IL-21 / IL-23 and gp130 blocking antibodies, further indicating that TCR directly phosphorylates STAT3 through Lck / Fyn, rather than affecting STAT3 phosphorylation through autocrine or paracrine mechanisms.

[0293] The interaction between Lck / Fyn and STAT3 was investigated by immunoprecipitation assay in the experimental method. The results are shown in FIG3A . The immunoprecipitation assay showed that Lck / Fyn can directly bind to STAT3.

[0294] According to the experimental method, the CHIP-qPCR experiment was performed to explore the recruitment of STAT3 to Th17-specific gene sites. As shown in Figure 3B, the CHIP-qPCR experiment showed that Srci1 treatment can significantly inhibit the recruitment of STAT3 to Th17-related gene sites.

[0295] Example 2 TCR stimulation induces phosphorylation of STAT3, but does not induce phosphorylation of STAT4, STAT5, and STAT6.

[0296] Naive CD4+ T cells were cultured according to the conditions described in Figures 7 and 9 , and inhibitors of DSMO, Scri1, or ITK were added to alter the intensity of TCR stimulation. Cells were harvested 48 hours later or on day 5 for intracellular cytokine staining or staining with an antibody against Y705-phosphorylated STAT3. As shown in Figures 7A-D , increasing the intensity of TCR stimulation increased STAT3 phosphorylation and promoted Th17 cell differentiation. As shown in Figures 8A-C , TCR stimulation had little effect on STAT4, STAT5, and STAT6 phosphorylation. As shown in Figures 9A-F , an ITK inhibitor did not affect TCR stimulation-induced STAT3 phosphorylation, despite suppressing Th17 cell differentiation.

[0297] Example 3 TCR stimulation-induced STAT3 phosphorylation is independent of the cytokine-JAK pathway.

[0298] Naive CD4+ T cells were cultured according to the conditions described in Figures 10A-G and treated with DMSO or Scri1. The mRNA expression of genes involved in the cytokine-JAK pathway was assessed by qRT-PCR, and the levels of receptors involved in the cytokine-JAK pathway were assessed by flow cytometry. As shown in Figures 10A-F, TCR stimulation alone did not induce the expression of Il21 or IL23R, and Scri1 treatment did not affect the expression of IL6RA, IL6ST, or IL21R, indicating that TCR stimulation is unlikely to induce STAT3 phosphorylation by affecting the expression of cytokines or cytokine receptors.

[0299] CD4+ naive T cells were cultured under the conditions indicated in Figure 10H, and DSMO, Scri1, Ruxolitinib, and Baricitinib were added. The cells were then stained with an antibody that recognizes phosphorylated STAT3 Y705. As shown in Figure 10H, STAT3 phosphorylation induced by TCR stimulation can be inhibited by Srci1 but not by a broad-spectrum inhibitor of JAK kinase, indicating that TCR stimulation directly activates STAT3 through Lck / Fyn, rather than affecting STAT3 phosphorylation through the cytokine-JAK pathway or inducing STAT3 phosphorylation in an autocrine or paracrine manner.

[0300] Example 4 Scri1 inhibits the differentiation of Th17 cells and promotes the differentiation of Treg cells by inhibiting the activity of Lck / Fyn

[0301] 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 converted Th17 cells into Treg cells, but had no effect on the differentiation of Th1 and Th2 mediated by STAT4 and STAT6. CD4+ naive T cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin-streptomycin, 55μM β-mercaptoethanol and 2mM glutamine. Approximately 400,000 CD4 naive T cells were seeded in 48-well plates and coated with 2μg / ml anti-mouse CD3 and 1μg / ml anti-mouse CD28, respectively. Optimal differentiation conditions were used to induce Th17 differentiation of naive CD4+ T cells by adding 2.5ng / ml TGFβ, 10ng / ml mouse IL-1β, 10ng / ml mouse IL-6, 10ng / ml mouse IL-23, 1μg / ml anti-IFNγ, and 1μg / ml anti-mouse IL-4. Standard differentiation conditions were used to induce Th17 differentiation of naive CD4+ T cells by adding 2.5ng / ml TGFβ, 20ng / ml mouse IL-6, 1μg / ml anti-IFNγ, and 1μg / ml anti-mouse IL-4. Differentiation of naive CD4+ T cells into Treg cells was induced by adding 0.5ng / ml TGFβ, 200U / ml mouse IL-2, 1μg / ml anti-IFNγ, and 1μg / ml anti-mouse IL-4. The addition of 10 ng / ml mouse IL-12 and 1 μg / ml anti-mouse IL-4 induced the differentiation of CD4+ naive T cells into Th1 cells. The addition of 20 ng / ml mouse IL-4 and 1 μg / ml anti-IFNγ induced the differentiation of CD4+ naive T cells into Th2 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 groups were supplemented with fresh culture medium containing the corresponding concentrations of small molecule compounds, while the control group was supplemented with fresh culture medium containing DMSO. 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 the levels of cytokine secretion by the cells. At the end of differentiation, T cells were stimulated again with coated anti-CD3 and anti-CD28 for 5 hours. The mRNA expression of the corresponding genes was detected by qRT-PCR.

[0302] The experimental results are shown in Figure 4A and Figure 4C. 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 same method was used under the standard differentiation conditions of Th17 to obtain results similar to those under the optimal differentiation conditions of Th17. As shown in Figure 4B and Figure 4D, the results of qRT-PCR showed that the mRNA expression level of Foxp3 was greatly increased, and the mRNA expression levels of Il17a, Il17f, 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. As shown in Figure 5A, under Treg differentiation conditions containing cytokines TGFβ and IL-2, Srci1 promoted Treg cell differentiation. As shown in Figure 5B, under Treg differentiation conditions containing cytokines TGFβ and IL-2, the administration of Srci1 promoted the increase in the mRNA expression levels of Treg-related genes. As shown in Figure 5C and Figure 5D, under the differentiation conditions of Th1, the experimental groups with the addition of 2.5μM and 5μM Scri1 showed an increase in IFNγ compared with the control group without the addition of 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 significantly higher IL-4 + There was no significant change in the percentage of cells, indicating that Scri1 did not affect Th2 differentiation. As shown in Figures 6A-C, point mutations in STAT3C were used to mimic activated STAT3, and the results showed that STAT3C reversed the effect of Scri1 on TH17 cell 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 the treatment of autoimmune diseases for application in autoimmune diseases caused by Th17 cells.

[0303] Example 5 Effect of Srci1 Inhibition of Lck / Fyn Activity on Gene Expression during Th17 Differentiation

[0304] T cells were differentiated under Th17 or Treg conditions, and Srci1 was added 24 hours later. After differentiation, cells were harvested for mRNA sequencing. As shown in Figure 11A, the results demonstrate that Srci1 treatment can transdifferentiate Th17 cells into Treg cells. As shown in Figures 11B-C, approximately 50% of the genes affected by Srci1 treatment are also affected by STAT3 deficiency. As shown in Figure 11D, Srci1 treatment downregulated the expression of Th17-related genes and upregulated the expression of Treg-related genes. As shown in Figure 11E, pathway enrichment analysis revealed that Srci1 treatment primarily affected the expression of cytokines and immune-related genes. As shown in Figures 12A-B, Srci1 treatment suppressed many direct STAT3 target genes associated with Th17 differentiation and upregulated many direct FOXP3 target genes. Figure 16A illustrates the identification of direct STAT3 target genes during Th17 cell differentiation. As shown in Figure 16B, KEGG pathway analysis was performed to characterize the gene expression changes induced by Srci1 treatment. As shown in Figure 16C, the process of determining FOXP3 direct target genes is shown. As shown in Figure 17A-B, Srci1 treatment has no significant effect on the expression of genes related to the TCR downstream signaling pathway and the mTOR signaling pathway.

[0305] Example 6 Srci1 does not affect the proliferation and survival of T cells

[0306] According to the manufacturer's instructions, CD4+ naive T cells were labeled using Cell Tracer Violet (Thermos Fisher #C34557), and then differentiated under Th17 or Treg differentiation conditions. After 24 hours, Srci1 or DMSO was added. After the differentiation was completed, intracellular cytokines and transcription factors were stained and then detected by flow cytometry. As shown in Figure 13A-B, Srci1 treatment did not affect the proliferation of T cells (the peak of Cell Tracer Violet was not affected by Srci1 treatment). In addition, CD4+ T cells were differentiated under Th17 or Treg conditions. After differentiation, cells were collected and stained with live / dead dye. The cells were then fixed, permeabilized, and stained for intracellular cytokines. As shown in Figures 13C-D, Srci1 treatment did not affect cell survival. As shown in Figures 14A-B, Srci1 treatment did not affect c-Myc expression or the mTOR signaling pathway. As shown in Figures 15A-B, constitutively activated AKT could not reverse the inhibitory effect of Srci1 on Th17 cell differentiation, indicating that Srci treatment does not affect Th17 cell differentiation by affecting the mTOR signaling pathway.

[0307] Example 7: Mutations in STAT3 that cause Th17 cell deficiency disrupt the interaction between STAT3 and Lck / Fyn

[0308] As shown in Figure 18A, the sites on STAT3 that can cause Th17 cell defects. The AlphaFold2 Multimer software was used to predict the binding of the SH2 / SH3 domain to STAT3. As shown in Figures 18B and 18C, the amino acid residues on the SH2 domain that bind to Lck / Fyn and STAT3 at positions 328-335 are conserved among multiple Src family members. The binding of STAT3 mutants to Lck / Fyn was explored according to the "Preparation of Retroviral Vectors and T Cell Infection" and "Co-immunoprecipitation" in the experimental method. As shown in Figures 18D-E, mutations in amino acids at positions 328-335 on STAT3 inhibited the binding of STAT3 and Lck / Fyn.

[0309] Example 8 Mutations at positions 328-335 on STAT3 affect the phosphorylation of STAT3 by Lck / Fyn, but do not affect the cytokine-induced phosphorylation of STAT3.

[0310] According to the steps shown in Figure 20A, LSK cells (rich in blood stem cells) were isolated from STAT3 conditional knockout mice and then overexpressed an empty vector, WT STAT3, STAT3 mutants STAT3 Dup228-230 and R335W. They were then transplanted into Rag1-deficient mice, and CD4+ naive T cells were isolated from them two months later for subsequent experiments. The CD4+ naive T cells obtained above were cultured under standardized conditions, and cells were collected after 48 hours for STAT3 phosphorylation staining or on the fifth day for intracellular cytokine and FOXP3 staining. As shown in Figures 20B-E, STAT3228-230dup and R335W significantly inhibited TCR stimulation-induced STAT3 phosphorylation without affecting cytokine (IL-6 / IL-23)-induced STAT3 phosphorylation. As shown in Figure 21A-E, the effects of peptide sequences containing STAT3 wild-type peptide and membrane-penetrating peptide on STAT3 phosphorylation and Th17 cell differentiation, wild-type STAT3 peptide promoted STAT3 phosphorylation and Th17 cell differentiation, while peptide containing mutation lost this ability. As shown in Figure 21F-H, in vitro kinase activity assays showed that Lck phosphorylated STAT3, and peptide containing STAT3 Dup 328-330 The peptide can promote Lck-mediated STAT3 phosphorylation, while the mutant peptide loses this ability.

[0311] As shown in Figures 19A-C, STAT3C or STAT3C containing a mutation at STAT3 328-335 (these vectors all contain the cell surface marker Thy1.1) was overexpressed in differentiating Th17 cells. After differentiation, intracellular staining and Thy.1.1 staining were performed, and cells overexpressing these vectors were circled using Thy1.1. The results showed that overexpression of STAT3C promoted the differentiation of Th17 cells, while the mutation at STAT3 328-335 did not affect the ability of STAT3C to promote Th17 cell differentiation, indicating that these mutations did not affect the DNA binding ability of STAT3; as shown in Figures 19D-F, STAT3C refers to STAT3 with amino acid mutations at positions 662 and 664 of STAT3, which is constitutively active (i.e., it can enter the cell nucleus and exert transcription factor activity independent of phosphorylation). We overexpressed STAT3C or STAT3C containing a mutation at STAT3 328-335 in differentiating Th17 cells, and then added Srci1 to the cell culture medium. Because STAT3C can directly enter the nucleus, it can directly reverse the effect of Srci1 on STAT3 phosphorylation, and STAT3C containing the STAT3 328-335 mutation can also reverse the effect of Srci1 on Th17 cell differentiation, which further indicates that the 328-335 mutation does not affect STAT3C's ability to reverse Srci1 on Th17 cell differentiation. However, when WT STAT3 or STAT3 328-335 mutants are directly overexpressed in STAT3KO CD4+T cells, as shown in Figure 19G-J, WT STAT3 can mediate the differentiation of Th17 cells, while the STAT3 328-335 mutation significantly affects the STAT3-mediated differentiation of Th17 cells. In summary, these experiments collectively reveal that the STAT3 328-335 mutation affects the phosphorylation of STAT3 induced by TCR stimulation, thereby affecting the differentiation of Th17 cells.

[0312] Example 9: The Lck / Fyn kinase inhibitor Scri1 significantly improves the symptoms of the EAE model

[0313] Eight-week-old C57BL / 6 mice were divided into groups of approximately 10. The mice were immunized twice with 300 μg of the myeloid oligodendritic cell glycoprotein MOG35-55 (MEVGWYRSPFSROVHLYRNGK) peptide emulsified in complete Freund's adjuvant (CFA). Pertussis toxin was then injected intraperitoneally to establish an EAE model. When clinical symptoms began to appear, DMSO or Scri1 were injected 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.

[0314] The purpose of this example is to study the therapeutic effect of Scri1 on autoimmune disease (EAE). The experiment effectively alleviated the clinical symptoms of EAE mice by administering Scri1 to EAE model mice, reduced the differentiation of Th17 cells, and converted Th17 cells into Treg cells, but had no effect on Th1 cells. As shown in Figure 22A-H, after the mice developed clinical symptoms of EAE, the disease score curve of the Scri1-treated group was flatter and the disease score was lower than that of the vehicle control group that was not administered Scri1, indicating that the clinical symptoms of EAE were effectively alleviated. In addition, the IL-17 in the experimental group administered with Scri1 decreased significantly. + 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.

[0315] Example 10 Single mutation STAT3 mutant Dup 328-330 Effects of R335W and R335W on Th17 differentiation and EAE disease

[0316] 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 h, cells were treated with STAT3 WT or Dup 328-330The CD4+T cells were infected with R335W and R335W encoding genes or empty vectors by centrifugation at 37°C and 2000 rpm 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+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.

[0317] The purpose of this example is to explore the 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 Figures 23A-D and 24A-G, 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 STAT3WT made mice IL-17 + A higher percentage of cells had FOXP3 + The percentage of cells was lower, indicating that STAT3WT could mediate the differentiation of Th17 cells, while Dup 328-330 , R335W cannot mediate the differentiation of Th17 cells. + There was no significant difference in the number of cells, indicating that there was no significant effect on the differentiation of Th1 cells.

[0318] Example 11 Effect of mutant STAT3 MUT on Th17 cell differentiation

[0319] Chemically synthesized STAT3 WT or STAT3 MUT peptides containing a membrane-penetrating peptide (10 μg / ml, using PBS as a blank control) were added to CD4+ T cells cultured under Th17 conditions to test their effects on Th17 cell differentiation. Fresh culture medium was added if necessary. After 5 days of differentiation, the above stimulation method was repeated and intracellular staining was performed.

[0320] 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 26A-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 the STAT3 MUT peptide containing the membrane-penetrating peptide had a significant inhibitory effect on the differentiation of Th17 cells and promoted the differentiation of Treg cells. These results indicate that the STAT3 double mutant containing the membrane-penetrating peptide may have a therapeutic effect on Th17 cell-induced autoimmune diseases.

[0321] Example 12 Prediction of the binding between the SH2 / SH3 domain on Lck / Fyn and STAT3

[0322] AlphaFold2 Multimer was used to predict the binding between the SH2 / SH3 domain on Lck / Fyn and STAT3. As shown in Figure 25A-E, the results showed that the SH2 domain on Lck / Fyn can bind to amino acids 328-335 on STAT3; while the SH3 domain does not bind to STAT3.

[0323] 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. A method for inhibiting Th17 cell differentiation and promoting Treg cell differentiation, the method being used for treating Th17 cell-related diseases, the method inhibiting or blocking the mutual binding of Lck / Fyn and STAT3, thereby inhibiting the phosphorylation of STAT3 by Lck / Fyn.

2. The method according to claim 1, characterized in that The method of inhibiting or blocking the mutual binding of Lck / Fyn and STAT3 includes using antibodies, small molecule compounds, polypeptides or other substances to inhibit the kinase activity of Lck / Fyn; or directly inhibiting the mutual binding of the SH2 domain on Lck / Fyn and the 320-340 position on STAT3; or inhibiting the binding of the kinase domain of Lck / Fyn with the domain near Y705 on STAT3; or inhibiting the conformational change of Lck / Fyn kinase, thereby inhibiting the phosphorylation of STAT3 by Lck / Fyn; and also includes using siRNA, shRNA, antibodies or their derivatives to inhibit the expression of Lck / Fyn or STAT3, thereby inhibiting the mutual binding of Lck / Fyn and STAT3.

3. The method according to claim 2, characterized in that The polypeptide, small molecule inhibitor or other substance that specifically inhibits the correct binding of Lck / Fyn to STAT3 used to directly inhibit the mutual binding between the SH2 domain on Lck / Fyn and the 320-340 position on STAT3; preferably, the polypeptide that specifically inhibits the correct binding of Lck / Fyn to STAT3 includes a STAT3 mutant or a fragment thereof, preferably, the STAT3 mutant or a fragment thereof includes a mutation in the 328-335 peptide segment compared to the wild-type STAT3; preferably, the STAT3 mutant or a fragment thereof includes the amino acid sequence shown in SEQ ID NO: 1; preferably, the STAT3 mutant or a fragment thereof is also fused with a cell-penetrating peptide; further preferably, the cell-penetrating peptide is located at the N-terminus or C-terminus of the STAT3 mutant or a fragment thereof; further preferably, the amino acid sequence of the cell-penetrating peptide is as shown in SEQ ID NO:

5.

4. The method according to claim 2, characterized in that: The inhibition of Lck / Fyn kinase activity uses a Lck / Fyn kinase inhibitor, and the Lck / Fyn kinase inhibitor is selected from 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, or one or more of their derivatives; the derivative refers to a compound formed by replacing atoms or atomic groups in the parent compound molecule with other atoms or atomic groups, but the compound formed after the atoms or atomic groups are replaced can still play the same function as the parent compound, especially a compound that can still inhibit the phosphorylation of STAT3 by Lck / Fyn.

5. The method according to claim 1, characterized in that The Th17 cell-related 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, inflammatory bowel disease, asthma, systemic lupus erythematosus, and / or other autoimmune diseases in which Th17 cells play an important pathogenic role, and the non-autoimmune disease is a cytokine storm generated during a Th17 cell-related infection or treatment; And / or, the subject of the method is a mammal, preferably, the mammal is a human.

6. A drug for preventing or treating Th17 cell-related immune diseases, the drug comprising a small molecule inhibitor of Lck / Fyn or a polypeptide shown in SEQ ID NO: 5; preferably, the small molecule inhibitor of Lck / Fyn is selected from Srci1, Dasatinib, Bosutinib, and Saracatinib.

7. Use of Lck / Fyn-mediated STAT3 activation pathway as a target in the preparation or screening of products, wherein the products block the Lck / Fyn-mediated STAT3 activation pathway and have any one or more of the following effects: (1) Inhibit the differentiation of Th17 cells; (2) Promote the transdifferentiation of Th17 cells into Treg cells; (3) Prevent or treat Th17 cell-related immune diseases.

8. Use of a blocker of Lck / Fyn-mediated STAT3 activation in preparing a product, characterized in that: The product has one or more of the following effects: (1) Inhibit the differentiation of Th17 cells; (2) Promote the transdifferentiation of Th17 cells into Treg cells; (3) Prevent or treat Th17 cell-related immune diseases.

9. The use according to claim 8, characterized in that The Lck / Fyn-mediated STAT3 activation blocker inhibits or blocks the binding of the Lck / Fyn kinase domain to the Y705 domain on STAT3; Alternatively, the Lck / Fyn-mediated STAT3 activation blocker is selected from: Lck / Fyn kinase inhibitors and derivatives thereof; substances that specifically inhibit the correct binding of Lck / Fyn to STAT3.

10. The use according to claim 9, 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 and their derivatives; 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 SH2 domain of Lck / Fyn to the 320-340 peptide segment 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.

11. The use according to claim 10, characterized in that The Lck / Fyn kinase inhibitor is selected from 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, or one or more of their derivatives; The substance that specifically inhibits the correct 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; Preferably, the STAT3 mutant or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 1; Preferably, the STAT3 mutant or fragment thereof is further fused with a cell-penetrating peptide; further preferably, the cell-penetrating peptide is located at the N-terminus or C-terminus of the STAT3 mutant or fragment thereof; further preferably, the amino acid sequence of the cell-penetrating peptide is as shown in SEQ ID NO:

5.

12. The use according to claim 8, characterized in that 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, inflammatory bowel disease, asthma and systemic lupus erythematosus, and / or other autoimmune diseases in which Th17 cells play an important pathogenic role, and the non-autoimmune disease is a cytokine storm generated during infection or treatment associated with Th17 cells; And / or, the product is used for mammals or T cells of the mammals.

13. Use of any of the following biomaterials in the preparation of products for preventing or treating Th17 cell-related 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; further preferably, the cell-penetrating peptide is located at the N-terminus or C-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; 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 has been integrated with the polynucleotide described in b.

14. A biomaterial, the biomaterial being selected from any one or more of the following: 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 or C-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; 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 has been integrated with the polynucleotide described in b.

15. A drug for preventing or treating Th17 cell-related immune diseases, comprising the biomaterial according to claim 14.

16. A method for treating Th17 cell-related immune diseases, comprising administering to a subject an effective amount of the medicament of claim 6, or the blocking agent for use in any one of claims 8 to 11, or the biomaterial of claim 14.

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