Thpatho and use thereof in treatment of autoimmune diseases

Abstract

A THPATHO and the use thereof in the treatment of autoimmune diseases. Specifically, the use of a metabolite of Bifidobacterium in the treatment of autoimmune diseases is provided. Bifidobacterium breve, Bifidobacterium faecale and Bifidobacterium adolescentis strains having a therapeutic effect are provided. The bifidobacteria can degrade 5-hydroxyindole to inhibit Thpatho expression and reduce the inflammatory factor secretion ability of Th17 cells, thus alleviating inflammations. A neutralizing antibody targeting Thpatho is provided, the antibody being capable of neutralizing IL17A high expression caused by Thpatho. Therefore, the Bifidobacterium strains and the antibody can treat autoimmune diseases mediated by Th17 cells.

Description

Thpatho and its application in the treatment of autoimmune diseases Technical Field

[0001] This invention relates to the field of treatment for autoimmune diseases. Specifically, this invention relates to THPATHO and its application in the treatment of autoimmune diseases. Background Technology

[0002] Current immunomodulatory drugs used for autoimmune diseases are broad-spectrum but non-disease-specific, and often cause side effects such as infections and malignant diseases. Therefore, there is an urgent need to develop novel or retargeted drugs from a new perspective.

[0003] Studies have found that Th17 cells are an important subset of helper T cells that play a crucial role in the body's fight against pathogenic infections and are regulated by the gut microenvironment. Gut microorganisms and their metabolites can induce the differentiation of pathogenic or non-pathogenic Th17 cells, thereby maintaining gut homeostasis or resisting pathogenic infections. However, excessive production of pathogenic Th17 cells in an inflammatory gut microenvironment can exacerbate tissue inflammation and autoimmune diseases. These studies indicate that the interaction between Th17 cells and gut microbiota promotes gut microecological balance.

[0004] IL-17A, a hallmark cytokine of Th17 cells, is considered a key factor mediating Th17 cell pathogenicity. However, studies in mouse models of enteritis have shown that IL-17A deficiency does not improve the progression of enteritis but rather exacerbates the inflammatory process. Similarly, clinical trials using IL-17A antibodies in Crohn's disease patients have failed, as the use of IL-17A antibodies worsened intestinal inflammation.

[0005] Therefore, there is a need in this field to develop treatment options for autoimmune diseases that target pathogenic Th17 cells. Summary of the Invention

[0006] The purpose of this invention is to provide THPATHO and its application in the treatment of autoimmune diseases.

[0007] In a first aspect of the invention, the use of Thpatho factor inhibitors in the preparation of compositions or formulations for the prevention and / or treatment of autoimmune diseases in subjects is provided.

[0008] In another preferred embodiment, the Thpatho factor inhibitor suppresses the expression of Thpatho in Th17 cells.

[0009] In another preferred embodiment, the Thpatho factor inhibitor is selected from the group consisting of: specific antibodies against Thpatho, siRNA encoding the Thpatho gene, small molecule drugs, bacterial agents, or combinations thereof.

[0010] In another preferred embodiment, the Thpatho factor inhibitor is a 5-hydroxyindole (5-HI) inhibitor.

[0011] In another preferred embodiment, the 5-HI inhibitor is selected from the group consisting of siRNAs of 5-HI expression-related genes, small molecule drugs, bacterial agents, or combinations thereof.

[0012] In another preferred embodiment, the 5-HI inhibitor is a probiotic.

[0013] In another preferred embodiment, the 5-HI inhibitor is Bifidobacterium or its metabolites.

[0014] In another preferred embodiment, the Bifidobacterium is selected from the group consisting of: Bifidobacterium breve, Bifidobacterium feces, Bifidobacterium adolescentis, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium pseudosporidis, Bifidobacterium catecholaria, Bifidobacterium lactis, Bifidobacterium longum, or combinations thereof.

[0015] In another preferred embodiment, the 16S rDNA sequence of the Bifidobacterium has ≥95% identity with the sequence shown in SEQ ID NO. 1, 2 or 3, preferably ≥98%, more preferably ≥99%, and even more preferably ≥99.9%.

[0016] In another preferred embodiment, the Bifidobacterium is Bifidobacterium faecale Zhulab-010, which was deposited on January 25, 2024, at the China Center for Type Culture Collection (CCTCC, Wuhan University, China) with accession number CCTCC NO.M2024202.

[0017] In another preferred embodiment, the Bifidobacterium is Bifidobacterium breve Zhulab-009, which was deposited on January 22, 2024 at the China Center for Type Culture Collection (CCTCC, Wuhan University, China) with accession number CCTCC NO.M2024150.

[0018] In another preferred example, Bifidobacterium adolescentis Zhulab-011 was deposited on January 25, 2024, at the China Center for Type Culture Collection (CCTCC, Wuhan University, China) with accession number CCTCC NO. M2024203.

[0019] In another preferred embodiment, the Thpatho factor inhibitor is a specific antibody against Thpatho, or its encoded nucleic acid, or its expression vector, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region, and the light chain comprising a light chain variable region, wherein the heavy chain variable region comprises the following complementarity-determining region (CDR):

[0020] The amino acid sequence is CDR-H1 as shown in SEQ ID No:4;

[0021] The amino acid sequence is CDR-H2 as shown in SEQ ID No:5;

[0022] The amino acid sequence is CDR-H3 as shown in SEQ ID No:6;

[0023] The light chain variable region includes the following complementary determinant regions (CDRs):

[0024] The amino acid sequence is CDR-L1 as shown in SEQ ID No:7;

[0025] The amino acid sequence is CDR-L2 as shown in the RTS.

[0026] The amino acid sequence is CDR-L3 as shown in SEQ ID NO:8, or

[0027] The heavy chain variable region includes the following complementary determinant region (CDR):

[0028] The amino acid sequence is CDR-H1 as shown in SEQ ID No:9;

[0029] The amino acid sequence is CDR-H2 as shown in SEQ ID No:10;

[0030] The amino acid sequence is CDR-H3 as shown in SEQ ID No:11;

[0031] The light chain variable region includes the following complementary determinant regions (CDRs):

[0032] The amino acid sequence is CDR-L1 as shown in SEQ ID No:12;

[0033] The amino acid sequence is CDR-L2 as shown in STS;

[0034] The amino acid sequence is CDR-L3 as shown in SEQ ID NO:13.

[0035] In another preferred embodiment, the HCVR amino acid sequence of the antibody is as shown in SEQ ID NO:14 or SEQ ID NO:16, or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0036] In another preferred embodiment, the LCVR amino acid sequence of the antibody is as shown in SEQ ID NO:15 or SEQ ID NO:17, or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0037] In another preferred embodiment, the antibody has an HCVR having an amino acid sequence as shown in SEQ ID No:14, and / or an LCVR having an amino acid sequence as shown in SEQ ID NO:15.

[0038] In another preferred embodiment, the antibody’s HCRV has an amino acid sequence as shown in SEQ ID No:16, and / or LCVR has an amino acid sequence as shown in SEQ ID NO:17.

[0039] In another preferred embodiment, the light chain and / or heavy chain of the antibody further include a constant region.

[0040] In another preferred embodiment, the constant region is a human-sourced constant region.

[0041] In another preferred embodiment, the light chain constant region of the antibody is derived from the human immunoglobulin kappa chain.

[0042] In another preferred embodiment, the heavy chain constant region of the antibody is derived from human IgG1.

[0043] In another preferred embodiment, the antibody is an animal-derived antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0044] In another preferred embodiment, the antibody is a single-chain antibody, a double-chain antibody, or an antigen-binding fragment. In another preferred embodiment, the autoimmune disease is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, or a combination thereof.

[0045] In another preferred embodiment, the inflammatory bowel disease is selected from the group consisting of ulcerative colitis, Crohn's disease, or a combination thereof.

[0046] In another preferred embodiment, the treatment of inflammatory bowel disease includes:

[0047] (i) Reduce weight loss;

[0048] (ii) Slows down the shortening of the colon;

[0049] (iii) Reduce the level of the inflammatory cytokine Thpatho;

[0050] (iv) Reduce the proportion and number of inflammatory cells in the gut.

[0051] In another preferred embodiment, the inflammatory cells are Th17 cells.

[0052] In another preferred embodiment, the Th17 cells are positive for IL-17A expression.

[0053] In another preferred embodiment, the mammal includes humans or non-human mammals.

[0054] In another preferred embodiment, the non-human mammals include rodents (such as mice and rats) and primates (such as monkeys).

[0055] In a second aspect of the invention, a Bifidobacterium is provided, wherein the 16S rDNA sequence of the Bifidobacterium has ≥95% identity with the sequence shown in SEQ ID NO. 1, 2 or 3, preferably ≥98%, more preferably ≥99%, and even more preferably ≥99.9%.

[0056] In another preferred embodiment, the Bifidobacterium is Bifidobacterium faecale Zhulab-010, which was deposited on January 25, 2024, at the China Center for Type Culture Collection (CCTCC, Wuhan University, China) with accession number CCTCC NO.M2024202.

[0057] In another preferred embodiment, the Bifidobacterium is Bifidobacterium breve Zhulab-009, which was deposited on January 22, 2024 at the China Center for Type Culture Collection (CCTCC, Wuhan University, China) with accession number CCTCC NO.M2024150.

[0058] In another preferred example, Bifidobacterium adolescentis Zhulab-011 was deposited on January 25, 2024, at the China Center for Type Culture Collection (CCTCC, Wuhan University, China) with accession number CCTCC NO. M2024203.

[0059] In a third aspect of the invention, a specific antibody against Thpatho is provided, the antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region, the light chain comprising a light chain variable region, wherein the heavy chain variable region comprises a complementarity-determining region (CDR):

[0060] The amino acid sequence is CDR-H1 as shown in SEQ ID No:4;

[0061] The amino acid sequence is CDR-H2 as shown in SEQ ID No:5;

[0062] The amino acid sequence is CDR-H3 as shown in SEQ ID No:6;

[0063] The light chain variable region includes the following complementary determinant regions (CDRs):

[0064] The amino acid sequence is CDR-L1 as shown in SEQ ID No:7;

[0065] The amino acid sequence is CDR-L2 as shown in the RTS.

[0066] The amino acid sequence is CDR-L3 as shown in SEQ ID NO:8, or

[0067] The heavy chain variable region includes the following complementary determinant region (CDR):

[0068] The amino acid sequence is CDR-H1 as shown in SEQ ID No:9;

[0069] The amino acid sequence is CDR-H2 as shown in SEQ ID No:10;

[0070] The amino acid sequence is CDR-H3 as shown in SEQ ID No:11;

[0071] The light chain variable region includes the following complementary determinant regions (CDRs):

[0072] The amino acid sequence is CDR-L1 as shown in SEQ ID No:12;

[0073] The amino acid sequence is CDR-L2 as shown in STS;

[0074] The amino acid sequence is CDR-L3 as shown in SEQ ID NO:13.

[0075] In another preferred embodiment, the HCVR amino acid sequence of the antibody is as shown in SEQ ID NO:14 or SEQ ID NO:16, or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0076] In another preferred embodiment, the LCVR amino acid sequence of the antibody is as shown in SEQ ID NO:15 or SEQ ID NO:17, or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0077] In another preferred embodiment, the antibody has an HCVR having an amino acid sequence as shown in SEQ ID No:14, and / or an LCVR having an amino acid sequence as shown in SEQ ID NO:15.

[0078] In another preferred embodiment, the antibody’s HCRV has an amino acid sequence as shown in SEQ ID No:16, and / or LCVR has an amino acid sequence as shown in SEQ ID NO:17.

[0079] In another preferred embodiment, the light chain and / or heavy chain of the antibody further include a constant region.

[0080] In another preferred embodiment, the constant region is a human-sourced constant region.

[0081] In another preferred embodiment, the antibody is an animal-derived antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0082] In another preferred embodiment, the antibody is a single-chain antibody, a double-chain antibody, or an antigen-binding fragment.

[0083] In another preferred embodiment, the light chain constant region of the antibody is derived from the human immunoglobulin kappa chain.

[0084] In another preferred embodiment, the heavy chain constant region of the antibody is derived from human IgG1.

[0085] In a fourth aspect of the invention, a polynucleotide is provided, said polynucleotide encoding an antibody as described in the third aspect of the invention.

[0086] In a fifth aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the fourth aspect of the invention.

[0087] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0088] In a sixth aspect of the invention, a genetically engineered host cell is provided, the host cell containing a vector as described in the fifth aspect of the invention, or having an exogenous polynucleotide as described in the fourth aspect of the invention integrated into its genome.

[0089] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0090] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0091] In a seventh aspect of the invention, a composition for preventing and / or treating autoimmune diseases is provided, the composition comprising:

[0092] (a) A safe and effective dose of a Thpatho factor inhibitor; and

[0093] (b) Pharmaceutically acceptable carriers.

[0094] In another preferred embodiment, the Thpatho factor inhibitor is selected from the group consisting of: specific antibodies against Thpatho, siRNA encoding the Thpatho gene, small molecule drugs, bacterial agents, or combinations thereof.

[0095] In another preferred embodiment, the Thpatho factor inhibitor is a 5-hydroxyindole (5-HI) inhibitor.

[0096] In another preferred embodiment, the 5-HI inhibitor is Bifidobacterium.

[0097] In another preferred embodiment, the Bifidobacterium is selected from the group consisting of: Bifidobacterium breve, Bifidobacterium feces, Bifidobacterium adolescentis, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium pseudosporidis, Bifidobacterium catecholaria, Bifidobacterium lactis, Bifidobacterium longum, or combinations thereof.

[0098] In another preferred embodiment, the 16S rDNA sequence of the Bifidobacterium has ≥95% identity with the sequence shown in SEQ ID NO. 1, 2 or 3, preferably ≥98%, more preferably ≥99%, and even more preferably ≥99.9%.

[0099] In another preferred embodiment, the Bifidobacterium is Bifidobacterium faecale Zhulab-010, which was deposited on January 25, 2024, at the China Center for Type Culture Collection (CCTCC, Wuhan University, China) with accession number CCTCC NO.M2024202.

[0100] In another preferred embodiment, the Bifidobacterium is Bifidobacterium breve Zhulab-009, which was deposited on January 22, 2024 at the China Center for Type Culture Collection (CCTCC, Wuhan University, China) with accession number CCTCC NO.M2024150.

[0101] In another preferred example, Bifidobacterium adolescentis Zhulab-011 was deposited on January 25, 2024, at the China Center for Type Culture Collection (CCTCC, Wuhan University, China) with accession number CCTCC NO. M2024203.

[0102] In another preferred embodiment, the Thpatho factor inhibitor is an antibody as described in the third aspect of the present invention, a polynucleotide as described in the fourth aspect of the present invention, a vector as described in the fifth aspect of the present invention, or a host cell as described in the sixth aspect of the present invention.

[0103] In another preferred embodiment, the composition is selected from the group consisting of: food compositions, health care compositions, pharmaceutical compositions, beverage compositions, feed compositions, or combinations thereof.

[0104] In another preferred embodiment, the composition is an oral formulation.

[0105] In another preferred embodiment, the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.

[0106] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of powders, granules, tablets, sugar-coated tablets, capsules, granules, suspensions, solutions, syrups, drops, sublingual tablets, or combinations thereof.

[0107] In another preferred embodiment, the food composition includes an emulsion product, a solution product, a powder product, or a suspension product.

[0108] In another preferred embodiment, the food composition includes dairy products, milk powder, or emulsions.

[0109] In another preferred embodiment, the liquid formulation is selected from the group consisting of solution products or suspension products.

[0110] In another preferred embodiment, the composition contains 1×10 7 -1×10 10 Bifidobacteria at cfu / mL, preferably 1×10⁻⁶ CFU / mL. 8 -

[0111] 1×10 9 Bifidobacteria cfu / mL, based on the total volume or total weight of the composition.

[0112] In another preferred embodiment, the composition contains 0.0001-99 wt%, preferably 0.1-90 wt%, of the Bifidobacterium, based on the total weight of the composition.

[0113] In another preferred embodiment, the composition is in a unit dosage form (a tablet, a capsule, or a vial), and the mass of the composition in each unit dosage form is 0.05-5g, preferably 0.1-1g.

[0114] In another preferred embodiment, the composition also contains other probiotics.

[0115] In another preferred embodiment, the probiotics are selected from the group consisting of lactic acid bacteria, Lactobacillus acidophilus, or combinations thereof.

[0116] In another preferred embodiment, the composition further contains other drugs for treating inflammatory bowel disease.

[0117] In another preferred embodiment, the other drugs for treating inflammatory bowel disease are selected from the group consisting of glucocorticoids, aminosalicylic acid, sulfasalazine, or combinations thereof.

[0118] In a fourth aspect of the invention, a method for preventing and / or treating mammalian autoimmune diseases in a subject is provided, comprising the steps of administering to the subject a Bifidobacterium as described in a second aspect of the invention, an antibody as described in a third aspect of the invention, a polynucleotide as described in a fourth aspect of the invention, a vector as described in a fifth aspect of the invention, or a host cell as described in a sixth aspect of the invention, or a Bifidobacterium as described in a seventh aspect of the invention.

[0119] In another preferred embodiment, the administration includes oral administration.

[0120] In another preferred embodiment, the dosage is 0.01-5g / 50kg body weight / day, more preferably 0.1-2g / 50kg body weight / day.

[0121] In another preferred embodiment, the object includes a human or a non-human mammal.

[0122] In another preferred embodiment, the non-human mammals include rodents (such as mice and rats) and primates (such as monkeys).

[0123] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0124] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0125] Figure 1A shows the differentially expressed genes in each CD4+ T cell subset.

[0126] Figure 1B shows the results of Thpatho mRNA content detection in different immune cells.

[0127] Figure 1C shows the expression level of Thpatho under the induction of different Th17 cell inducing factors.

[0128] Figure 2 shows the results of screening for Thpatho metabolites in vitro. A: Intracellular Thpatho content after the addition of different metabolites; B: Flow cytometry analysis and statistics. Compared with the control group (22.9%), 5-hydroxyindole significantly increased Thpatho secretion in T cells (41.5%).

[0129] Figure 3 shows the in vivo validation experiment of the regulatory effect of metabolites on Thpatho. A: Statistical graph of mouse colon length, B: Statistical graph of Thpatho content, C: Flow cytometry graph, D: Proportion of IL-17A+ cells and IFNγ+ cells in CD4+ T cells. Compared with the control group, the colon of mice with enteritis in the experimental group injected with 5-HI was shortened, the Thpatho content was increased, and the proportion of IL-17A+ cells in CD4+ T cells was also increased, indicating that 5-hydroxyindole has the potential to induce pathogenic Th17 differentiation and secretion of the inflammatory factor Thpatho.

[0130] Figure 4 shows the metabolic capacity of Bifidobacteria for 5-hydroxyindole, revealing that Bifidobacteria such as Bifidobacterium breve can significantly degrade 5-hydroxyindole.

[0131] Figure 5 shows the relative abundance of Bifidobacteria in mouse feces, illustrating the colonization status of Bifidobacteria in mice. Specific qPCR analysis of Bifidobacterium breve (A), Bifidobacterium feces (B), and Bifidobacterium adolescentis (C) in mouse feces showed that gavage administration of these different Bifidobacteria increased the level of Bifidobacteria in the mouse intestines, suggesting that these Bifidobacteria can all colonize in mice.

[0132] Figure 6 shows the effect of Bifidobacterium colonization on intestinal immunity. It displays the expression map of the Thpatho gene in the colon tissue of mice after modeling, indicating that mice colonized with Bifidobacterium showed decreased inflammatory factors compared to mice in the PBS gavage group.

[0133] Figures 7A-7D show the effects of Bifidobacterium colonization on intestinal inflammation. Figure 7A shows the weight loss of mice after modeling, indicating that the weight loss of mice colonized with Bifidobacterium was not significant compared with the PBS gavage group. Figure 7B is a statistical graph of colon length in mice with TNBS enteritis in different groups. Compared with the PBS group, the colon length of mice colonized with Bifidobacterium was longer, indicating that Bifidobacterium has a certain protective effect against TNBS enteritis in mice. Figure 7C shows the flow cytometry analysis and statistical graph of IL-17A and IFNγ expression, and Figure 7D is an HE staining image of colon sections. The results show that, compared with the control, the colon of mice colonized with Bifidobacterium was longer, the content of inflammatory factors was lower, and the proportion of pathogenic Th17 cells in CD4+ T cells was also lower.

[0134] Figures 8A-8D show the screening and validation of anti-mouse Thpatho neutralizing antibodies. Figure 8A shows the inhibitory effect of the antibody clone on the increase in IL-17A expression induced by Thpatho. Figure 8B shows the clinical scores of experimental autoimmune encephalomyelitis (EAE) mice after treatment with antibody clone 8. Figure 8C shows the results of staining observation of demyelination in the central nervous system of EAE mice after treatment. Figure 8D shows the results of cell infiltration detection in the central nervous system of EAE mice after treatment.

[0135] Figure 9 shows that the anti-human Thpatho neutralizing antibody can neutralize the increase in IL-17A expression induced by human Thpatho. Detailed Implementation

[0136] Through extensive and in-depth research, the inventors have developed, for the first time, a gut microbiota and its application in the treatment of autoimmune diseases. This invention, through screening, discovered that Thpatho is an important factor in triggering Th17 cell-related autoimmune diseases, and further found that 5-HI promotes Thpatho expression. After screening and verification, this invention discovered that metabolites of Bifidobacterium, represented by *Bifidobacterium breve*, can degrade 5-HI, thereby treating autoimmune diseases such as inflammatory bowel disease. The strains provided by this invention have strong intestinal colonization capacity and good safety, and have the potential to be used in probiotic colonization combined with antibody therapy to improve the efficacy of autoimmune disease treatment and extend patient survival. Based on these findings, this invention was completed.

[0137] the term

[0138] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0139] As used herein, “containing,” “having,” or “including” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0140] Inflammatory bowel disease

[0141] As used herein, the term "inflammatory bowel disease" refers to a chronic inflammatory bowel disease influenced by multiple factors. The Bifidobacterium strains of this invention can alleviate inflammatory bowel disease by reducing Thpatho secretion through the degradation of 5-hydroxyindole. The inflammatory bowel diseases treated by this invention include, but are not limited to, Crohn's disease and ulcerative colitis.

[0142] As used in this article, the term "Thpatho" refers to a Th17 cell-specific inflammatory cytokine, one of the factors that cause Th17 cell-related autoimmune diseases. Specifically, "Th" is short for T helper cells, and "Patho" is short for pathogenic factor. Therefore, "Thpatho" refers to the cytokine that makes helper T cells pathogenic.

[0143] Therefore, in this invention, the use of Thpatho inhibitors in the treatment of autoimmune diseases (such as inflammatory bowel disease) has been screened, discovered, and provided.

[0144] As used herein, the term "5-hydroxyindole" refers to a compound with the structure shown below. 5-hydroxyindole can promote the proportion of pathogenic Th17 cells in colonic lymphocytes, increase Thpatho content, and aggravate enteritis in mice.

[0145] Bifidobacteria

[0146] As used herein, the term "Bifidobacterium" refers to a Gram-positive, non-motile, rod-shaped, sometimes forked, strictly anaerobic genus of bacteria. This invention, through in vitro screening and in vivo validation, has found that strains of the genus *Bifidobacterium* produce metabolites capable of degrading 5-hydroxyindole. The *Bifidobacterium* used in this invention may include, but is not limited to, *Bifidobacterium breve*, *Bifidobacterium adolescentis*, *Bifidobacterium fecalith*, or combinations thereof.

[0147] In one embodiment, the Bifidobacterium of the present invention is *Bifidobacterium breve*. This Bifidobacterium strain was isolated from BBE plates derived from healthy volunteers and was capable of anaerobic culture in GAM broth. In in vitro screening, this strain was able to efficiently metabolize 5-hydroxyindole. The *Bifidobacterium breve* of the present invention was deposited on January 22, 2024, at the China Center for Type Culture Collection (CCTCC) (Wuhan, China), accession number: CCTCC NO. M2024150, and classified as *Bifidobacterium breve* Zhulab-009. Its 16S rDNA sequence is as follows:

[0148] In one embodiment, the Bifidobacterium of the present invention is *Bifidobacterium adolescentis*. This Bifidobacterium strain was isolated from BBE plates derived from healthy volunteers and was capable of anaerobic culture in GAM broth. In in vitro screening, this strain was able to efficiently metabolize 5-hydroxyindole. The *Bifidobacterium adolescentis* of the present invention was deposited on January 25, 2024, at the China Center for Type Culture Collection (CCTCC) (Wuhan, China), accession number: CCTCC NO.M 2024203, and classified as *Bifidobacterium adolescentis* Zhulab-011. Its 16S rDNA sequence is as follows:

[0149] In one embodiment, the Bifidobacterium of the present invention is *Bifidobacterium faecale*. This Bifidobacterium strain was isolated from BBE plates derived from healthy volunteers and was capable of anaerobic culture in GAM broth. In in vitro screening, this strain was able to efficiently metabolize 5-hydroxyindole. The *Bifidobacterium faecale* of the present invention was deposited on January 25, 2024, at the China Center for Type Culture Collection (CCTCC) (Wuhan, China), accession number: CCTCC NO. M2024202, and classified as *Bifidobacterium faecale* Zhulab-010. Its 16S rDNA sequence is as follows:

[0150] Filtering methods

[0151] The screening steps for pathogenic factors and therapeutic bacteria in this invention are as follows:

[0152] 1. Identification of inflammatory factors specifically secreted by pathogenic Th17 cells:

[0153] Different CD4 T cell subsets were differentiated using an in vitro CD4 T cell differentiation system, and their transcriptomes were sequenced, including Th0, Th1, Th2, Th9, Th17, and Treg. Transcriptome analysis of these subsets revealed distinct transcriptional expression profiles, including different effector molecules and transcription factors. Further analysis of Th17 cell-specific genes identified 20 genes with the most significant expression differences. These genes specifically expressed in Th17 cells included secreted cytokines, intracellular transcription factors, and cell membrane surface molecules. Among the Th17 cell-specific genes, one secreted factor—

[0154] Thpatho is highly expressed in Th17 cells. To further validate the transcriptome sequencing results, different CD4 T cell subsets were differentiated in vitro, and qPCR was performed on these cells. Consistent with the RNA sequencing results, the qPCR results showed that Thpatho was specifically highly expressed under Th17 cell differentiation conditions, while its expression was very low or absent in other cell subsets.

[0155] Th17 cell differentiation is regulated by multiple cytokines. To further investigate which cytokines regulate Thpatho expression, initial CD4 T cells were treated with different cytokines and combinations thereof, and Thpatho expression was detected by qPCR. The results showed that among different Th17 cell inducing factors, IL-6 could induce Thpatho expression, while IL-23 or TGFβ1 alone could not induce Thpatho expression. However, the synergistic effect of IL-6 and TGFβ1 induced a higher level of Thpatho expression, demonstrating that differentiated Th17 cells exhibit higher and more specific Thpatho expression.

[0156] 2. Establish gut microbiota banks from healthy volunteers and IBD patients:

[0157] Single colonies were isolated and cultured from the feces of healthy volunteers and IBD patients: Volunteer fecal samples were preserved in 20% glycerol phosphate buffer and serially diluted to 10⁻⁶. -5 10 -6 10 -7 The bacteria were plated on GAM broth, BBE, RCM, MRS, TSB, BHI, and Columbia blood agar plates. Single clones were picked and transferred to the corresponding liquid culture medium. Species were determined by 16S sequencing. The bacteria were stored in glycerol at -80°C, and the bacterial supernatant was collected and cryopreserved to establish a human intestinal microbial bank.

[0158] 3. In vitro screening of Thpatho metabolites with specific regulation:

[0159] CD4+ was isolated from the spleen of a mouse with Thpatho fluorescence reporting. T cells: Spleen tissue was cleaned of surface fat, added with phosphate-buffered saline solution, and ground into a cell suspension. Residual tissue was filtered through a steel mesh. Cells were collected at the bottom of the tube by centrifugation at 500g, and red blood cells were removed by adding erythrocyte lysis buffer. The remaining cells were then subjected to CD4+... T-cell magnetic bead sorting kit isolates CD4+ cells T cells. Adding anti-CD3 to activate CD4+ cells in vitro. T cells were divided into 96-well plates. Over one hundred metabolites from endogenous, microbial, and dietary sources were added at the same concentration for stimulation, and the cells were cultured under Th17 differentiation conditions. After 4 days of incubation, T cells were collected from the wells, and their DNA was extracted. qPCR was used to detect changes in Thpatho content in each well of the T cells.

[0160] 4. In vivo verification of the regulatory effect of metabolites on Thpatho:

[0161] Mice with enteritis and transfused with CD45RBhi T cells were intravenously injected every two days with a metabolite and a solvent to dissolve the metabolite, at a dose of 30 mg / kg. The mice were weighed weekly. After six weeks, the mice were sacrificed, and their weight changes and colon length were statistically analyzed. Thpatho content in colon tissue was detected by qPCR, and Th17 differentiation in lymphocytes promoting colon differentiation was detected by flow cytometry.

[0162] 5. Obtain the metabolite 5-hydroxyindole:

[0163] Bacterial-derived 5-hydroxyindole (5-HI) can be produced by the degradation of tryptophan by its own TnaA enzyme and is also present in vivo. In in vitro screening, this invention found that 5-hydroxyindole strongly promotes Thpatho expression, and this promoting effect is gradient-dependent. In in vivo validation, 5-hydroxyindole exacerbated enteritis in mice and increased Thpatho levels and Th17 cell counts in colonic lymphocytes.

[0164] 6. In vitro screening of strains that specifically degrade metabolites:

[0165] Glyceryl bacteria from the bacterial bank were inoculated into EP tubes containing 1 ml of liquid culture medium. After 48 hours of successful bacterial recovery, 100 μL of the bacterial culture was added to liquid culture medium containing 5-hydroxyindole and incubated in the dark for 24 hours. All bacterial supernatants were collected, and mass spectrometry was used to detect changes in metabolite levels. It was found that all Bifidobacterium strains had the ability to metabolize 5-hydroxyindole.

[0166] 7. Safety Inspection:

[0167] Representative strains of Bifidobacterium breve, Bifidobacterium adolescentis, and Bifidobacterium faecale were colonized in mice. The weight of the mice was recorded daily. After the mice were sacrificed, the organs of the mice were weighed to assess the safety of each candidate strain.

[0168] 8. Detect the colonization effect of the strain in mouse models:

[0169] C57 / B6J mice were administered ABX for one week, followed by gavage of 0.8 OD 200 μL bacterial solution three times a week. Fecal samples were collected, and the bacteria were lysed with 20% SDS. Bacterial DNA was extracted using phenol-chloroform, and PCR was performed using strain-specific primers with a universal 16S primer as an internal control to assess colonization efficiency. Further validation was achieved through 16S sequencing of mouse feces.

[0170] 9. Detection of intestinal 5-hydroxyindole and Thpatho in colonized strains

[0171] For mice that successfully colonized, their feces were collected, and the Thpatho content was detected by qPCR and compared with the uncolonized control group.

[0172] 10. IBD treatment efficacy assessment:

[0173] Candidate and control strains were colonized in mice with trinitrobenzenesulfonic acid (TNBS)-induced enteritis, and mouse body weight was measured weekly. After six weeks, the mice were sacrificed, and their body weight changes and colon length were statistically analyzed. Thpatho content in colon tissue was detected by qPCR, and Th17 differentiation in colonic lymphocytes was detected by flow cytometry to evaluate the efficacy of the functional bacteria against IBD.

[0174] Antibody

[0175] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0176] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0177] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0178] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0179] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0180] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0181] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0182] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0183] Currently, the DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0184] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0185] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0186] The main advantages of this invention include:

[0187] 1) The strains Bifidobacterium breve, Bifidobacterium adolescentis, and Bifidobacterium faecale provided by this invention were isolated from healthy volunteers, are easy to culture in vitro, and have good colonization ability in mouse intestines.

[0188] 2) The metabolites produced by the strain provided by this invention can degrade 5-hydroxyindole and inhibit the secretion of the inflammatory factor Thpatho by Th17 cells, thus improving the efficacy of autoimmune diseases.

[0189] 3) In vivo experiments in mice confirmed that the strain of the present invention can reduce the content of 5-hydroxyindole and Thpatho in mice and has a certain tendency to alleviate IBD inflammation in mice with TNBS-induced enteritis.

[0190] 4) This invention screened anti-Thpatho neutralizing antibodies and verified through in vivo experiments that they could reduce the increase in IL-17A expression induced by Thpatho.

[0191] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0192] Example 1: Identification of inflammatory factors specifically secreted by pathogenic Th17 cells

[0193] Mouse spleen tissue was cleaned of surface fat, added with phosphate-buffered saline solution, and ground into a cell suspension. Residual tissue was filtered through a steel mesh. Cells were collected at 500g by centrifugation and red blood cell lysis buffer was added to remove red blood cells. The remaining cells were then used to isolate CD4+ T cells using a CD4+ T cell magnetic bead sorting kit. Anti-CD3 was added to activate CD4+ T cells in vitro, and the cells were then separated into 96-well plates. Different CD4+ T cell subsets were differentiated using an in vitro CD4+ T cell differentiation system with the addition of different cytokines. Transcriptome sequencing was performed on the different CD4+ T cell subsets, including Th0, Th1, Th2, Th9, Th17, and Treg. Transcriptome analysis of different cell subsets revealed different transcriptional expression profiles, including different effector molecules and transcription factors. Further analysis of genes specifically expressed in Th17 cells identified 20 genes with the most significant expression differences. These genes specifically expressed in Th17 cells included secreted cytokines, intracellular transcription factors, and cell membrane surface molecules. Among the genes specifically expressed by Th17 cells, a secreted factor, Thpatho (Figure 1A), was identified. This factor can promote the development of Th17 cell-mediated autoimmune diseases and exhibits high expression levels in pathogenic Th17 cells. To further validate the transcriptome sequencing results, different CD4+ T cell subsets were differentiated in vitro, and qPCR was performed on these cells using primers for Thpatho.

[0194] RT-PCR (Real-Time PCR):

[0195] The kits used were SYBR GREEN (Novazia, Q311) and CFX384 (BIO-RAD, C1000 touch).

[0196] qPCR primers; 384 qPCR plate; sealing film

[0197] Mixing system (5ul system):

[0198] Apply 5 μL per well to a 384 plate. Avoid direct sunlight throughout the process, as prolonged application time can lead to evaporation.

[0199] Seal the plate with a membrane, centrifuge, and load it into the PCR machine. Perform real-time PCR (reaction on ice). Detect changes in Thpatho content in each well of T cells.

[0200] Consistent with the transcriptome sequencing results, the qPCR results (Figure 1B) showed that Thpatho was specifically highly expressed under Th17 cell differentiation conditions, while it was expressed at very low levels or not at all in other cell subpopulations.

[0201] Th17 cell differentiation is regulated by a variety of cytokines. To further investigate which cytokines regulate Thpatho expression, different cytokines and combinations of cytokines were used to treat initial CD4 T cells, and Thpatho expression was detected by qPCR.

[0202] Experimental results showed that among different Th17 cell inducing factors, IL-6 could induce Thpatho expression, while IL-23 or TGFβ1 alone could not induce Thpatho expression (Figure 1C). However, the synergistic effect of IL-6 and TGFβ1 could induce a higher level of Thpatho expression, demonstrating that differentiated Th17 cells have higher and more specific Thpatho expression (Figure 1C).

[0203] Example 2: In vitro screening of Thpatho metabolites with specific regulation

[0204] CD4+ was isolated from the spleen of Throphosphorus mice (from the USTC Laboratory Animal Center). T cells: Spleen tissue was cleaned of surface fat, added with phosphate-buffered saline solution, and ground into a cell suspension. Residual tissue was filtered through a steel mesh. Cells were collected at the bottom of the tube by centrifugation at 500g, and red blood cells were removed by adding erythrocyte lysis buffer. The remaining cells were then subjected to CD4+... T-cell magnetic bead sorting kit isolates CD4+ cells T cells. Adding anti-CD3 to activate CD4+ cells in vitro. T cells were divided into 96-well plates. Each plate was stimulated with the same concentration of over one hundred metabolites from endogenous, microbial, and dietary sources, and cultured under Th17 differentiation conditions. After 4 days of incubation, T cells were collected from the wells, and cellular RNA was extracted.

[0205] 1. Transfer the 96-well cell sample to a 1.5 ml EP tube, centrifuge, and add 500 μL of Trizol (TIANGEN DP424) to mix thoroughly. For suspended cells, discard the supernatant after centrifugation, add Trizol, mix well, and transfer to a 1.5 ml EP tube (not necessarily RNase-free). Add 0.2 ml of chloroform (1 / 5 the amount of Trizol) to 1 ml of Trizol, then vortex vigorously for 15 seconds and incubate at room temperature for 5 minutes. Centrifuge at 12000 g for 15 minutes at 4°C, transfer approximately 200 μL of the supernatant to a new RNase-free EP tube, add an equal volume of isopropanol to precipitate the RNA; mix thoroughly, and incubate at 4°C for 10 minutes to precipitate the RNA (if the amount of RNA is large, the 10-minute incubation period is not necessary); centrifuge at 12000 g for 15-30 minutes at 4°C, discard the supernatant, add 1 ml of 75% ethanol (prepared with DEPC water) to wash the RNA precipitate, and centrifuge at 7500 g for 3 minutes at 4°C; dry the RNA. Carefully discard the supernatant after centrifugation, then centrifuge for another 5 seconds to collect the liquid on the tube wall to the bottom of the tube. Remove the liquid with an RNase-free pipette tip, open the centrifuge tube cap and dry the RNA for 15 minutes (be careful of RNase contamination). After the RNA precipitate becomes transparent, add an appropriate amount of DEPC water, dissolve it thoroughly, mix well, centrifuge, and store at -80℃ for long-term storage.

[0206] 2. Reverse transcription (RNA-reverse transcription): Using a reverse transcription kit (Novizan, R323)

[0207] Reverse transcription: Removal of genomic gDNA

[0208] Prepare the reaction mixture (4 μL system) according to the following components.

[0209] 42℃ for 2 min / 37℃ for 5 min

[0210] 4℃∞

[0211] Reverse transcription (5ul system)

[0212] The reverse transcription procedure:

[0213] 37℃ for 15 minutes

[0214] 85℃ for 5 seconds

[0215] 4℃ 5min

[0216] Dilute the cDNA product 10-fold with ddH2O. Store at 4°C for short-term storage and -20°C for long-term storage.

[0217] As shown in Figure 2-A, statistical analysis of qPCR results revealed that almost all metabolites in the laboratory metabolite library could promote the secretion of the inflammatory factor Thpatho by Th17 cells. The addition of most metabolites increased Thpatho levels by 50%-150%, with bacterial-derived 5-hydroxyindole showing the strongest promoting effect, approaching 180%. To further confirm the promoting effect of 5-hydroxyindole on Thpatho, flow cytometry was used for further verification. The results are shown in Figure 2-B. Compared with the DMSO group, 5-hydroxyindole significantly increased the secretion of Thpatho by T cells, and this promoting effect showed a gradient-dependent effect. Since 5-hydroxyindole is poorly soluble in water but readily soluble in DMSO, DMSO was used as the solvent in the experiment.

[0218] The results of Example 2 showed that 5-hydroxyindole could promote the secretion of the inflammatory cytokine Thpatho by Th17 cells in vitro. The following examples further verify whether 5-hydroxyindole plays the same role in vivo.

[0219] Example 3: In vivo verification of the regulatory effect of metabolites on Thpatho

[0220] CD45RBhi CD4 T cell transfusion-induced enteritis models (including ulcerative colitis and Crohn's disease) are a common method for studying the role of CD4 T cells in the development of enteritis and are also one of the classic models simulating human inflammatory bowel disease (IBD). Transfused CD45RBhi CD4 T cells, in T and B cell-deficient Rag1... - / - In mice, different effector cell subsets differentiate, thereby promoting the development of enteritis. In particular, Th17 cells play an important role in this model.

[0221] Using flow cytometry, CD4+CD25-CD45RBhigh T cells from wild-type mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were sorted and transfused (intraperitoneally injected) into the three groups of Rag1 cells mentioned above. - / - In mice, the number of cells transferred per mouse was 5 x 10-1 5 Inducing a mouse enteritis model.

[0222] Specifically, spleens and lymph nodes from mice (C57BL / 6 mice, 6-8 weeks old) were thoroughly ground in a sterile steel mesh to prepare a single-cell suspension. The single-cell suspension was added to a 15ml centrifuge tube and centrifuged at 500g for 5 minutes. The supernatant was discarded, and 2ml of erythrocyte lysis buffer was added to each spleen. The cells were lysed at room temperature for 5 minutes. 8ml of 1x PBS was added to each spleen to terminate the reaction. The cells were centrifuged at 500g for 5 minutes, and the pellet was resuspended in 1ml of MACS buffer (1x PBS contains 0.5% FBS and 2Mm EDTA). The pellet was filtered through a 200-mesh filter and cell counting was performed. CD4+ T cell enrichment (MACS) was performed using the MojoSort™ Mouse CD4 T Cell Separation Kit (BD). The enriched cells were centrifuged, counted, and labeled with antibodies. CD4+CD25-CD45RBhi cells were then sorted using BD Aria III. T cells; purity test (>95% indicates good sorting effect); sorted cells were centrifuged at 500g for 5 min in 15ml centrifuge tubes, the supernatant was discarded, and the cells were resuspended in pre-cooled 1x PBS for counting; the required number of cells for transfusion was taken, one Rag1 per cell. - / - Transfuse 5 x 10⁵ cells (ip), or 200 μL, into mice. Record the initial body weight before transfusion. Monitor mouse body weight weekly. When the mouse's body weight drops to 70-80%, collect the mouse's intestines for analysis. When the body weight drops below 90%, monitor body weight every 0.5 weeks.

[0223] Mice with enteritis, transfused with CD45RBhi T cells, were intravenously injected with metabolites and DMSO every two days at a dose of 30 mg / kg. Six weeks later, the mice were sacrificed, and their body weight changes were statistically analyzed. Colon length was measured, and the intestinal contents were washed with PBS. A portion of the colon tissue was pre-extracted, and RNA was extracted. PCR was performed using Thpatho-specific primers, with a 16S universal primer as an internal control, to detect Thpatho levels in the mice. The intestine was then cut into 1-2 cm segments and placed in 25 ml of pre-digestion solution. The mixture was incubated at 37°C and 220 rpm for 20 min. The resulting culture medium was filtered through a 200-mesh steel mesh. The intestinal segments were added to the pre-digestion solution for further processing. Centrifugation of the filtered medium yielded intestinal epithelial cells and intraepithelial lymphocytes. This process was repeated twice. The remaining tissue was added to a digestion solution containing collagenase and DNase, and filtration yielded intestinal lamina propria lymphocytes. Intraepithelial lymphocytes and lamina propria lymphocytes were labeled with specific fluorescent antibodies, and the secretion of INF-γ and IL-17A was detected by flow cytometry.

[0224] The experimental results are shown in Figure 3. Mice injected intravenously with 5-hydroxyindole showed a shorter colon length compared to mice injected intravenously with DMSO (Figure 3-A, the colon length shortens when there is inflammation in the mouse colon), and also had higher levels of the inflammatory factor Thpatho (Figure 3-B, the Thpatho level increases when there is inflammation in the mouse colon). Intestinal lamina propria lymphocytes were obtained by digesting the pretreated colon with a digestive solution containing collagenase II. Simultaneously, mouse mesenteric lymph nodes were collected and ground through a 200-mesh filter to prepare a single-cell suspension. Flow cytometry analysis of the expression of IL-17A+ cytokines in the intestinal lamina propria lymphocytes and mesenteric lymph nodes revealed a significant increase in the proportion and number of inflammatory Th17 cells in the intestine of mice injected intravenously with 5-hydroxyindole (Figures 3-C and 3-D). The above data indicate that intravenous injection of 5-hydroxyindole during enteritis in mice mainly exacerbates weight loss caused by intestinal inflammation and protects against intestinal lymphocyte infiltration by promoting the secretion of the inflammatory factor Thpatho by pathogenic Th17 cells.

[0225] The results of Example 3 showed that intravenous administration of 5-hydroxyindole to mice increased the levels of the inflammatory cytokine Thpatho, thereby exacerbating the Th17 cell-mediated intestinal inflammatory response and significantly upregulating the expression of IL-17A+ at the site of inflammation. The following examples will verify whether strains capable of degrading 5-hydroxyindole can alleviate Th17 cell-mediated autoimmune diseases.

[0226] Example 4: Establishment of a gut microbiota bank

[0227] Single colonies were isolated and cultured from the feces of healthy volunteers and IBD patients: Volunteer fecal samples were preserved in 20% glycerol phosphate buffer and serially diluted to 10⁻⁶. -5 10 -6 10 -7 The bacteria were plated on GAM broth, BBE, RCM, MRS, TSB, BHI, and Columbia blood agar plates. Single clones were picked and transferred to the corresponding liquid culture medium. Species were determined by 16S sequencing. The bacteria were stored in glycerol at -80°C, and the bacterial supernatant was collected and cryopreserved. A human gut microbiota bank was established.

[0228] Example 5: In vitro screening of strains that specifically degrade metabolites

[0229] Different bacteria were selected from the bacterial library. One day in advance, glycerol-containing bacteria were cultured in 1 ml of GAM broth. After 36 hours, the growth of the strains was assessed. Strains with good growth were centrifuged and diluted with phosphate buffer to an OD600 of 0.8. They were then inoculated into GAM broth containing 5-hydroxyindole. After 24 hours of anaerobic incubation in the dark, the cultures were centrifuged, and the supernatant was collected. Acetonitrile was added at a 1:1 volume ratio. Magnesium sulfate (0.2 g / ml) and sodium acetate (0.05 g / ml) were then added. The mixture was vortexed and shaken for 10 minutes, followed by centrifugation at 3000 rpm for 10 minutes. 750 μL of the supernatant was collected and centrifuged at 12000 rpm for 3 minutes to prepare a mass spectrometry sample. The 5-hydroxyindole content in the bacterial culture was measured after 24 hours of incubation.

[0230] Mass spectrometry data processing: The concentration of 5-hydroxyindole remaining after 24 hours of anaerobic incubation in the dark is called the initial concentration. The concentration of 5-hydroxyindole in each bacterial culture after 24 hours is called the final concentration. Subtracting the final concentration from the initial concentration gives the concentration reduction over 24 hours. Dividing this by the initial concentration gives the metabolic rate of 5-hydroxyindole for each strain. A higher metabolic rate indicates that the strain degrades more 5-hydroxyindole in 24 hours, i.e., a stronger metabolic capacity.

[0231] The experimental results are shown in Figure 4. The results show that all Bifidobacterium strains have a good metabolic effect on 5-hydroxyindole in vitro.

[0232] Example 6: Verification of strain colonization ability and safety

[0233] This bacterium was isolated from fecal samples of healthy individuals. Fresh fecal samples were added to an equal volume of phosphate buffer containing 20% ​​glycerol, and then serially diluted to 10⁻⁶ with anaerobic phosphate buffer. 6 The bacteria were spread onto a plate and cultured anaerobically on bile aescin agar (BBE) containing vitamin K1 and heme chloride for 48 hours to isolate colonies of the strain.

[0234] Single colonies were picked and cultured in GAM broth for 36 hours. After centrifugation, the cultures were diluted with phosphate buffer to an OD600 of 0.8 and administered to mice via gavage in 200 μL volumes three times a week.

[0235] Mice aged 6-8 weeks were given a quadruple antibiotic regimen to purge their intestinal flora. 0.25g of ampicillin, neomycin, metronidazole, and 0.125g of vancomycin hydrochloride were added to every 250ml of water. Afterward, the water was replaced with regular water, and mice were administered strain Hg79 via gavage. Mice that were given antibiotics and administered strain Hg5593 via gavage served as the control group.

[0236] Mice were administered the strains via gavage for one week, with daily weight recording. Fecal DNA was extracted from each group of mice. 200 μL of SDS, 200 μL of disruption beads, 400 μL of PCR-A, and 400 μL of phenol-chloroform were added to the fecal samples, which were then thoroughly mixed using a tissue homogenizer. DNA was extracted and diluted to 2 ng / μL. PCR was performed using specific primers Hg79 and Hg5593, with a universal 16S primer as an internal control, to detect colonization. After successful colonization, mice were euthanized, and their organs were weighed to assess the safety of each candidate strain.

[0237] The experimental results are shown in Figure 5. The levels of the corresponding strains in the intestines of the three groups of mice colonized with different strains increased significantly, indicating successful colonization after bacterial clearance. After euthanizing the mice, no changes were found in the morphology and weight of any organs in the four groups, proving the safety of the three strains.

[0238] Example 6 shows that after the intestinal flora of mice was cleared by gavage with a quadruple antibiotic (ABX), short-lived Bifidobacterium, adolescent Bifidobacterium, and fecal Bifidobacterium strains were successfully colonized, and the mice all had good health.

[0239] Example 7: Bifidobacteria significantly inhibit intestinal Thpatho expression

[0240] Feces from successfully colonized mice and control mice that did not undergo colonization were collected into EP tubes for DNA extraction. 200 μL of SDS, 200 μL of disruption beads, 400 μL of PCR-A, and 400 μL of phenol-chloroform were added to each tube, and the mixture was thoroughly homogenized using a tissue homogenizer. DNA was extracted and diluted to 2 ng / μL. Thpatho-specific primer PCR was performed with Hprt as an internal control to detect Thpatho content.

[0241] The experimental results are shown in Figure 6. Compared with the control group mice administered PBS by gavage, the Thpatho levels in mice colonized with the three types of Bifidobacteria were decreased. The results indicate that colonization of mice with Bifidobacteria reduces their Thpatho levels, which may play a protective role in mouse inflammation models. In the following examples, mouse models were used to further verify the anti-inflammatory bowel disease effects of the strains.

[0242] Example 8: Evaluation of IBD alleviation effect in mouse model

[0243] TNBS-infected mice were treated with quadruple antibiotics for one week. Starting from day 2, the control group was administered 200 μL of phosphate-buffered saline by gavage, while the experimental group was administered 200 μL of different Bifidobacteria with an OD600 of 0.8 by gavage, three times a week, with mouse weight recorded weekly. In week 6, mice were sacrificed, and after measuring colon length, the intestinal contents were washed with PBS, and the intestinal tract was dissected. A portion of the colon tissue was removed beforehand, and RNA was extracted. Thpatho-specific primers were used for PCR, with Hprt as an internal control, to detect Thpatho levels in the mice. The intestine was then cut into 1-2 cm segments and placed in 25 ml of pre-digestion solution, shaken at 37°C and 220 rpm for 20 min. The resulting culture medium was filtered through a 200-mesh steel mesh. The intestinal segments were added to the pre-digestion solution for subsequent processing. Centrifugation of the filtered culture medium yielded intestinal epithelial cells and intraepithelial lymphocytes. This process was repeated twice. The remaining tissue was added to a digestion solution containing collagenase and DNase, and filtration yielded intestinal lamina propria lymphocytes. Intraepithelial lymphocytes and lamina propria lymphocytes were labeled with specific fluorescent antibodies, and the secretion of INF-γ and IL-17A was detected by flow cytometry.

[0244] The experimental results are shown in Figure 7. Mice colonized with Bifidobacterium experienced slower weight loss (Figure 7A, weight loss was observed in mice with enteritis) and longer colons (Figure 7B, colon length was shortened in mice with colon inflammation). Intestinal lamina propria lymphocytes were obtained by digesting the pretreated colon with a digestive solution containing collagenase II. Simultaneously, mesenteric lymph nodes from mice were collected and ground through a 200-mesh filter to prepare single-cell suspensions. Flow cytometry analysis of IL-17A+ cytokine expression in intestinal lamina propria lymphocytes and mesenteric lymph nodes revealed a significant decrease in both the proportion and number of inflammatory Th17 cells in the intestine of mice colonized with Bifidobacterium (Figure 7C). The above results indicate that when Bifidobacteria are present in the intestines during enteritis in mice, they mainly inhibit the differentiation of Th17 cells, thereby suppressing the production of the inflammatory factor Thpatho secreted by pathogenic Th17 cells, reducing weight loss caused by intestinal inflammation, and preventing intestinal lymphocyte infiltration. HE staining of colon sections confirmed the same conclusion, showing that inflammatory infiltration in the intestinal tissue of TNBS mice colonized with Bifidobacteria was reduced (Figure 7D).

[0245] The results of Example 8 indicate that Bifidobacteria play an important role in Th17-mediated autoimmune diseases by degrading 5-hydroxyindole in vivo, thereby inhibiting the differentiation of Th17 cells under inflammatory conditions and suppressing the secretion of the inflammatory factor Thpatho by pathogenic Th17 cells.

[0246] Example 9: Screening and Validation of Mouse-Derived Thpatho Neutralizing Antibodies

[0247] After synthesizing and purifying murine Thpatho, it was injected into rabbits as an antigen. Affinity screening identified the 10 antibodies with the highest affinity. These antibodies were then isolated and purified for functional screening. Thpatho can promote IL-17A production in Th17 cells. These 10 antibodies were then added to Thpatho along with... In CD4 cells, the IL-17A content was detected by flow cytometry after 4 days of culture under Th17 differentiation conditions. After screening, clone 8 was found to significantly reduce the increase in IL-17A expression induced by Thpatho (Figure 8A). This result indicates that we obtained a Thpatho neutralizing antibody through screening.

[0248] Next, the Thpatho effect was tested in vivo. An experimental autoimmune encephalomyelitis (EAE) model was established by twice immunizing C57BL / 6J mice with the myelin oligodendrocyte glycoprotein (MOG35-55) peptide to determine whether the neutralizing antibody could attenuate the effects of Thpatho. 100 mg of inactivated BCG lyophilized powder was added to 10 mL of IFA to prepare a complete Freund's adjuvant (CFA); 4 mg of MOG35-55 peptide was dissolved in 1.33 mL of phosphate buffer to prepare a 3 g / L peptide solution; the MOG35-55 peptide solution (3 g / L) was mixed with CFA at a 1:1 ratio and emulsified repeatedly using a syringe to prepare a water-in-oil antigen emulsion. PTX solution: 50 μg of PTX was dissolved in 1 mL of PBS to prepare a 50 mg / L PTX stock solution; the concentration used was 1 mg / L. Eight-week-old female SPF-grade C57BL / 6J mice were randomly assigned to receive neutralizing antibodies and a human IgG isotype control group after one week of acclimatization. Both groups received subcutaneous injections of 100 μL of the emulsion at two points in the bilateral groin. PTX (1 mg / L) was administered intraperitoneally at 0 and 48 hours post-immunization (200 μL). Twenty-five days after the neuroantigen injection, mice were monitored daily, including EAE scores, cytokine measurements, central nervous system demyelination, and cell infiltration (Figures 8B-D). We found that compared to the isotype control group, mice injected with neutralizing antibodies exhibited lower EAE scores, less central nervous system demyelination and cell infiltration, and less production of inflammatory factors IFNγ and IL-17A.

[0249] The results of Example 9 indicate that the murine Thpatho neutralizing antibody has high affinity and can effectively alleviate the aggravating effect of Thpatho on autoimmune diseases.

[0250] The neutralizing antibody sequences are as follows, with bold text from front to back representing CDR1 to CDR3, the constant region being the human kappa chain and human IgG1.

[0251] HC:

[0252] LC:

[0253] Example 10: Screening and Validation of Human Thpatho Neutralizing Antibodies

[0254] After synthesizing and purifying human Thpatho, it was injected into rabbits as an antigen. Affinity screening identified the three antibodies with the highest affinity. These antibodies were then isolated and purified for functional screening. Human Thpatho can promote IL-17A production by Th17 cells. These antibodies were then added to human cells along with Thpatho. In CD4 cells, after 7 days of culture under Th17 differentiation conditions, the IL-17A content was detected by flow cytometry. Screening revealed that all three clones used in the experiment significantly reduced the increase in IL-17A expression induced by Thpatho (Figure 9). The results indicate that a human Thpatho neutralizing antibody was obtained through screening, which can neutralize the effects of human Thpatho.

[0255] The neutralizing antibody sequences are as follows, with bold text from front to back representing CDR1 to CDR3, the constant region being the human kappa chain and human IgG1.

[0256] HC:

[0257] LC:

[0258] Table 1 Antibody Sequences

[0259] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of a Thpatho factor inhibitor in the preparation of a composition or formulation for the prevention and / or treatment of autoimmune diseases in subjects; preferably, the Thpatho factor inhibitor is selected from the group consisting of: 5-hydroxyindole (5-HI) inhibitors, specific antibodies against Thpatho, siRNA of the Thpatho-encoding gene, small molecule drugs, bacterial agents, or combinations thereof.

2. The use as described in claim 1, characterized in that, The 5-HI inhibitor is selected from the following group: siRNA of 5-HI expression-related genes, small molecule drugs that degrade 5-HI, bacterial agents, or combinations thereof.

3. The use as described in claim 2, characterized in that, The 5-HI inhibitor is Bifidobacterium or its metabolites; preferably, the Bifidobacterium is selected from the group consisting of: Bifidobacterium breve, Bifidobacterium feces, Bifidobacterium adolescentis, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium pseudosporidis, Bifidobacterium catecholaria, Bifidobacterium lactis, Bifidobacterium longum, or combinations thereof.

4. The use as described in claim 3, characterized in that, The 16S rDNA sequence of the Bifidobacterium has ≥95% identity with the sequence shown in SEQ ID NO. 1, 2 or 3, preferably ≥98%, more preferably ≥99%, and even more preferably ≥99.9%.

5. The use as described in claim 3, characterized in that, The Bifidobacteria mentioned herein comprise one or more strains selected from the group consisting of: a) Bifidobacterium faecale Zhulab-010, which was deposited at the China Center for Type Culture Collection (CCTCC, Wuhan University, China) on January 25, 2024, with accession number CCTCC NO. M2024202； b) Bifidobacterium breve Zhulab-009, which was deposited on January 22, 2024 at the China Center for Type Culture Collection (CCTCC, Wuhan University, China), with accession number CCTCC NO. M2024150; and c) Bifidobacterium adolescentis Zhulab-011, deposited on January 25, 2024 at the China Center for Type Culture Collection (CCTCC, Wuhan University, China), with accession number CCTCC NO. M2024203。 6. The use as described in claim 1, characterized in that, The Thpatho factor inhibitor is a specific antibody against Thpatho, or its encoded nucleic acid, or its expression vector, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region, and the light chain comprising a light chain variable region, wherein the heavy chain variable region comprises the following complementarity-determining region (CDR): The amino acid sequence is CDR-H1 as shown in SEQ ID No:4; The amino acid sequence is CDR-H2 as shown in SEQ ID No:5; The amino acid sequence is CDR-H3 as shown in SEQ ID No:6; The light chain variable region includes the following complementary determinant regions (CDRs): The amino acid sequence is CDR-L1 as shown in SEQ ID No:7; The amino acid sequence is CDR-L2 as shown in the RTS. The amino acid sequence is CDR-L3 as shown in SEQ ID NO:8, or The heavy chain variable region includes the following complementary determinant region (CDR): The amino acid sequence is CDR-H1 as shown in SEQ ID No:9; The amino acid sequence is CDR-H2 as shown in SEQ ID No:10; The amino acid sequence is CDR-H3 as shown in SEQ ID No:11; The light chain variable region includes the following complementary determinant regions (CDRs): The amino acid sequence is CDR-L1 as shown in SEQ ID No:12; The amino acid sequence is CDR-L2 as shown in STS; The amino acid sequence is CDR-L3 as shown in SEQ ID NO:

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7. A type of Bifidobacterium, characterized in that, The 16S rDNA sequence of the Bifidobacterium has ≥95% identity with the sequence shown in SEQ ID NO. 1, 2 or 3, preferably ≥98%, more preferably ≥99%, and even more preferably ≥99.9%.

8. The Bifidobacterium as described in claim 7, characterized in that, The Bifidobacterium mentioned is a strain selected from the following group: a) Bifidobacterium faecale Zhulab-010, which was deposited at the China Center for Type Culture Collection (CCTCC, Wuhan University, China) on January 25, 2024, with accession number CCTCC NO. M2024202； b) Bifidobacterium breve Zhulab-009, which was deposited on January 22, 2024 at the China Center for Type Culture Collection (CCTCC, Wuhan University, China), with accession number CCTCC NO. M2024150; and c) Bifidobacterium adolescentis Zhulab-011, deposited on January 25, 2024 at the China Center for Type Culture Collection (CCTCC, Wuhan University, China), with accession number CCTCC NO. M2024203。 9. A specific antibody against Thpatho, said antibody comprising a heavy chain and a light chain, said heavy chain comprising a heavy chain variable region, said light chain comprising a light chain variable region, wherein, The heavy chain variable region includes the following complementary determinant regions (CDRs): The amino acid sequence is CDR-H1 as shown in SEQ ID No:4; The amino acid sequence is CDR-H2 as shown in SEQ ID No:5; The amino acid sequence is CDR-H3 as shown in SEQ ID No:6; The light chain variable region includes the following complementary determinant regions (CDRs): The amino acid sequence is CDR-L1 as shown in SEQ ID No:7; The amino acid sequence is CDR-L2 as shown in the RTS. The amino acid sequence is CDR-L3 as shown in SEQ ID NO:8, or The heavy chain variable region includes the following complementary determinant region (CDR): The amino acid sequence is CDR-H1 as shown in SEQ ID No:9; The amino acid sequence is CDR-H2 as shown in SEQ ID No:10; The amino acid sequence is CDR-H3 as shown in SEQ ID No:11; The light chain variable region includes the following complementary determinant regions (CDRs): The amino acid sequence is CDR-L1 as shown in SEQ ID No:12; The amino acid sequence is CDR-L2 as shown in STS; The amino acid sequence is CDR-L3 as shown in SEQ ID NO:

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10. A composition for the prevention and / or treatment of autoimmune diseases, characterized in that, The composition comprises: (a) A safe and effective dose of a Thpatho factor inhibitor; and (b) Pharmaceutically acceptable carriers.

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