Bispecific fusion protein based on Th17 immune axis regulation and application thereof in combined treatment of inflammatory bowel disease and psoriasis

Abstract

The invention discloses a bispecific fusion protein based on Th17 immune axis regulation and application of the bispecific fusion protein in combined treatment of inflammatory bowel disease and psoriasis. The fusion protein is formed by sequentially connecting a human IL-23 receptor (IL-23R) extracellular functional structural domain, a flexible connecting peptide and a human trefoil factor 3 (TFF3). According to the invention, IL-23R is specifically combined with IL-23 highly expressed at a focus part to block a cascade reaction of a Th17 inflammation axis, and TFF3 with a mucosa repair function is delivered to damaged tissues in a targeted manner. Experiments show that in a DSS-induced ulcerative colitis model and an IMQ-induced psoriasis model, the fusion protein is obviously superior to a single-target drug in the aspects of reducing disease activity index (DAI), improving skin lesion score (PASI) and promoting histopathological repair, has a synergistic effect of anti-inflammation and repair, and provides a new strategy for treatment of autoimmune diseases.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and genetic engineering, and more specifically, to a bispecific fusion protein (IL-23R-TFF3) based on Th17 immune axis regulation, its preparation method, and its application in the preparation of drugs for treating inflammatory bowel disease and psoriasis. Background Technology

[0002] Inflammatory bowel disease (IBD, including ulcerative colitis and Crohn's disease) and psoriasis are two typical chronic, relapsing autoimmune diseases that severely impact patients' quality of life. Recent studies have confirmed that the IL-23 / Th17 immune axis plays a central driving role in the pathogenesis of these diseases. IL-23, by binding to its receptor IL-23R, stimulates Th17 cell differentiation and the secretion of inflammatory factors such as IL-17 and IL-22, leading to disruption of the intestinal mucosal barrier or excessive proliferation of keratinocytes in the skin, triggering persistent tissue damage and inflammatory responses.

[0003] Currently, treatment strategies targeting this pathway primarily rely on monoclonal antibodies (such as ustekinumab and gusejinumab) that target IL-23 or IL-17. While these biologics can effectively suppress the inflammatory response, clinical practice has revealed that some patients exhibit primary non-response or secondary resistance. More importantly, simply blocking inflammatory factors cannot actively promote the regeneration and repair of damaged tissues (such as ulcerated intestinal mucosa or damaged skin barriers), resulting in suboptimal mucosal healing, a key indicator for predicting the long-term prognosis of IBD patients.

[0004] Trefoil factor 3 (TFF3) is a small peptide mainly secreted by goblet cells in the intestine. It possesses excellent properties in promoting epithelial cell migration and mucosal repair, and also shows potential in skin wound healing. However, the short half-life of natural TFF3 and its lack of targeting of inflammatory lesions limit its application as a systemic drug. Therefore, developing a dual-function drug that can precisely block the IL-23 / Th17 inflammatory axis and target the delivery of repair factors to the lesion site is a pressing technical challenge in the treatment of IBD and psoriasis. Summary of the Invention

[0005] To address the issues of existing technologies where single anti-inflammatory treatments cannot simultaneously address tissue repair and where repair factors lack targeting specificity, this invention provides a bispecific fusion protein based on Th17 immune axis regulation, along with its preparation method and applications.

[0006] The technical solution adopted in this invention is as follows: This invention provides a bispecific fusion protein, which is formed by fusing the extracellular ligand-binding domain of IL-23R with the full-length sequence of TFF3 through a flexible linker using genetic engineering technology. Specifically, the IL-23R terminus acts as a "decoy receptor," competitively binding to the high concentration of IL-23 at the lesion site, blocking its downstream transmission of pro-inflammatory signals; the TFF3 terminus, guided by IL-23R, accumulates on the surface of damaged tissue, promoting epithelial migration and barrier remodeling.

[0007] In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 3, which includes an optimized (GGGGS)4 linker and a C-terminal 6xHis purification tag.

[0008] The present invention also provides a method for preparing the above-mentioned fusion protein, comprising constructing a recombinant prokaryotic expression vector containing a synthetic gene, transforming it into an Escherichia coli host, inducing expression at low temperature, separating and purifying it using Ni-NTA affinity chromatography, and obtaining a biologically active fusion protein by gradient dialysis refolding.

[0009] Compared with the prior art, the present invention has at least the following beneficial effects: Dual Mechanism, Synergistic Effect: This invention is the first to fuse an IL-23 antagonist with a TFF3 repair factor, achieving simultaneous "anti-inflammatory" and "repair" effects. Animal experiments have confirmed that in IBD and psoriasis models, the efficacy (DAI score, PASI score, and pathological repair) of the fusion protein is significantly better than using anti-IL-23 antibodies or TFF3 proteins alone, demonstrating a synergistic effect of "1+1>2".

[0010] Targeted delivery and precise treatment: By leveraging the high expression of IL-23 in inflamed tissues, the fusion protein can actively target the lesion site, improving the local bioavailability of TFF3 in damaged tissues, while potentially reducing the side effects caused by nonspecific distribution throughout the body.

[0011] The structure is stable and easy to prepare: the fusion protein designed with computer simulation is structurally stable, and the linkers do not interfere with the independent folding of the functional domains at both ends. It employs prokaryotic system expression and one-step purification, a mature process with controllable costs, and possesses good drug-like properties and industrialization prospects. Attached Figure Description

[0012] Figure 1 This is a three-dimensional homology model of the bispecific fusion protein (IL-23R-TFF3) of this invention. The results show that the IL-23R domain and the TFF3 domain are effectively connected by flexible linkers, each maintaining an independent and stable native folding conformation.

[0013] Figure 2 This is the elution peak pattern (AKTA pattern) of the fusion protein of this invention purified by Ni-NTA affinity chromatography. The figure shows that a single symmetrical main peak was obtained under specific imidazole concentration elution, indicating high protein purity and thorough separation of impurities.

[0014] Figure 3 This is an SDS-PAGE gel electrophoresis image of the purified fusion protein of this invention. Lane 1 is the protein marker, and lane 2 is the purified fusion protein. The results show a clear band at approximately 50 kDa, consistent with the theoretical molecular weight of 47.77 kDa, indicating a purity greater than 90%.

[0015] Figure 4 This is a dynamic change graph of the Disease Activity Index (DAI) in each group of mice in the DSS-induced ulcerative colitis model in Example 2. The results show that the fusion protein group (Group F) is significantly better than the model group and each positive control group in inhibiting disease progression and reducing DAI scores.

[0016] Figure 5 This is a dynamic change graph of PASI scores in mice of each group in the IMQ-induced psoriasis-like model in Example 3. The results showed that the fusion protein group (Group D) had a rapid onset of action and sustained improvement in skin lesion symptoms, with the final score being significantly lower than that of the anti-IL-23 monoclonal antibody treatment group. Detailed Implementation

[0017] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0018] Example 1: Preparation of a bispecific fusion protein (IL-23R-TFF3) Based on the full-length sequence of IL-23R (interleukin-23 receptor precursor [Homo sapiens], NCBI Reference Sequence: NP_653302.2) recorded in NCBI, its extracellular functional domain was determined after removing the signal peptide portion: GITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGG KKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSLFFHKTPETVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRG (SEQ ID NO:1); According to NCBI records, the full-length sequence of TFF3 (trefoil factor 3 [Homo sapiens]) is as follows: MLGLVLALLSSSSAEEYVGLSANQCAVPAKDRVDCGYPHVTPKECNRGCCFDSRIPGVPWCFKPLQEAECTF (SEQ ID NO: 2); Sequence design of a HIS-tagged bispecific fusion protein (IL-23R-TFF3): GITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEV TCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKTI IYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSLFFHKTPETVPQVTSKAFQHDTWNSGLTVASISTGHLT SDNRGGGGGSGGGGSGGGGSGGGGSMLGLVLALLSSSSAEEYVGLSANQCAVPAKDRVDCGYPHVTPKECNNRGCCFDSRIPGVPWCFKPLQEAECTFHHHHHH (SEQ ID NO:3); The three-dimensional structural homology model of the bispecific fusion protein IL-23R-TFF3 (SEQ ID NO:3) was performed using the Swiss-Model. The model was constructed based on the extracellular domain of IL-23R (template PDB: 6N5J) and TFF3 (template PDB: 1TFF), respectively, with the (GGGGS)4 flexible linker connecting them. Results are shown in [Figure number missing]. Figure 1 .

[0019] The predicted results show that the IL-23R domain maintains the typical type I fold of a cytokine receptor, and the key ligand-binding residues are conformably intact; the TFF3 domain forms a stable cloverleaf structure containing three conserved disulfide bonds, with a well-exposed active loop region; the linker exhibits a random coil conformation, without interfering with the independent folding of the functional domains at both ends. The overall structure is stable and the spatial arrangement is reasonable, consistent with the expected design, indicating that this fusion protein is expected to simultaneously retain IL-23 binding capacity and epithelial repair activity.

[0020] Construction of expression vector and strain screening: Based on the amino acid sequence of the bispecific fusion protein shown in SEQ ID NO:3, codon optimization targeting E. coli preferences was commissioned to Nanjing GenScript Biotech Co., Ltd. to improve its expression efficiency in the prokaryotic system. The optimized coding sequence has a 6-hitidine (6xHis-Tag) sequence at the 3' end to facilitate subsequent affinity chromatography purification.

[0021] The optimized full-length gene fragment was synthesized artificially by Nanjing Genscript Biotech Co., Ltd., with Nco I and Xho I restriction sites introduced at both ends, respectively. The synthesized gene fragment and the expression vector pET-28a(+) (MerckMillipore) were double-digested, and the products were recovered and ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into E. coli DH5α competent cells, and positive clones were selected using kanamycin (50 μg / mL) resistant plates. The plasmid was extracted and sequenced, and the results showed that the inserted fragment sequence was completely consistent with the design. The successfully constructed recombinant expression plasmid was named pET28a-IL23R-Linker-TFF3.

[0022] Induction of fusion protein expression: The correctly sequenced recombinant plasmid pET28a-IL23R-Linker-TFF3 was transformed into the expression host bacterium E. coli BL21(DE3). A single colony was picked and inoculated into 5 mL of LB broth containing 50 μg / mL kanamycin, and cultured overnight at 37°C with shaking at 220 rpm. The next day, the seed culture was transferred to 1 L of fresh LB broth (containing 50 μg / mL kanamycin) at a 1:100 ratio, and cultured at 37°C with shaking until the optical density (OD600) reached 0.6-0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to the medium to a final concentration of 0.5 mM to initiate induction. Considering the large molecular weight of the fusion protein and the presence of disulfide bonds, the induction temperature was adjusted to 20°C, and the culture was performed at low rotation speed (180 rpm) for 16 hours to promote soluble protein expression and reduce inclusion body formation. After induction, the bacterial pellet was collected by centrifugation at 4°C and 6000 rpm for 15 minutes, washed once with PBS, and stored at -80°C for later use.

[0023] Protein extraction and Ni column purification: The collected bacterial cells were resuspended at a ratio of 1:10 (w / v) in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0, containing 1 mM PMSF). The cells were lysed using an ultrasonic cell disruptor under ice bath conditions (3 seconds on, 5 seconds off, 400 W for 30 minutes) until the bacterial suspension was clear and no longer viscous. The lysed suspension was centrifuged at 12000 rpm for 30 minutes at 4°C, and the supernatant was collected. Affinity chromatography purification using Ni-NTA agarose resin (Qiagen): First, the column was equilibrated with 5 column volumes of lysis buffer. The supernatant was then loaded at a flow rate of 1 mL / min. After loading, contaminating proteins were washed sequentially with wash buffer containing 20 mM and 50 mM imidazole until the absorbance of the effluent at 280 nm returned to baseline. Figure 2 .

[0024] Finally, the target protein was eluted with an elution buffer (50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH 8.0). The elution peak fractions were collected separately. The collected elution fractions were analyzed by SDS-PAGE gel electrophoresis. The eluates with a purity greater than 90% were combined and loaded into a dialysis bag with a molecular weight cut-off of 10 kDa. The buffer was exchanged and imidazole was removed against PBS buffer (pH 7.4) at 4°C. See Figure 3 .

[0025] Figure 3 The results showed that a clear specific band was visible at a molecular weight of approximately 50 kDa, which was roughly equivalent to the expected theoretical molecular weight of 47.77 kDa.

[0026] Example 2 Evaluation of the Therapeutic Effect of Bispecific Fusion Protein (IL-23R-TFF3) in a DSS-Induced Mouse Model of Ulcerative Colitis 1. Experimental Materials and Animals: Male C57BL / 6 mice, 6 - 8 weeks old, weighing 20 ± 2 g, were purchased from Vital River Laboratory Animal Technology Co., Ltd. (SCXK (Beijing) 2023 - 0006); dextran sulfate sodium (DSS, MW: 36000 - 50000 Da) was purchased from MP Biomedicals; Positive control drug 1: anti-mouse IL-23p19 monoclonal antibody G23 - 8 (AbsoluteAntibody, catalog number: Ab00413 - 2.0); Positive control drug 2: recombinant human TFF3 protein (Cloud-Clone / USCnK, catalog number: RPB656Hu02); Test drug: the IL-23R-TFF3 fusion protein prepared in Example 1.

[0027] 2. Model Construction and Drug Administration Grouping: Sixty mice were randomly divided into 6 groups, with 10 mice in each group: Group A: normal control group (drinking distilled water); Group B: model control group (PBS); Group C: positive control group 1 (anti-IL-23p19 monoclonal antibody, 5 mg / kg); Group D: positive control group 2 (rTFF3, 5 mg / kg); Group E: combined drug administration group (anti-IL-23p19 monoclonal antibody 2.5 mg / kg + rTFF3 2.5 mg / kg, non-fused state); Group F: high-dose fusion protein group (IL-23R-TFF3, equimolar dose conversion, 5 mg / kg).

[0028] Except for group A, mice in the other groups were allowed free access to 3% DSS aqueous solution for 7 consecutive days to induce an acute ulcerative colitis model, and then switched to drinking distilled water for 3 days to recover. On days 1, 3, 5, 7 and 9 after modeling, the corresponding drugs were administered via intraperitoneal injection, while group B was given an equal volume of PBS.

[0029] Disease Activity Index (DAI) score: During the experiment, the mice were monitored at a fixed time each day (9:00-10:00 AM). The DAI score was calculated using a comprehensive scoring method, which was the sum of the scores of three indicators: percentage of body weight loss, stool characteristics, and blood in stool, divided by 3 (i.e., DAI = (body weight loss score + stool characteristics score + blood in stool score) / 3). The specific scoring criteria are shown in Table 1.

[0030] Table 1 Disease Activity Index (DAI) Scoring Criteria Note: If the stool characteristics or rectal bleeding are between two levels, the score can be increased by 0.5 points.

[0031] Figure 4 The results showed that throughout the experimental period, mice in group A (normal control) were in good condition, with their DAI scores remaining close to baseline. In contrast, in group B (model control), the DAI score progressively increased with the duration of DSS induction, peaking on day 7, manifesting as severe weight loss, diarrhea, and gross bloody stools. Recovery was slow after DSS withdrawal (day 10), indicating a successful establishment of the ulcerative colitis model with significant pathological features. Groups C (anti-IL-23 monoclonal antibody) and D (rTFF3 protein) showed significantly lower DAI scores than group B on days 7 and 10 (P<0.05), confirming that blocking the IL-23 inflammatory pathway or supplementing TFF3 can alleviate colitis symptoms to some extent. However, while group C showed better improvement in bleeding and inflammation, it was slightly less effective in restoring weight (reflecting absorption function). Although group D promoted repair, its effect on controlling diarrhea and bloody stools during the acute inflammatory peak (Days 5-7) was weaker than the antibody group, indicating the limitations of single-mechanism treatment.

[0032] Colon length measurement: Mice were sacrificed on day 10 of the experiment, and the entire colon was dissected and the length from the cecum to the anus was measured. Colon shortening is a direct indicator of the severity of colitis.

[0033] The results showed that the average colon length in group A was 8.2±0.4 cm; in group B it was significantly shortened to 4.5±0.3 cm. Group F showed the best recovery in colon length, reaching 7.1±0.5 cm, which was better than group C (6.2±0.4 cm) and group D (5.8±0.3 cm).

[0034] Histopathological examination and scoring (0-4 point system): Distal colon tissue (0.5-2 cm from the anus) was taken, fixed in 4% paraformaldehyde for 24 hours, routinely embedded in paraffin, sectioned (4 μm), and stained with H&E. Under double-blind conditions, a comprehensive pathological score was performed focusing on "crypt structure integrity" (reflecting the repair effect of TFF3) and "depth of inflammatory cell infiltration" (reflecting the anti-inflammatory effect of anti-IL-23). ​​The scoring criteria are as follows:

[0035] 0 points (normal): The colonic mucosal epithelium is intact, the crypts are neatly arranged, and there is no inflammatory cell infiltration in the lamina propria; 1 point (mild): The basal 1 / 3 of the crypts are damaged or disappeared, and a small number of scattered inflammatory cells are visible in the lamina propria; 2 points (moderate): The basal 2 / 3 of the crypts are missing, goblet cells are reduced, inflammatory cell infiltration extends to the submucosa, accompanied by mild edema; 3 points (severe): Extensive destruction of crypt structure, with only surface epithelium remaining, extensive infiltration of inflammatory cells, and significant thickening of the mucosa; 4 points (extremely severe): The crypts and epithelial structures are completely lost, and extensive mucosal erosion or ulceration occurs, with transmural inflammatory infiltration (involving the muscle layer or serosal layer).

[0036] Table 2. Pathological damage scores of colon tissue in mice of each group. Note: Compared with group B, *P<0.05; compared with group C or group D, #P<0.05; compared with group E, &P<0.05.

[0037] Group A (normal group) showed clear and intact colonic mucosal structure with abundant goblet cells, and a score of 0. Group B (model group) showed typical pathological changes of severe colitis, with a score of 3.65 ± 0.35. Microscopic examination revealed extensive destruction of crypt structures, epithelial shedding and ulceration, and inflammatory cells penetrating the submucosa and reaching the muscular layer (transmural inflammation), confirming the successful establishment of the DSS-induced intestinal injury model.

[0038] Group C (anti-IL-23 monoclonal antibody) had a score of 2.45, mainly showing a reduction in the depth of inflammatory cell infiltration (from transmural to submucosal), but the repair of crypt structures was relatively delayed, indicating that anti-inflammatory treatment alone was insufficient to rapidly reverse tissue damage. Group D (rTFF3) had a score of 2.60, with the advantage of more residual crypt structures and better preservation of goblet cells than Group B, but due to the lack of a potent anti-inflammatory mechanism, tissue edema and inflammatory infiltration remained significant. Group E (combined drug administration) had a score of 1.95, showing additive efficacy. Group F (fusion protein group) had the lowest pathological score, only 1.15 ± 0.25, significantly better than all control groups (P<0.05).

[0039] Detection of inflammatory cytokine levels in colon tissue (ELISA method): Mice were sacrificed on day 10 of the experiment, and a portion of diseased colon tissue was collected, accurately weighed, and mixed with pre-cooled PBS (containing protease inhibitors) to prepare a 10% tissue homogenate using a tissue homogenizer. The homogenate was centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The total protein concentration was determined using the BCA method for standardization. The levels (pg / mg protein) of inflammatory cytokines IL-6, TNF-α, IL-17A, and IL-22 in the colon tissue homogenate were measured using an enzyme-linked immunosorbent assay (ELISA) kit (purchased from R&D Systems) strictly following the instructions.

[0040] Table 3. Results of Detection of Inflammatory Factor Levels in Colon Tissue of Mice in Each Group Note: Compared with Group B, * P<0.05; compared with group C or group D, # P<0.05; compared with group E, & P<0.05.

[0041] Table 3 shows that the levels of IL-6, TNF-α, IL-17A, and IL-22 in the colon tissue of mice in group B (model group) were significantly higher than those in group A (normal group) (P<0.001), indicating that DSS successfully induced a severe intestinal inflammatory response characterized by Th17 immune axis activation. Group C (anti-IL-23 monoclonal antibody) significantly reduced the expression levels of downstream effector factors IL-17A and IL-22 by specifically blocking IL-23, thereby inhibiting the cascade release of IL-6 and TNF-α. Although group D (rTFF3) mainly played a repair role, it also reduced the level of inflammatory factors to some extent by improving the intestinal mucosal barrier function and reducing the invasion of intestinal antigens, but its anti-inflammatory effect was significantly weaker than that of group C. Group F (fusion protein group) had the strongest inhibitory effect on the above four inflammatory factors, and its index levels were significantly lower than those in group E (combination drug group) (P<0.05). In particular, regarding the markers of the Th17 pathway, IL-17A and IL-22, the levels in group F (58.74 and 46.33 pg / mg, respectively) were close to those in the normal group and significantly lower than those in group C, which used antibodies alone. This result strongly supports the concept of this invention—that the TFF3 terminus of the fusion protein not only plays a repair role but also acts as an "anchor molecule" to target and enrich the IL-23R decoy receptor on the damaged mucosal surface (a region rich in TFF3 binding sites). This locally high concentration of "decoy receptors" can more efficiently neutralize IL-23 at the lesion site, thus demonstrating superior efficacy in blocking the inflammatory cascade compared to systemic free antibodies (group C) or simple drug mixtures (group E).

[0042] The experimental data above strongly demonstrate that the bispecific fusion protein IL-23R-TFF3 is not a simple functional additive, but rather achieves a synergistic effect of "1+1>2" through molecular structural fusion. Its mechanism lies in using the IL-23R terminus as a "navigation point" to precisely deliver the repair-functional TFF3 to the most severely inflamed lesion site. This effectively inhibits the Th17 inflammatory axis while significantly accelerating the reconstruction and healing of damaged intestinal mucosal epithelium, demonstrating significantly superior performance in reducing disease activity compared to existing single-target drugs and physical therapy regimens.

[0043] Example 3: Therapeutic effect of bispecific fusion protein (IL-23R-TFF3) in IMQ-induced mouse psoriasis-like skin lesion model Experimental animals and materials: Eight-week-old male BALB / c mice, weighing 22±2g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed in an SPF-grade environment with free access to food and water. Modeling reagent: 5% imiquimod cream (Imiquimod, IMQ, trade name: Aldara, 3M Pharmaceuticals).

[0044] Positive control: Anti-mouse IL-23p19 monoclonal antibody (same as in Example 2).

[0045] Test drug: IL-23R-TFF3, a bispecific fusion protein prepared in Example 1.

[0046] Model construction and experimental grouping: 32 mice were randomly divided into 4 groups of 8 mice each. The day before the experiment, the villi on the back of all mice in an area of ​​about 2cm × 3cm were removed.

[0047] Group A (normal control group): The back was coated with an equal amount of petroleum jelly, and PBS was injected into the peritoneum daily.

[0048] Group B (model control group): 62.5 mg of 5% IMQ cream was applied to the back daily for 7 consecutive days, and PBS was injected intraperitoneally daily.

[0049] Group C (positive control - anti-IL-23 group): IMQ was applied to the back, and anti-IL-23p19 monoclonal antibody (5 mg / kg) was injected intraperitoneally daily.

[0050] Group D (fusion protein group): IMQ was applied to the back, and IL-23R-TFF3 fusion protein (equimolar dose, 5 mg / kg) was injected intraperitoneally daily.

[0051] Psoriasis Skin Lesion Area and Severity Index (PASI) scoring: During the experiment, changes in the skin on the backs of mice were observed daily. A modified version of the clinical PASI scoring criteria was used, with independent scoring for three indicators: erythema, scaling, and thickening. Figure 5 The PASI score of psoriasis-like skin lesions in each group of mice changed over time.

[0052] Scoring criteria (0-4 points): 0 points: asymptomatic; 1 point: mild; 2 points: moderate; 3 points: severe; 4 points: very severe.

[0053] Cumulative score: Total score = Erythema score + Scaling score + Thickening score (maximum 12 points).

[0054] Figure 5 The results showed that the score of group B (model group) increased rapidly with the induction time of IMQ, reaching the level of moderate to severe skin lesions (score > 6) on day 4 and remaining high on day 7 (9.80 ± 0.80), which is consistent with the typical characteristics of an acute psoriasis-like model. Group C (anti-IL-23) showed no significant difference from the model group on day 2, and only showed a statistically significant inhibitory effect on day 4. This is consistent with the kinetic characteristics of antibody drugs, which mainly inhibit the downstream inflammatory cascade by blocking upstream cytokines, and have a relatively delayed onset of action. Group D (fusion protein) showed an inhibitory trend on day 2 of early administration (1.20 ± 0.25), which was significantly lower than that of the model group (2.15). This indicates that the TFF3 terminus of the fusion protein may have already begun to exert a direct protective and homeostatic effect on stimulated keratinocytes before the inflammation has fully erupted, thus delaying the initial formation of skin lesions. By the end of the experiment (Day 7), the PASI score of group D (3.20) was not only significantly better than that of the model group, but also reduced by about 29% compared with group C (4.50).

[0055] Epidermal thickness measurement: Mice were sacrificed on day 8, and skin lesions on the back were excised, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned (4 μm), and stained with H&E. Under a microscope (100×), five fields of view were randomly selected from each section to measure the vertical distance (μm) from the stratum corneum to the basal layer, reflecting the degree of acanthosis.

[0056] Group A (normal): The epidermis is thin, the structure is clear, and the thickness is about 15-20 μm.

[0057] Group B (model): significant epidermal hyperplasia (acanthosis), parakeratosis, and extensive inflammatory cell infiltration in the dermis, with the average thickness surging to 95.6±8.2 μm.

[0058] Group C (anti-IL-23): Epidermal thickening was somewhat inhibited, with a thickness of 52.3±5.5 μm, and inflammatory infiltration was reduced.

[0059] Group D (fusion protein): The epidermal thickness further decreased to 35.4±4.1 μm, the basal cells were neatly arranged, and the stratum corneum structure was better restored, approaching the normal skin morphology.

[0060] Table 4. Comparison of epidermal thickness data of mice in each group (day 8) Note: Compared with group B, *P<0.05; compared with group C, # P<0.05.

[0061] Table 4 shows that in the IMQ-induced psoriasis model, the IL-23R-TFF3 fusion protein (Group D) was significantly more effective than the model group, and superior to the current mechanism-positive anti-IL-23 monoclonal antibody (Group C) in improving skin lesion scores and inhibiting epidermal thickening. These results confirm that the fusion protein can more effectively control Th17-mediated skin pathological changes through a dual mechanism of "blocking inflammation" and "promoting repair."

[0062] Detection of key inflammatory factors in skin lesion tissue: Mice were sacrificed on day 8 of the experiment, and skin lesion tissue from the test area on the back was excised and flash-frozen in liquid nitrogen. Tissue samples were accurately weighed, and RIPA lysis buffer containing PMSF protease inhibitor was added. The samples were mechanically homogenized on ice. Centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. Total protein concentration was determined using a BCA kit for standardization. The levels (pg / mg protein) of inflammatory factors IL-6, TNF-α, IL-17A, and IL-22 in the skin lesion tissue homogenate were measured using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) strictly following the manufacturer's instructions.

[0063] Table 5. Results of Detection of Inflammatory Factor Levels in Skin Lesions of Mice in Each Group Note: Compared with Group B, * P<0.05; compared with group C, # P<0.05.

[0064] Table 5 shows that the IMQ-induced psoriasis model exhibited a typical high-inflammatory state. In group B, the levels of IL-17A and IL-22 were more than 10-fold higher than in the normal group A (P<0.001), confirming that the model was IL-23 / Th17 pathway dependent. Group C (anti-IL-23 monoclonal antibody) significantly reduced the levels of downstream effector factors IL-17A and IL-22 (approximately 46%) by neutralizing upstream IL-23, demonstrating the effectiveness of the positive control drug. Group D (IL-23R-TFF3 fusion protein) showed the best inhibitory effect on all measured indicators, with significantly lower cytokine levels than group C (P<0.05). The levels of IL-17A and IL-22 in group D decreased to 118.45 pg / mg and 95.68 pg / mg, respectively, with an inhibition rate of approximately 74%, significantly better than group C. This indicates that the fusion protein more thoroughly blocks the pathogenic pathway. The significant reduction in IL-6 and TNF-α (nearly half the normal level) suggests a positive feedback effect from tissue repair. Since TFF3 promotes keratinocyte repair and epidermal barrier reconstruction, it reduces the release of "danger signals" (DAMPs) from damaged keratinocytes, thereby further reducing the local broad-spectrum inflammatory response; this indicates that the IL-23R-TFF3 fusion protein does not rely solely on "anti-inflammatory" effects in the psoriasis model. By precisely "trapping" high concentrations of IL-23 in lesions at the IL-23R terminus, combined with the rapid repair of the epidermal barrier at the TFF3 terminus, the fusion protein achieves a dual blockade of the psoriasis inflammatory loop, and its efficacy is significantly superior to single-target antibody drugs at the molecular level.

[0065] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A bispecific fusion protein based on Th17 immune axis regulation, characterized in that, The fusion protein comprises, from the N-terminus to the C-terminus, the extracellular functional domain of human interleukin-23 receptor (IL-23R), a linker peptide, and the amino acid sequence of human trefoil factor 3 (TFF3); preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO:

3.

2. An isolated nucleic acid molecule encoding the bispecific fusion protein of claim 1.

3. A recombinant expression vector containing the nucleic acid molecule of claim 2, characterized in that, The preferred vector is the prokaryotic expression vector pET-28a(+).

4. A host cell comprising the recombinant expression vector of claim 3, characterized in that, The preferred host cell is Escherichia coli BL21(DE3).

5. The use of the bispecific fusion protein of claim 1 in the preparation of a medicament for treating IL-23 / Th17 pathway-mediated autoimmune diseases, characterized in that, The autoimmune diseases mentioned include inflammatory bowel disease (IBD) and psoriasis.

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