Antibody for treating ulcerative colitis and application thereof

Abstract

The invention belongs to the technical field of antibodies, particularly discloses an antibody for treating ulcerative colitis and application of the antibody, and particularly relates to a fully human single-chain antibody (ScFv) specifically combined with human IL-23 and application of the fully human single-chain antibody in treatment of ulcerative colitis. According to the antibody, a fully human bacteriophage ScFv antibody library is constructed, high-affinity IL-23 specific ScFv clone is obtained through three rounds of panning, and the antibody is named as P23A. ELISA detection shows that EC50 of the P23A to IL-23 is 10 nM, and the P23A has high affinity. In a TNBS-induced humanized mouse IBD model, the P23A can significantly improve weight loss of a mouse, reduce the score of a disease activity index (DAI) and recover the length of damaged intestinal tissues, and the treatment effect is equivalent to that of a positive control drug Guselkumab. The antibody provided by the invention has the advantages of high specificity, low immunogenicity and the like, and can be used for preparing medicines for treating ulcerative colitis and other IL-23 related diseases.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to an antibody for treating ulcerative colitis and its application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease that primarily affects the colonic mucosa and submucosa, clinically manifesting as diarrhea, abdominal pain, and bloody, mucus-containing stools. In recent years, the incidence of UC has been rising globally, becoming one of the major diseases seriously impacting human health. Current treatments for UC mainly include 5-aminosalicylic acid derivatives, glucocorticoids, immunosuppressants, and biologics; however, a significant proportion of patients respond poorly to existing treatments or develop drug resistance, highlighting the urgent need to develop more effective therapeutic drugs.

[0003] Studies have shown that interleukin-23 (IL-23) plays a crucial role in the pathogenesis of ulcerative colitis (UC). IL-23 is a heterodimeric cytokine composed of p19 and p40 subunits, primarily produced by activated dendritic cells and macrophages. IL-23 activates Th17 cells and innate lymphocytes, promoting the secretion of pro-inflammatory factors such as IL-17 and IL-22, thereby leading to intestinal mucosal barrier disruption and inflammatory responses. Clinical studies have found that IL-23 expression is significantly elevated in the intestinal tissue of UC patients and is positively correlated with disease activity. Biologics targeting IL-23, such as Ustekinumab (targeting the IL-12 / 23 p40 subunit) and Guselkumab (targeting the IL-23 p19 subunit), have shown good therapeutic effects in clinical trials, confirming IL-23 as an ideal target for UC treatment.

[0004] Phage display technology is an important method for screening high-affinity antibodies. By constructing a large-capacity antibody library and conducting multiple rounds of screening, antibodies with high specificity and affinity can be obtained. Fully human antibodies have advantages such as low immunogenicity and high safety, making them an ideal choice for therapeutic antibodies. Single-chain antibodies (ScFv) are small molecule antibodies composed of the variable region (VH) of the antibody heavy chain and the variable region (VL) of the light chain linked by flexible linker peptides. They have advantages such as small molecular weight, strong penetration, and ease of genetic engineering modification.

[0005] This invention constructs a fully human phage ScFv antibody library, screens and obtains the high-affinity IL-23-specific ScFv antibody P23A from an antibody library derived from ulcerative colitis patients, and verifies its therapeutic effect in a TNBS-induced humanized mouse IBD model, providing a new option for the treatment of UC. Summary of the Invention

[0006] This invention first provides a fully human single-chain antibody (ScFv) that specifically binds to human IL-23, the heavy chain variable region sequence of which is shown in SEQ ID NO:39 or SEQ ID NO:41, and the light chain variable region sequence of which is shown in SEQ ID NO:40 or SEQ ID NO:42. In some embodiments, the heavy chain variable region sequence is shown in SEQ ID NO:39, and the light chain variable region sequence is shown in SEQ ID NO:40.

[0007] In some embodiments, its EC50 for IL-23 is 10 nM.

[0008] The present invention also provides a nucleic acid molecule encoding the above-mentioned fully human single-chain antibody.

[0009] The present invention also provides an expression vector comprising the above-described nucleic acid molecules.

[0010] The present invention also provides a host cell comprising the above-described expression vector.

[0011] The present invention also provides a pharmaceutical composition comprising the above-described fully human single-chain antibody and a pharmaceutically acceptable carrier.

[0012] The present invention also provides the application of the above-mentioned fully human single-chain antibody in the preparation of drugs for treating ulcerative colitis.

[0013] The present invention also provides the application of the above-mentioned fully human single-chain antibody in the preparation of drugs for treating IL-23-mediated inflammatory diseases.

[0014] Finally, the present invention provides a method for treating ulcerative colitis, comprising administering a therapeutically effective amount of the above-mentioned fully human single-chain antibody to a patient in need.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The P23A antibody provided by the present invention is a fully human single-chain antibody with advantages such as low immunogenicity and high safety, which can reduce the generation of anti-drug antibodies during treatment and improve the safety of long-term use.

[0016] (2) P23A antibody has a high affinity for IL-23 (EC50=10nM), which can effectively block the IL-23 signaling pathway and inhibit the production of downstream pro-inflammatory factors, thereby reducing intestinal inflammatory response.

[0017] (3) In the TNBS-induced humanized mouse IBD model, P23A can significantly improve disease symptoms, including reversing weight loss, reducing DAI scores, and restoring the length of damaged intestinal tissue. The therapeutic effect is comparable to that of the marketed drug Guselkumab.

[0018] (4) Single-chain antibodies have a small molecular weight (about 25kDa), which has better tissue penetration and can reach the site of inflammation more effectively to exert their effects.

[0019] (5) The antibody production process provided by the present invention is simple, can be efficiently expressed in CHO cells, and can be purified to obtain a high-purity product, which is suitable for large-scale production.

[0020] (6) This antibody can not only be used to treat ulcerative colitis, but also to other IL-23-mediated inflammatory diseases, such as Crohn's disease, psoriasis, and psoriatic arthritis, and has broad clinical application prospects. Attached Figure Description

[0021] Figure 1 This is a gel image of nucleic acid from overlap PCR products.

[0022] Figure 2 This is a screening diagram for IL-23-specific monoclonal phages.

[0023] Figure 3 This is an SDS-PAGE image of P23A after expression and purification via CHO-K1.

[0024] Figure 4 This is a graph showing the antigen-binding activity of P23A.

[0025] Figure 5 This is a graph showing the changes in mouse body weight.

[0026] Figure 6 This is a mouse DAI score chart.

[0027] Figure 7 This is a statistical chart of mouse intestinal length. Detailed Implementation

[0028] 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.

[0029] Example 1: Construction of a fully human phage ScFv antibody library (1) Isolation of peripheral blood lymphocytes and extraction of total RNA Peripheral blood (200 ml) was collected from patients with ulcerative colitis in a blood collection bag containing anticoagulant. Solarbiopharmaceutical human peripheral blood lymphocyte separation solution (catalog number P8900) was used to separate the blood samples. The specific steps are as follows: 200 ml of DPBS was added to the blood sample and mixed thoroughly. The samples were then aliquoted into 15 ml vials. The amounts of reagents required for subsequent steps are described based on 15 ml of diluted blood. 15 ml of separation solution was added to a 50 ml centrifuge tube. The diluted blood was carefully spread evenly on the surface of the separation solution, ensuring a clear liquid interface. Centrifuged using a horizontal rotor at 500 g for 40 min at room temperature. After centrifugation, the white membrane layer cells were carefully aspirated into a 15 ml centrifuge tube. 10 ml of cell washing buffer was added to thoroughly wash the cells, and the tube was centrifuged at 250 g at room temperature for 10 min. The supernatant was discarded, and the cells were resuspended in 5 ml of cell washing buffer. The tube was centrifuged at 250 g at room temperature for 10 min. The supernatant was discarded again, and the cells were resuspended in 5 ml of cell washing buffer. The tube was centrifuged at 250 g at room temperature for 10 min. After discarding the supernatant, resuspend the cells in 5 ml of DPBS and count them using a hemocytometer. After counting, aliquot the cell suspension into 1.5 ml EP tubes, centrifuge at 250 g at room temperature for 10 min, discard the supernatant, and use the cell pellet for total RNA extraction. The total RNA extraction procedure using the QIAGEN RNA Extraction Kit (catalog number 74104) is as follows: Add approximately 1 × 10⁻⁶ lymphocytes... 7 Add 500 μL Trizol to the sample and mix thoroughly. Incubate at room temperature for 5 min. Add 100 μL chloroform, shake well for 30 s, and incubate at room temperature for 5 min. Centrifuge at 12000 g for 15 min at 4 °C. Transfer the supernatant to a new RNase-Free EP tube and add 600 μL anhydrous ethanol. Add 600 μL of the mixture to the RNeasy column in the kit, centrifuge at 12000 g for 30 s, and discard the waste liquid. Repeat the above steps until all the mixed liquid has been loaded. After discarding all the waste liquid, centrifuge the empty tube once. Transfer the RNeasy column to a new 1.5 mL collection tube, add 60 μL RNase-Free water, incubate at room temperature for 3 min, centrifuge at 12000 g for 1 min, transfer the eluent to a new RNase-Free EP tube, and take 2 μL for concentration detection using a NanoDrop 2000C.

[0030] (2) cDNA synthesis of the whole genome The complete genome cDNA was synthesized using ThermoFisher's RevertAid RT reverse transcription kit (catalog number K1691). The specific steps are as follows: Place a clean nuclease-free tube on ice and add the components listed in Table 1.

[0031] Table 1. Reverse Transcription System

[0032] After mixing thoroughly, incubate at 42°C for 60 min and then at 70°C for 5 min. The reverse transcription product can be used directly for subsequent PCR or aliquoted and stored at -80°C.

[0033] (3) PCR amplification Using the cDNA synthesized in (2) as a template, PCR amplification of the light chain variable region and the heavy chain variable region was performed. The primer sequences are shown in Table 2. The reaction system was: 5 μL cDNA, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 10 μL of 2×PCR TaqMastermix, and RNase-free ddH2O to a final volume of 20 μL. The reaction program was: 95℃ for 5 min; 95℃ for 15 s, gradient cooling from 65-55℃ for 15 s, 72℃ for 20 s, 40 cycles; 72℃ for 10 min. The amplified products were subjected to nucleic acid electrophoresis, and the bands around 400 bp were collected by gel extraction. After gel recovery, the products were stored at -20℃ for later use.

[0034] Table 2. Primer sequences for the first round of PCR

[0035] The products of the first round of PCR were used as templates for the second round of PCR. The primer sequences for the second round of PCR are shown in Table 3. The second round VH upstream primer was modified from the first round primer by adding an SfiI restriction site. The second round VH downstream primer was modified from the first round primer by adding a flexible linker (Gly4Ser). The 5' end of the second round Vκ upstream primer was modified by adding a sequence complementary to the VH downstream linker. The downstream primer was modified by adding a NotI restriction site and a stop codon. The reaction system was: 30 ng template, 5 μL 10 μM forward primer, 5 μL 10 μM reverse primer, 150 μL 2×PCR Taq Mastermix, and RNase-free ddH2O to a final volume of 300 μL. The reaction program was: 95℃ for 5 min; 95℃ for 15 s, 55℃ for 15 s, 72℃ for 30 s, 30 cycles; 72℃ for 10 min. The amplified products were subjected to nucleic acid electrophoresis, and the band around 350 bp was collected by gel extraction. After gel recovery, the product was stored at -20℃ for later use. Table 3. Primer sequences for the second round of PCR

[0036] Overlap PCR was performed using the second-round PCR product as a template. Primers VH-2F and Vκ / λ-2R were used, and the complete ScFv gene was formed through base complementation in the linker region. The reaction mixture consisted of: 20 ng template, 5 μL of 10 μM forward primer, 5 μL of 10 μM reverse primer, 150 μL of 2×PCR Taq Mastermix, and RNase-free ddH2O to a final volume of 300 μL. The reaction program was: 95℃ for 5 min; 95℃ for 15 s, 55℃ for 15 s, 72℃ for 30 s, 30 cycles; 72℃ for 10 min. The amplified product was subjected to nucleic acid electrophoresis, and the band around 750 bp was collected by gel extraction. After gel recovery, the product was stored at -20℃ for later use. Figure 1 As shown.

[0037] (4) Construction of ScFv phage display library The overlap PCR product and the phage display vector pSEX were double-digested with SfiI and NotI at 37°C for 4 h. The digested products were recovered by 1% agarose gel electrophoresis and then by an Omega gel recovery kit. Concentration was determined using a NanoDrop 2000C. Ligation was performed at a vector:insert ratio of 1:10 at 16°C overnight. The ligation product was electroporated into XL-1 Blue competent cells, and after recovery in antibiotic-free medium at 37°C and 220 rpm for 1 h, the cells were plated (containing ampicillin and tetracycline). After overnight culture, bacteria were scraped from the medium using a cell scraper, mixed with 50% glycerol, and stored at -80°C.

[0038] (5) Calculation of library capacity and determination of library diversity 1 ng, 10 ng, and 100 ng of pSEX plasmid were placed on ice and electroporated using XL-1 Blue competent cells. The cells were counted the following day, and the transformation efficiency was calculated to be 1*10^6. 9CFU / ug. Take 100 μL of phage library culture to package phages, add 100 mL of antibiotic-containing liquid medium, incubate at 220 rpm at 37°C for 1 h until OD595 = 0.5, add 20 mol / L hyperphage, incubate at 37°C at 120 rpm for 1 h, centrifuge at 4000 rpm at room temperature for 15 min to collect the cells, resuspend the precipitate in 100 mL of the aforementioned medium, and incubate overnight at 30°C at 220 rpm. The next day, centrifuge the culture to collect the supernatant, add 25 mL of PEG8000 / NaCl (20% PEG8000, 2.5 mol / L NaCl), incubate on ice for 2 h, centrifuge at 4000 rpm at 4°C for 1 h. Discard the supernatant, resuspend the precipitate in 2 mL of DPBS, let stand at room temperature for 15 min, centrifuge at 10000 rpm at 4°C for 15 min, collect the supernatant, dilute 1 μL for phage titer testing, and aliquot the remainder for storage at -80°C. The phage titer was observed the following day, and the library capacity was calculated to be approximately 1*10. 8 This capacity is sufficient to meet subsequent screening needs.

[0039] Example 2: Panning of IL-23-specific ScFv phages Remove the fully human ScFv phage library from the -80℃ freezer and thaw it rapidly on ice. Take 10 μL of the thawed phage library and add it to 10 mL of XL-1 Blue bacterial culture in logarithmic growth phase (OD595 = 0.5). Mix gently and incubate at 37℃ for 30 min to allow the phage to fully infect the host bacteria. Transfer the infected bacterial culture to 100 mL of 2×YT medium containing antibiotics and incubate at 37℃ and 220 rpm with shaking for 12–16 h to amplify the phage. After incubation, transfer the bacterial culture to a 50 mL sterile centrifuge tube and centrifuge at 4℃ and 8000 rpm for 10 min, collecting the supernatant. Add 1 / 5 volume of PEG8000 / NaCl solution to the supernatant, mix gently, and incubate at 4℃ for 2 h to precipitate. Centrifuge at 4℃ and 10000 rpm for 20 min, discard the supernatant, and collect the phage pellet. Resuspend the phage pellet in 2 mL of TBS buffer, transfer it to a 1.5 mL sterile centrifuge tube, centrifuge at 12,000 rpm for 5 min at 4 °C to remove residual bacterial fragments, and the supernatant is the revived phage library, which should be stored at 4 °C for later use.

[0040] Take a 96-well microplate and add 100 μL of recombinant human IL-23 protein diluted with carbonate coating buffer (concentration 10 μg / mL) to each well, and coat overnight at 4°C. Set up negative control wells and add 100 μL of carbonate coating buffer without IL-23 protein to each well. The next day, discard the coating buffer in the wells, add 200 μL of blocking buffer to each well, and block at 37°C for 2 hours to block non-specific binding on the microplate surface. Discard the blocking buffer and wash the microplate three times with TBST buffer, incubating for 3 minutes each time before discarding the washings. Add 100 μL of revived phage library to the wells coated with IL-23 protein, and add 100 μL of phage library diluted with TBST buffer (same concentration as the experimental group) to the negative control wells, and incubate at 37°C for 2 hours. Discard the phage solution in the wells. Wash the ELISA plate vigorously 10 times with TBST buffer, and finally wash twice with TBS buffer to remove residual Tween-20. Add 100 μL of 0.1 mol / L Glycine-HCl solution (pH=2.2) to each well and incubate at room temperature for 10 min to elute the phage bound to the IL-23 protein. Immediately transfer the eluent to a sterile centrifuge tube pre-filled with 10 μL of 1 mol / L Tris-HCl solution (pH=8.0), mix gently, and neutralize the pH of the eluent. Take 50 μL of the eluted phage solution and determine the titer of the eluted phage according to the aforementioned method for phage library titer determination. Record the phage titer after the first round of panning. The remaining eluted phage solution was added to 10 mL of XL-1Blue bacterial culture in logarithmic growth phase (OD595 = 0.5), incubated at 37°C for 30 min, and then transferred to 100 mL of 2×YT medium containing antibiotics. The culture was incubated overnight at 37°C with shaking at 200 rpm for phage amplification. Following the aforementioned phage library recovery method, the amplified phage was purified to obtain the first-round panned phage for the second round of panning. In the second round of panning, the concentration of the coating antigen recombinant human IL-23 protein was reduced to 5 μg / mL to improve panning specificity. The blocking procedure was the same as in the first round. The blocking solution was discarded, and the ELISA plate was washed three times with TBST buffer. 100 μL of the first-round panned phage (titer ≥ 10¹) was added to the wells coated with IL-23 protein. 0 The negative control wells were treated the same as in the first round, incubated at 37°C for 1.5 h. The number of washes was increased to 15. The remaining steps were the same as in the first round, yielding phages amplified in the second round of panning, which were used for the third round of panning. In the third round of panning, the concentration of the coated antigen recombinant human IL-23 protein was reduced to 2 μg / mL, the number of washes was increased to 20, and the remaining steps were the same as in the second round of panning. The phages amplified in the third round of panning were used for subsequent single-clone screening.

[0041] Example 3: Screening and identification of IL-23-specific ScFv phage monoclonals The phages amplified after the third round of panning were serially diluted 10-fold with TBST buffer. 3 10 4 10 5 10 6 Take 10 μL of each dilution of phage solution and add it to 100 μL of XL-1 Blue bacterial culture in logarithmic growth phase (OD595=0.5). Incubate at 37°C for 15 min. Spread the incubated bacterial culture evenly on LB agar plates containing 100 μg / mL Amp, with three replicates for each dilution. Incubate at 37°C upside down for 12-16 h. Select 50 single colonies from each plate and inoculate them into 96-well cell culture plates containing 100 μL of 2×YT medium (containing 100 μg / mL Amp). Incubate at 37°C with shaking at 220 rpm for 6 h. Add 100 μL of 2×YT medium (containing 100 μg / mL Amp and 1 mmol / L IPTG) to each well and incubate at 37°C with shaking at 220 rpm for 16 h to induce ScFv protein expression. Centrifuge the culture plate at 3000 rpm for 10 min at 4℃, collect the supernatant (monoclonal phage supernatant), and store at 4℃ for later use. Screen for specific monoclonal phages using ELISA by coating with recombinant human IL-23 protein (5 μg / mL) overnight at 4℃. Set up negative control wells (coated with irrelevant protein, 5 μg / mL) and blank control wells (coated with coating buffer only), with three replicates for each control well. Discard the coating solution, add 200 μL of blocking buffer to each well, and block at 37℃ for 2 h. Discard the blocking solution and wash the ELISA plate three times with TBST buffer. Add 100 μL of monoclonal phage supernatant to each well and incubate at 37℃ for 1 h. Discard the phage supernatant in the wells, wash the ELISA plate five times with TBST buffer, incubating for 3 min each time before discarding the washing buffer. Add 100 μL of HRP-labeled anti-M13 phage antibody diluted 1:10000 with blocking buffer to each well and incubate at 37°C for 1 h. Discard the secondary antibody solution, wash the ELISA plate 5 times with TBST buffer, and finally wash twice with TBS buffer. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 10 min. After the positive control wells show a clear blue color, add 100 μL of 1 mol / L H2SO4 solution to each well to stop the reaction. Measure the absorbance of each well at 450 nm using an ELISA reader. Use 2.1 times the OD450 value of the blank control wells as the positive threshold. Monoclonal phages with an OD450 value greater than the threshold and significantly higher than the negative control wells are considered IL-23-specific positive monoclonal phages. Experimental results are shown below. Figure 2Monoclonal clones 1 and 10 exhibited strong IL-23 binding activity, making them suitable for further sequence analysis.

[0042] Example 4: Sequence analysis of IL-23-specific ScFv phage monoclonal samples Colonies containing single clones 1 and 10 were inoculated into 5 mL of LB medium containing 100 μg / mL Amp and cultured overnight at 37°C with shaking at 220 rpm. The plasmid was extracted and sequenced the following day. The sequencing primer sequences were as follows: upstream primer GAA TTTTCT GTA TGA GCA GTT GCT CA, downstream primer CTA TGA CCA TGA TTA CGC CAA GCT T. Analysis of the sequencing results yielded the IL-23-specific ScFv gene sequence, as shown in Table 4.

[0043] Table 4. IL-23-specific ScFv sequences

[0044] Example 5: Expression of IL-23-specific ScFv Codon optimization was performed based on the amino acid sequence of ScFv clone 1 to adapt it to mammalian cells. The corresponding DNA sequence was synthesized by Genewiz, and its sequence information is as follows: SEQ ID No. 43. This sequence was constructed into the vector pcDNA3.1, and the plasmid was extracted. The vector contains a His tag for subsequent protein purification.The obtained plasmid was transfected into CHO-K1 cells using PEI for protein expression. The expressed protein was labeled P23A, and its amino acid sequence is shown in SEQ ID No. 44: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGFGLEWVSAISGSGGSTYYADSVKGRKTISRDNSKNTLYLQMNSLRAEDATVYYCARDPYGDYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK. The cell supernatant was collected and filtered through a 0.22 μm filter membrane to remove impurities. The Ni-NTA column was washed with Binding Buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0). The sample was slowly added to the column and incubated at 4 °C for 1 h. Impurities were washed away with Washing Buffer (20 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole, pH 8.0). Elution was performed stepwise with Elution Buffer (20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 8.0), and the elution peaks were collected. The purified product was analyzed by SDS-PAGE, and the results are shown below. Figure 3 As shown, there is a single band at 25 kDa. The molecular weight of P23A is consistent with that of fully human ScFv, and the purity is good with no impurities.

[0045] Example 6: Antigen-binding activity of IL-23-specific ScFv The antigen-binding activity of P23A was detected by ELISA, using the same method as described above. The coating antigen was recombinant human IL-23 protein (concentration 1 μg / mL). The experimental results are as follows. Figure 4 As shown in the figure, the EC50 is 10 nM, indicating that P23A has a high affinity for human IL-23 and is suitable for use as a therapeutic antibody.

[0046] Example 7: Evaluation of the therapeutic effect of P23A in a TNBS-induced humanized mouse IBD model We purchased TNBS-induced humanized IBD model mice from Nanmo Biotechnology, and after 7 days of acclimatization, they were randomly divided into groups as shown in Table 5.

[0047] Table 5. Grouping of Animal Experiments

[0048] All drugs were administered via intraperitoneal injection once daily, with the first administration date recorded as day 1. Mouse weight was monitored throughout the administration period, with mice weighed at the same time each day to avoid influencing results due to weight fluctuations after feeding or drinking. Scores were assigned to three dimensions: "weight loss," "fecal characteristics," and "fecal bloodiness," with each dimension ranging from 0 to 4 points. The DAI total score was calculated by summing the scores from the three dimensions, with a total score range of 0-12. 0 points: no enteritis symptoms; 1-4 points: mild enteritis; 5-8 points: moderate enteritis; 9-12 points: severe enteritis. On day 4, mice were euthanized, and intestinal length was measured and statistically analyzed. Figure 5 The graph shows the changes in mouse body weight. From day 0 to day 1, the body weight of mice in each group did not change much. Starting from day 2, the body weight of mice in group A gradually decreased, while the body weight of mice in groups B and C gradually increased after treatment. Figure 6 The DAI score chart shows that the DAI scores of groups B and C decreased after treatment, indicating a reduction in disease severity. Figure 7 According to the statistical analysis of mouse intestinal length, P23A and Guselkumab can effectively restore damaged intestinal tissue in mice and improve intestinal shortening caused by enteritis.

[0049] 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 fully human single-chain antibody (ScFv) that specifically binds to human IL-23, characterized in that, The heavy chain variable region sequence is shown in SEQ ID NO:39 or SEQ ID NO:41, and the light chain variable region sequence is shown in SEQ ID NO:40 or SEQ ID NO:

42.

2. The fully human single-chain antibody according to claim 1, characterized in that, Its heavy chain variable region sequence is shown in SEQ ID NO:39, and its light chain variable region sequence is shown in SEQ ID NO:

40.

3. The fully human single-chain antibody according to claim 1 or 2, characterized in that, Its EC50 for IL-23 is 10 nM.

4. A nucleic acid molecule encoding the fully human single-chain antibody according to any one of claims 1-3.

5. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, It includes the expression vector as described in claim 5.

7. A pharmaceutical composition, characterized in that, It comprises the fully human single-chain antibody as described in any one of claims 1-3 and a pharmaceutically acceptable vector.

8. The use of the fully human single-chain antibody according to any one of claims 1-3 in the preparation of a medicament for treating ulcerative colitis.

9. The use of the fully human single-chain antibody according to any one of claims 1-3 in the preparation of a drug for treating IL-23-mediated inflammatory diseases.

10. A method for treating ulcerative colitis, characterized in that, This includes administering a therapeutically effective amount of the fully human single-chain antibody according to any one of claims 1-3 to patients in need.

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