An antibody for treating ulcerative colitis and use thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antibodies, and particularly relates to an antibody for treating ulcerative colitis and application thereof. BACKGROUND

[0002] Ulcerative colitis (UC) is a chronic nonspecific inflammatory bowel disease, mainly involving the colonic mucosa and submucosa, and clinically manifested as diarrhea, abdominal pain, mucous and purulent bloody stool and the like. In recent years, the incidence of UC has shown an upward trend worldwide, and has become one of the major diseases seriously affecting human health. At present, the treatment of UC mainly includes 5-aminosalicylic acid drugs, glucocorticoids, immunosuppressants and biological agents, but a considerable proportion of patients still have poor response to the existing treatment or develop drug resistance, and it is urgent to develop more effective therapeutic drugs.

[0003] Studies have shown that interleukin-23 (IL-23) plays a key role in the pathogenesis of UC. IL-23 is a heterodimeric cytokine composed of p19 and p40 subunits, mainly produced by activated dendritic cells and macrophages. IL-23 activates Th17 cells and innate lymphocytes, promotes the secretion of pro-inflammatory factors such as IL-17 and IL-22, and thus leads to intestinal mucosal barrier destruction and inflammatory response. Clinical studies have found that the expression of IL-23 in the intestinal tissue of UC patients is significantly increased, and is positively correlated with the disease activity. Biological agents targeting IL-23 such as Ustekinumab (targeting IL-12 / 23 p40 subunit) and Guselkumab (targeting IL-23 p19 subunit) have shown good therapeutic effect in clinical trials, confirming that IL-23 is an ideal target for the treatment of UC.

[0004] Phage display technology is an important means of screening high-affinity antibodies, and through the construction of large-capacity antibody library, specific and high-affinity antibodies can be obtained through multiple rounds of panning. Fully human antibodies have the advantages of low immunogenicity and high safety, and are an ideal choice for therapeutic antibodies. Single-chain antibody (ScFv) is a small molecule antibody composed of antibody heavy chain variable region (VH) and light chain variable region (VL) connected by a flexible linker peptide, and has the advantages of small molecular weight, strong penetration and easy genetic engineering modification.

[0005] The present application constructs a fully human phage ScFv antibody library, and screens a 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 choice for the treatment of UC. SUMMARY

[0006] The present application provides a full human single-chain antibody (ScFv) specifically binding to human IL-23, wherein the heavy chain variable region sequence is shown as SEQ ID NO: 39 or SEQ ID NO: 41, and the light chain variable region sequence is shown as SEQ ID NO: 40 or SEQ ID NO: 42

[0007] In some embodiments, the heavy chain variable region sequence is shown as SEQ ID NO: 39, and the light chain variable region sequence is shown as SEQ ID NO: 40.

[0008] In some embodiments, the EC50 of the antibody to IL-23 is 10 nM.

[0009] The present application also provides a nucleic acid molecule encoding the full human single-chain antibody.

[0010] The present application also provides an expression vector comprising the nucleic acid molecule.

[0011] The present application also provides a host cell comprising the expression vector.

[0012] The present application also provides a pharmaceutical composition comprising the full human single-chain antibody and a pharmaceutically acceptable carrier.

[0013] The present application also provides the use of the full human single-chain antibody in the preparation of a medicament for treating ulcerative colitis.

[0014] The present application also provides the use of the full human single-chain antibody in the preparation of a medicament for treating IL-23-mediated inflammatory diseases.

[0015] The present application also provides a method for treating ulcerative colitis, comprising administering to a patient in need thereof a therapeutically effective amount of the full human single-chain antibody.

[0016] Compared with the prior art, the present application has at least the following advantages:

[0017] (1) The P23A antibody provided by the present application is a full human single-chain antibody, which has the advantages of low immunogenicity, high safety, etc., can reduce the generation of anti-drug antibodies during treatment, and improve the safety of long-term medication.

[0018] (2) The P23A antibody has high affinity to IL-23 (EC50=10 nM), can effectively block the IL-23 signaling pathway, and inhibit the production of downstream pro-inflammatory factors, thereby reducing intestinal inflammatory response.

[0019] (3) In the TNBS-induced humanized mouse IBD model, P23A can significantly improve the symptoms of the disease, including reversing body weight loss, reducing DAI score, restoring damaged intestinal tissue length, and the treatment effect is comparable to the marketed drug Guselkumab.

[0020] (4) The single-chain antibody has a small molecular weight (about 25 kDa) and better tissue penetration, and can more effectively reach the inflammatory site to play a role.

[0021] (5) The antibody production process provided by the present application is simple, can be expressed by CHO cells, and high-purity products can be obtained after purification, which is suitable for large-scale production.

[0022] (6) The antibody can not only be used for treating ulcerative colitis, but also can be applied to other IL-23-mediated inflammatory diseases, such as Crohn's disease, psoriasis, psoriatic arthritis, etc., and has a wide clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is an overlap PCR product nucleic acid gel map.

[0024] Figure 2 is an IL-23-specific monoclonal phage screening diagram.

[0025] Figure 3 is an SDS-PAGE diagram of P23A expressed by CHO-K1 and purified.

[0026] Figure 4 is a detection diagram of the antigen binding activity of P23A.

[0027] Figure 5 is a mouse weight change diagram.

[0028] Figure 6 is a mouse DAI score diagram.

[0029] Figure 7 is a mouse intestinal length statistical diagram. DETAILED DESCRIPTION

[0030] In order to make the technical problems, technical solutions and advantages to be solved by the present application more clear, specific embodiments will be described in detail below with reference to the drawings.

[0031] Example 1, Construction of Human Whole Phage ScFv Antibody Library

[0032] (1) Isolation of peripheral blood lymphocytes and extraction of total RNA

[0033] Collect 200ml peripheral blood of ulcerative colitis patients in a blood collection bag containing anticoagulant, use Sulebao human peripheral blood lymphocyte separation medium (item number P8900) to separate the collected blood sample, the specific steps are as follows: add 200ml DPBS to the blood sample and mix evenly, then divide according to 15ml / branch, and the amount of reagent required in the subsequent steps is described according to 15ml diluted blood. Add 15ml separation medium to a 50ml centrifuge tube, carefully lay the diluted blood on the surface of the separation medium and keep the liquid interface clear. Centrifuge using a horizontal rotor at 500g, 40min, room temperature. After centrifugation, carefully aspirate the white membrane layer cells into a 15ml centrifuge tube, add 10ml cell washing solution to wash the cells thoroughly, 250g centrifuge at room temperature for 10min. Discard the supernatant, add 5ml cell washing solution to resuspend the cells, 250g centrifuge at room temperature for 10min, add 5ml cell washing solution to resuspend the cells again, 250g centrifuge at room temperature for 10min. After discarding the supernatant, resuspend the cells with 5ml DPBS, count using a blood cell counting plate. After counting, the cell suspension is divided into 1.5ml EP tubes, 250g centrifuged at room temperature for 10min, the supernatant is discarded, and the cell pellet is used for total RNA extraction. The total RNA extraction uses the RNA extraction kit (item number 74104) of QIAGEN, and the operation steps are as follows: add 500ul Trizol to the extracted lymphocytes (about 1×10 7

[0034] (2) Whole gene cDNA synthesis

[0035] Use ThermoFisher's RevertAid RT reverse transcription kit (item number K1691) to synthesize whole gene cDNA, and the specific steps are as follows: place a clean nuclease-Free tube on ice, and add the components in Table 1 to it.

[0036] Table 1, reverse transcription system​

[0037]

[0038] After mixing evenly, incubate at 42℃ for 60 min and at 70℃ for 5 min. The reverse transcription product can be directly used for subsequent PCR or can be aliquoted and stored at -80℃.

[0039] (3) PCR amplification

[0040] The cDNA synthesized in (2) was used as a template for PCR amplification of the light chain variable region and the heavy chain variable region. The primer sequences are shown in Table 2. The reaction system was as follows: 5 μl of cDNA, 10 uM of forward primer 1 μl, 10 uM of reverse primer 1 μl, 2 x PCR Taq Mastermix 10 μl, and RNase-free ddH2O to make up to 20 μl. The reaction program was as follows: 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 product was subjected to nucleic acid electrophoresis, and the band near 400 bp was collected by gel cutting and stored at -20℃ for standby.

[0041] Table 2, primer sequences for the first round of PCR

[0042]

[0043] The product of the first round of PCR was used as a template for the second round of PCR. The primer sequences for the second round of PCR are shown in Table 3. The second round of VH upstream primer was added with a SfiI enzyme cutting site based on the first round, the second round of VH downstream primer was added with a flexible linker (Gly4Ser) based on the first round, the 5' end of the second round of VK upstream primer was added with a sequence complementary to the VH downstream linker, and the downstream primer was added with a NotI enzyme cutting site and a stop codon. The reaction system was as follows: 30 ng of template, 10 uM of forward primer 5 μl, 10 uM of reverse primer 5 μl, 2 x PCR Taq Mastermix 150 μl, and RNase-free ddH2O to make up to 300 μl. The reaction program was as follows: 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 near 350 bp was collected by gel cutting and stored at -20℃ for standby.

[0044] Table 3, primer sequences for the second round of PCR

[0045]

[0046] The second round of PCR products were used as templates for overlap PCR. The primers were VH-2F and VK / K-2R, and the complete ScFv gene was formed through base complementation of the linker region. The reaction system was: 20 ng of template, 5 ul of 10 uM forward primer, 5 ul of 10 uM reverse primer, 150 ul of 2x PCR Taq Mastermix, and RNase-free ddH2O to make up to 300 ul. The reaction program was: 95°C for 5 min; 95°C for 15 s, 55°C for 15 s, 72°C for 30 s, 30 cycles; 72°C for 10 min. The obtained amplification products were subjected to nucleic acid electrophoresis, and the band near 750 bp was collected and stored at -20°C for later use. Figure 1

[0047] (4) Construction of ScFv phage display library

[0048] The overlap PCR product and the phage display vector pSEX were double-digested using Sfil and Notl, and the enzyme digestion conditions were 37°C for 4 h. The enzyme digestion products were recovered using 1% agarose gel electrophoresis, and the Omega gel recovery kit was used for recovery, and the concentration was determined using NanoDrop2000C. The ratio of vector:insert was 1:10 for ligation, and the ligation conditions were 16°C overnight. The ligation product was electroporated into XL-1 Blue competent cells, and after 1 h of recovery at 37°C and 220 rpm in an antibiotic-free medium, it was plated (containing ampicillin and tetracycline). After overnight culture, the bacteria on the medium were scraped with a cell scraper, 50% glycerol was added, and it was mixed evenly and stored in a -80°C refrigerator.

[0049] (5) Calculation of library capacity and determination of library diversity

[0050] 1 ng, 10 ng, and 100 ng of pSEX plasmid were placed in an ice bath, and XL-1 Blue competent cells were used for electroporation. The next day, the bacteria were counted, and the transformation efficiency was calculated to be 1*10 9 ​CFU / ug. Take 100ul phage library bacteria solution to package phage, add 100ml liquid medium containing antibiotics, 220rpm 37℃ recover 1h to OD595=0.5, add 20MOI hyperphage, 37℃ 120rpm infection 1h, 4000rpm room temperature centrifugation 15min to collect bacteria, resuspend the precipitate with 100ml of the above-mentioned culture medium, 30℃ 220rpm overnight culture. The next day, centrifuge the bacteria solution to collect the supernatant, add 25ml PEG8000 / NaCl (20% PEG8000, 2.5mol / L NaCl), ice bath 2h, 4000rpm 4℃ centrifugation 1h. Discard the supernatant, resuspend the precipitate with 2ml DPBS, room temperature standing 15min, 10000rpm 4℃ centrifugation 15min, take the supernatant, 1ul dilution for plating to detect phage titer, the rest is stored at -80℃. The next day, observe the phage titer, calculate the library capacity is 1*10 8 The capacity can meet the subsequent screening requirements.

[0051] Example 2, IL-23 specific ScFv phage screening

[0052] Take the whole human ScFv phage library from the -80℃ refrigerator, quickly thaw on ice. Take 10μL of the thawed phage library and add it to 10mL of XL-1 Blue bacteria solution in the logarithmic growth phase (OD595=0.5), mix gently, and incubate at 37℃ for 30min to allow the phage to fully infect the host bacteria. Transfer the infected bacteria solution to 100mL of 2×YT medium containing antibiotics, and incubate at 37℃ with 220rpm shaking for 12-16h for phage amplification. After the culture is completed, transfer the bacteria solution to a 50mL sterile centrifuge tube, centrifuge at 8000rpm for 10min at 4℃ to collect the supernatant. Add 1 / 5 volume of PEG8000 / NaCl solution to the supernatant, mix gently, and let it stand at 4℃ for 2h. Centrifuge at 10000rpm for 20min at 4℃, discard the supernatant, and collect the phage precipitate. Resuspend the phage precipitate with 2mL TBS buffer, transfer it to a 1.5mL sterile centrifuge tube, centrifuge at 12000rpm for 5min at 4℃ to remove residual bacterial debris, and the supernatant is the recovered phage library, which is stored at 4℃ for future use.

[0053] Take 96-well enzyme-coated plates, add 100 μL of recombinant human IL-23 protein diluted with carbonate coating buffer (concentration of 10 μg / mL) to each well, and coat at 4°C overnight. Set up negative control wells, add 100 μL of carbonate coating buffer without IL-23 protein to each well. The next day, discard the coating solution in the enzyme-coated plate wells, add 200 μL of blocking solution to each well, and block at 37°C for 2 h to block non-specific binding on the surface of the enzyme-coated plate wells. Discard the blocking solution and wash the enzyme-coated plate with TBST buffer 3 times, discard the washing solution after standing for 3 min each time. Add 100 μL of the recovered phage library to the IL-23 protein-coated wells, and add 100 μL of the phage library diluted with TBST buffer (same concentration as the experimental group) to the negative control wells, and incubate at 37°C for 2 h. Discard the phage solution in the wells, wash the enzyme-coated plate vigorously with TBST buffer 10 times, and finally wash with TBS buffer 2 times 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 IL-23 protein. Immediately transfer the eluate to a sterile centrifuge tube pre-added with 10 μL of 1 mol / L Tris-HCl solution (pH=8.0), mix gently, and neutralize the pH of the eluate. Take 50 μL of the eluted phage solution, determine the titer of the eluted phage according to the method for determining the titer of the phage library described above, and record the phage titer after the first round of selection. Add the remaining eluted phage solution to 10 mL of XL-1 Blue bacterial solution (OD595=0.5) in the logarithmic growth phase, incubate at 37°C for 30 min, transfer to 100 mL of 2×YT medium containing antibiotics, and incubate at 37°C with 200 rpm shaking overnight for phage amplification. Purify the amplified phage according to the method for recovering the phage library described above to obtain the first round of selection and amplification of the phage for the second round of selection. The concentration of the coated antigen, recombinant human IL-23 protein, is reduced to 5 μg / mL in the second round of selection to improve the specificity of the selection. The blocking operation is the same as the first round. Discard the blocking solution and wash the enzyme-coated plate with TBST buffer 3 times. Add 100 μL of the first round of selection and amplification of the phage (titer ≥10¹ 0 pfu / mL) to the IL-23 protein-coated wells, and treat the negative control wells the same as the first round, and incubate at 37°C for 1.5 h. The number of washes is increased to 15 times. The remaining steps are the same as the second round of selection to obtain the second round of selection and amplification of the phage for the third round of selection. The concentration of the coated antigen, recombinant human IL-23 protein, is reduced to 2 μg / mL in the third round of selection, the number of washes is increased to 20 times, and the remaining steps are the same as the second round of selection. The third round of selection and amplification of the phage is used for subsequent monoclonal screening.

[0054] Example 3, Screening and identification of IL-23 specific ScFv phage monoclonal

[0055] The third round of amplified phage was taken and diluted with TBST buffer in 10-fold gradient, with dilution factors of 10 3 , 10 4 , 10 5 , 10 6 . 10 μL of phage solution at each dilution was added to 100 μL of XL-1 Blue bacteria solution in the logarithmic growth phase (OD595=0.5), and incubated at 37°C for 15 min. The incubated bacteria solution was evenly coated on LB solid medium plates containing 100 μg / mL Amp, with 3 repeats for each dilution, and incubated at 37°C for 12-16 h. Single colonies were selected from the plates, with 50 single colonies selected from each plate, and inoculated into 96-well cell culture plates containing 100 μL of 2×YT medium containing 100 μg / mL Amp, and incubated at 37°C and 220 rpm for 6 h. 100 μL of 2×YT medium containing 100 μg / mL Amp and 1 mmol / L IPTG was added to each well, and incubated at 37°C and 220 rpm for 16 h to induce ScFv protein expression. The plates were centrifuged at 4°C and 3000 rpm for 10 min, and the supernatant was collected as the single monoclonal phage supernatant, which was stored at 4°C for later use. ELISA was used to screen specific monoclonal phage, and recombinant human IL-23 protein (5 μg / mL) was used for coating overnight at 4°C. Negative control wells (coated with irrelevant protein at a concentration of 5 μg / mL) and blank control wells (only coated with buffer) were set up, with 3 repeats for each control well. The coating solution was discarded, 200 μL of blocking solution was added to each well, and incubated at 37°C for 2 h. The blocking solution was discarded, and the enzyme-labeled plate was washed with TBST buffer 3 times. 100 μL of monoclonal phage supernatant was added to each well, and incubated at 37°C for 1 h. The phage supernatant in the wells was discarded, and the enzyme-labeled plate was washed with TBST buffer 5 times, with the washing solution discarded after 3 min of standing each time. 100 μL of HRP-labeled anti-M13 phage antibody diluted 1:10000 with blocking solution was added to each well, and incubated at 37°C for 1 h. The secondary antibody solution was discarded, and the enzyme-labeled plate was washed with TBST buffer 5 times, and finally washed with TBS buffer 2 times. 100 μL of TMB color developing solution was added to each well, and incubated at room temperature for 10 min. After the positive control wells turned blue, 100 μL of 1 mol / L H2SO4 solution was added to each well to stop the reaction. The absorbance value of each well at 450 nm was detected by an enzyme-labeled instrument. The OD450 value of the blank control well was taken as the positive judgment threshold, and the monoclonal phage with an OD450 value greater than the threshold and significantly higher than that of the negative control well was determined as IL-23 specific positive monoclonal phage. The experimental results are shown in Table 1. Figure 2No. 1 and No. 10 monoclonal have strong IL-23 binding activity, and are suitable for further sequence analysis.

[0056] Example 4, Sequence analysis of IL-23 specific ScFv phage monoclonal

[0057] The colonies of No. 1 and No. 10 monoclonal were inoculated into 5 mL LB medium containing 100 μg / mL Amp, and cultured at 37°C, 220 rpm overnight. The next day, plasmid was extracted and sequenced, and the sequence of the sequencing primer was as follows: upstream primer GAA TTTTCT GTA TGA GCA GTT GCT CA, downstream primer CTA TGA CCA TGA TTA CGC CAA GCT T. The sequencing results were analyzed to obtain the sequence of IL-23 specific ScFv gene, as shown in Table 4.

[0058] Table 4, IL-23 specific ScFv sequence

[0059]

[0060] Example 5, Expression of IL-23 specific ScFv

[0061] The amino acid sequence of ScFv clone 1 was codon-optimized for mammalian cells and the corresponding DNA sequence was synthesized by Genewiz, the sequence information is as follows SEQ ID No 43: GAAGTGCAACTGGTGGAGAGTGGGGGAGGTCTGGTGCAACCTGGAGGCTCTCTCAGGCTGTCATGCGCCGCCTCAGGCTTTACCTTTTCCAGTTATGCAATGCACTGGGTTAGGCAGGCGCCTGGCTTTGGGCTGGAATGGGTCAGCGCTATTTCCGGGTCTGGGGGAAGTACCTACTACGCCGACTCCGTGAAGGGGAGAAAAACCATCAGCCGCGACAACTCAAAGAACACGCTGTACCTCCAAATGAATTCACTGCGGGCGGAAGACGCCACCGTCTACTACTGCGCTAGAGACCCCTATGGCGACTACTTCGATTACTGGGGTCAAGGAACCCTTGTGACTGTGTCTTCCGGAGGAGGGGGCAGTGGCGGTGGCGGATCCGGAGGAGGAGGCAGCGATATCCAGATGACCCAGTCACCAAGTAGTCTTAGTGCGAGTGTGGGAGATCGGGTCACCATCACCTGTAGAGCTAGCCAAGACATTAGCAACTATCTCAATTGGTATCAGCAAAAGCCCGGCAAGGCCCCCAAGCTGTTGATCTACTACACCAGCCGACTTCACAGCGGAGTGCCTTCTCGATTTTCAGGCTCAGGGTCTGGCACTGACTTCACCTTGACAATAAGTTCCCTGCAGCCCGAGGACTTTGCTACATATTACTGCCAGCAGTATAGTACGGTTCCTTGGACCTTTGGTCAAGGTACAAAGGTGGAGATAAAA. It was constructed on the vector pcDNA3.1 and the plasmid was extracted, the vector contains a His tag for subsequent protein purification.The obtained plasmid was used to transfect CHO-K1 cells using PEI for protein expression. The expressed protein was labeled as P23A, and the amino acid sequence was as shown in SEQ ID No 44: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGFGLEWVSAISGSGGSTYYADSVKGRKTISRDNSKNTLYLQMNSLRAEDATVYYCARDPYGDYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK. The cell supernatant was collected and filtered using a 0.22 μm filter 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. The impurities were washed away using Washing Buffer (20 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole, pH 8.0). The product was eluted stepwise using Elution Buffer (20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 8.0), and the elution peak was collected. The purified product was detected by SDS-PAGE, and the results are shown in Figure 3 As shown in the figure, there is a single band at 25 kDa, and the molecular weight of P23A is consistent with that of a full human ScFv, and the purity is good without impurity bands.

[0062] Example 6, antigen binding activity of IL-23 specific ScFv

[0063] The antigen binding activity of P23A was detected by ELISA, and the method was the same as described above. The coating antigen was recombinant human IL-23 protein (concentration of 1 μg / mL), and the experimental results are shown in Figure 4 As shown in the figure, the EC50 was 10 nM, indicating that P23A had high human IL-23 affinity and was suitable for use as a therapeutic antibody.

[0064] Example 7, evaluation of the therapeutic effect of P23A in a TNBS-induced humanized mouse IBD model

[0065] TNBS-induced humanized IBD model mice were purchased from Nanmou Biological Company, and were randomly grouped after 7 days of acclimation. The grouping is shown in Table 5.

[0066] Table 5, grouping of animal experiments

[0067]

[0068] All drugs were administered by intraperitoneal injection, the administration frequency was once a day, and the first administration day was recorded as day 1. During the administration period, the body weight changes of mice were monitored, and the body weight of mice was weighed at the same time every day to avoid the influence of body weight fluctuation after eating and drinking on the results. Three dimensions of "body weight loss", "fecal characteristics" and "blood in stool" were scored respectively, and each dimension was scored from 0 to 4. DAI total score calculation: add the scores of the three dimensions to get the final DAI score, and the total score range is 0-12. 0: no intestinal inflammation; 1-4: mild intestinal inflammation; 5-8: moderate intestinal inflammation; 9-12: severe intestinal inflammation. On day 4, the mice were euthanized and the length of the intestine was measured and counted. Figure 5 The figure is the body weight change graph of mice, and the body weight of mice in each group from day 0 to day 1 changes little, and the body weight of mice in group A shows a gradual downward trend from day 2, and the body weight of mice in groups B and C gradually increases after treatment. Figure 6 The figure is the DAI score graph, and the DAI score of groups B and C decreases after treatment, and the severity of the disease decreases. Figure 7 The figure is the length of the mouse intestine, and P23A and Guselkumab can effectively restore the damaged intestinal tissue of mice and improve the intestinal shortening caused by intestinal inflammation.

[0069] The above is the preferred embodiment of the present application, it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A fully human single-chain antibody that specifically binds to human IL-23, characterized in that, 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.

2. A nucleic acid molecule encoding the fully human single-chain antibody of claim 1.

3. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that, It includes the expression vector as described in claim 3.

5. A pharmaceutical composition, characterized in that, It comprises the fully human single-chain antibody as described in claim 1 and a pharmaceutically acceptable vector.

6. The use of the fully human single-chain antibody according to claim 1 in the preparation of a drug for treating ulcerative colitis.

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