Anti-canine IL-31 antibody and application thereof

Abstract

The invention relates to an anti-canine IL-31 antibody or an antigen binding part thereof and application thereof, and belongs to the technical field of antibody preparation. The invention relates to an anti-canine interleukin-31 antibody or an antigen binding part thereof, amino acid for coding the antibody or the antigen binding part thereof, polynucleotide, a carrier containing the polynucleotide, a host cell containing the polynucleotide or the carrier, a method for preparing and purifying the antibody and application of the antibody or the antigen binding part thereof. The anti-canine IL-31 antibody disclosed by the invention can be specifically combined with a canine IL-31 antigen, and can effectively relieve symptoms such as pruritus, allergy, nasal polyp or asthma and the like. The mutated anti-canine IL-31 antibody also has a corresponding function of combining with the antigen canine IL-31, and also can be used for treating IL-31 related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and in particular to an antibody against canine IL-31 and a use thereof. Background Art

[0002] Atopic dermatitis (AD) is a hereditary inflammatory and itchy allergic skin disease. Severe itching brings more inconvenience to the lives of pets and owners, especially secondary hair loss and erythema, which are of particular concern to pet owners. Approximately 450 million dogs in the world suffer from chronic or lifelong dermatitis.

[0003] The occurrence of atopic dermatitis is the result of the combined action of a series of cytokines, among which IL-31 (interleukin-31, IL-31) plays an important role in the occurrence of itching in skin diseases. IL31 is a cytokine mainly produced by activated T helper cells, and can also be expressed by mast cells or macrophages. As early as 2004, the IL-31 gene sequence had been cloned by scientists, and there have been many related studies on IL-31. According to research, IL-31 binds to a composite receptor composed of IL-31RA (interleukin 31 receptor A, IL31RA) and OSMR (Recombinant Oncostatin M Receptor, OSMR). After the receptor is activated, it phosphorylates through the STAT (signal transducer and activator of transcription, STAT) of the JAK (janus kinase) receptor, resulting in upregulation of target genes and causing itching behavior.

[0004] More and more researchers are investing in the study of treating atopic dermatitis by blocking the IL-31 pathway. Lokivetmab (Cytopoint, Zoetis) is currently the only caninized monoclonal antibody approved for the treatment of canine-related dermatitis. Its target is canine IL-31 and it is injected once every four to eight weeks. According to clinical trials, Lokivetmab can significantly reduce the itching symptoms of dogs and significantly improve the skin condition of dogs, which shows that IL-31 monoclonal antibodies are effective in treating specific dermatitis. Although some products in the prior art have shown certain effects in the treatment of animal atopic dermatitis, the high cost of these products limits the treatment of animal atopic dermatitis. Therefore, the development of more canine IL-31 antibodies has become a problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an anti-canine interleukin-31 antibody and its application. The IL-31 monoclonal antibody developed by the present invention can be used for the treatment of atopic dermatitis, as well as allergies, nasal polyps, asthma and other diseases.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In the first aspect, the present invention provides an anti-canine interleukin-31 antibody or an antigen-binding portion thereof, wherein the amino acid sequence of the heavy chain CDR1 of the antibody is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 3; the amino acid sequence of the light chain CDR1 of the antibody is shown in SEQ ID NO: 4, the amino acid sequence of the light chain CDR2 is AAS, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 5.

[0008] As a preferred embodiment of the first aspect, the nucleotide sequence of the heavy chain CDR1 of the antibody is shown in SEQ ID NO: 8, the nucleotide sequence of the heavy chain CDR2 is shown in SEQ ID NO: 9, and the nucleotide sequence of the heavy chain CDR3 is shown in SEQ ID NO: 10; the nucleotide sequence of the light chain CDR1 of the antibody is shown in SEQ ID NO: 11, the nucleotide sequence of the light chain CDR2 is GCTGCATCC, and the nucleotide sequence of the light chain CDR3 is shown in SEQ ID NO: 12.

[0009] As a preferred embodiment of the first aspect, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO: 6; the light chain amino acid sequence of the antibody is shown as SEQ ID NO: 7.

[0010] As a preferred embodiment of the first aspect, the heavy chain nucleotide sequence of the antibody is shown as SEQ ID NO: 13; the light chain nucleotide sequence of the antibody is shown as SEQ ID NO: 14.

[0011] As a preferred embodiment of the first aspect, it also includes a canine IgGA heavy chain framework structure, and a canine Igκ light chain constant region framework structure.

[0012] As a preferred embodiment of the first aspect, the amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO: 19; and the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO: 20.

[0013] As a preferred embodiment of the first aspect, the antibody or antigen-binding portion thereof is a mutant of the interleukin-31 antibody, and the mutant is any one of the following or a combination of any one of the following heavy chains and light chains:

[0014] 1) mutating the amino acid D at position 6 in the heavy chain CDR1 sequence GYNFTDNS as shown in SEQ ID NO: 1 to amino acid E, amino acid N or amino acid V;

[0015] 2) mutating the amino acid E at position 4 in the heavy chain CDR2 sequence INTETGEP as shown in SEQ ID NO: 2 to amino acid D;

[0016] 3) mutating the amino acid E at position 7 in the heavy chain CDR2 sequence INTETGEP as shown in SEQ ID NO: 2 to amino acid D, amino acid Q, amino acid L or amino acid T;

[0017] 4) mutating the amino acid D at position 4 in the heavy chain CDR3 sequence AKGDYEY as shown in SEQ ID NO: 3 to amino acid E, amino acid K or amino acid I;

[0018] 5) mutating the amino acid E at position 6 in the heavy chain CDR3 sequence AKGDYEY as shown in SEQ ID NO: 3 to amino acid D, amino acid N or amino acid Q;

[0019] 6) mutating the amino acid E at position 4 in the light chain CDR1 sequence ESVENYGISF as shown in SEQ ID NO: 4 to amino acid D;

[0020] 7) The amino acid E at position 5 in the light chain CDR3 sequence QQSKEVPWT as shown in SEQ ID NO: 5 is mutated to amino acid D.

[0021] In a second aspect, the present invention further provides a kit for detecting canine interleukin-31, the kit comprising the anti-canine interleukin-31 antibody or antigen-binding portion thereof as described in the first aspect.

[0022] In a third aspect, the present invention also provides the use of the anti-canine interleukin-31 antibody or its antigen-binding portion as described in the first aspect or the kit for detecting canine interleukin-31 as described in the second aspect in the preparation of a pharmaceutical composition for treating, preventing and / or alleviating IL-31-related diseases.

[0023] As a preferred embodiment of the third aspect, the IL-31-related disease is selected from pruritus, allergy, nasal polyps or asthma; preferably, the pruritus is selected from atopic dermatitis, eczema, psoriasis, scleroderma and pruritus; preferably, the allergy is selected from allergic dermatitis, summer eczema, urticaria, airway inflammatory diseases, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease and inflammatory processes caused by autoimmunity.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The anti-canine IL-31 antibody disclosed in the present invention can specifically bind to canine IL-31, and has an affinity that is not weaker than that of the commercial product certomin. It has been verified that it has good biological activity in cells and animals, and can effectively alleviate the related symptoms of model animals in a canine pruritus model. These related diseases include pruritus, allergies, nasal polyps or asthma; pruritus includes atopic dermatitis, eczema, psoriasis, scleroderma and pruritus; allergies include allergic dermatitis, summer eczema, urticaria, airway inflammatory diseases, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease and inflammatory processes caused by autoimmunity. At the same time, after the antibody is caninized, it has no obvious immunogenicity. At the same time, the mutated anti-canine IL-31 antibody also has the corresponding binding function with the antigen canine IL-31, and can also treat diseases related to IL-31. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the electrophoresis diagram of the recombinant protein canine IL-31 antigen;

[0027] Figure 2 This is a schematic diagram of the results of the phosphorylation inhibition test of IL-31 signaling pathway by different antibodies in Example 4;

[0028] Figure 3 This is a schematic diagram of the pruritus model established in Example 5, showing the concentration of injected IL-31 protein and the pruritus score results;

[0029] Figure 4 This is a schematic diagram of the results of the efficacy test of the mouse 3G12 antibody in experimental dogs in Example 6;

[0030] Figure 5 This is a schematic diagram of the affinity comparison results of ceratopin and caninized 3G12 antibody in Example 9;

[0031] Figure 6 This is a schematic diagram of the results of the in vivo efficacy test of the caninized 3G12 antibody in experimental dogs in Example 10;

[0032] Figure 7 This is a schematic diagram of the affinity test results of the caninized 3G12 antibody mutants in Example 11;

[0033] Figure 8 This is a schematic diagram of the results of the phosphorylation inhibition experiment of the caninized 3G12 antibody mutant in Example 11. DETAILED DESCRIPTION

[0034] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0035] Example 1. Antigen preparation

[0036] The canine IL-31 amino acid sequence (NP_001159386.1) was retrieved from NCBI and the gene sequence was inferred and synthesized. The prokaryotic expression system pET30a(+) was used to construct a vector and express the recombinant protein canine IL-31. The molecular size of the recombinant protein was verified after expression and purification (predicted 17.79 kDa, see Figure 1 ) and other information are correct, it is used for mouse immunization.

[0037] Example 2. Immunization of mice

[0038] The immunized mice were Bal b / c (purchased from: Experimental Animal Management Center of Southern Medical University), 6 weeks old. The purified recombinant canine IL-31 was used as an immunogen to immunize mice. A batch of 20 mice were immunized for 3 times. The immunization process was as follows:

[0039] 1. Immunize mice with 50 μg of immunogen per mouse for the first immunization;

[0040] 2. Three weeks later, the second immunization was performed with 25 μg of immunogen per mouse;

[0041] 3. Two weeks later, three immunizations were performed, with the mice being immunized with 25 μg of the immunogen per mouse;

[0042] 4. One week after the third immunization, blood was collected from the eyelids and the titer of the mice after immunization was tested by indirect ELISA. The mice with higher titers were selected as mice for subsequent spleen cell fusion experiments.

[0043] Example 3. Screening of monoclonal cell lines

[0044] 1. Mouse spleen cell fusion:

[0045] 1.1 Spleen cell collection: Remove the spleen under sterile conditions, peel off the membranous tissue, place it in incomplete culture medium, use a syringe to extract the incomplete culture medium and inject it into the spleen to release spleen cells, grind the remaining spleen tissue with a cell sieve, collect the cells, and count them.

[0046] 1.2SP2 / 0 cell collection: SP2 / 0 cells are round and translucent. Collect the cells in a 50 ml centrifuge tube and count them.

[0047] 1.3 Adjust the ratio of SP2 / 0 cells and spleen cells to 1:3-1:10.

[0048] 1.4 Take out the PEG1450 preheated at 37℃, rotate the centrifuge tube, add 1ml of PEG1450 solution to the mixed cell culture container in step 1.3 within 60s, and let it stand for 1min; slowly add 3ml of DMEM medium within 3min, then gradually increase the dropping speed, and add DMEM medium to a total volume of 30ml within 5min; put it in a 37℃ water bath and let it stand for 5min.

[0049] 1.5 Centrifuge at 1000 rpm for 5 min, remove the supernatant, add appropriate amount of HAT medium to resuspend the cells, plate 200 μl per well in a 96-well cell culture plate (the 96-well plate had been pre-added with peritoneal macrophages the day before, 10,000 cells / well), and culture in a 37°C 5% carbon dioxide incubator.

[0050] 2. Indirect ELISA screening positive wells

[0051] 2.1 Coating: Dilute canine IL-31 fusion protein with 1× coating buffer to a 2 μg / ml coating solution, add 50 μL of coating solution to each well of the test plate, and incubate at 2-8°C for at least 12 hours.

[0052] 2.2 Blocking: Wash the coated plate 4 times with TBST, remove the remaining solution, add blocking solution, 100 μL per well, seal, and place at 37°C for 2 hours; wash the plate 4 times with TBST and set aside.

[0053] 2.3 Sample incubation: Take the supernatant of the cell culture plate after fusion for detection, use the immunized mouse serum as the positive control, the cell culture medium as the negative control, and PBS as the blank control. Load 100 μL into each well and incubate at 37°C for 1 hour.

[0054] 2.4 Secondary antibody incubation: Wash the detection plate 4 times with TBST, dilute the goat anti-mouse secondary antibody (Selvi, HRP*Goat Anti-Mouse IgG (H+L)) at 1:10000 in blocking solution, load 100 μL into each well, incubate at 37°C for 1 hour.

[0055] 2.5 Color development and termination: Wash the test plate 4 times with TBST, remove the remaining liquid, add color development solution, 100 μL per well, and leave at room temperature for 10 minutes; add 50 μL of stop solution to all wells of the ELISA reaction plate.

[0056] 2.6 Reading: The absorbance value was read at 450 nm using a microplate reader.

[0057] 2.7 Select the wells with higher test values ​​as suspected positive wells. After repeated testing the next day, the wells with higher positive values ​​are regarded as positive wells. After the above tests, 13 positive wells including 1G2, 3G12, 4G4, 5D4, 6F3, 6F5, 7F9, 8A2, 9D8, 12B3, 15A4, 16B6 and 19G11 were screened.

[0058] 3. Monoclonalization

[0059] 3.1 First round of monoclonal cloning: Collect cells from the above positive wells, dilute the cells to single cell / 200 μL using monoclonal cell culture medium, add the cell suspension to a 96-well cell culture plate, and after 8 days of culture, detect using the above indirect ELISA method.

[0060] 3.2 Second round of monocloning: Analyze the results of the first round of subcloning ELISA test. If the positive well rate is 100%, it can be determined that the cell purity is sufficient. Otherwise, select the positive wells for the next round of monocloning. The second round of monocloning operation is the same as the first round of monocloning.

[0061] 3.3 After multiple rounds of monocloning of the above positive well cells, if the purity of the hybridoma cells reaches 100%, the monocloning can be terminated.

[0062] The cell lines obtained by the above screening were named 1G2, 3G12, 4G4, 5D4, 6F3, 6F5, 7F9, 8A2, 9D8, 12B3, 15A4, 16B6 and 19G11 cell lines according to the plate number and well number, and the corresponding antibodies were named 1G2, 3G12, 4G4, 5D4, 6F3, 6F5, 7F9, 8A2, 9D8, 12B3, 15A4, 16B6 and 19G11 antibodies.

[0063] Example 4. Phosphorylation inhibition assay of IL-31 signaling pathway

[0064] IL-31 regulates the expression of related genes through signal pathways, thereby affecting the itching behavior of dogs, and neutralizing IL-31 can block the itching behavior. It is known that the composite receptor composed of IL-31 receptor A (IL-31RA) and oncostatin M receptor (OSMR) mediates Janus kinase phosphorylation and activation of the STAT signal cascade after binding to IL-31. Therefore, the present invention establishes a cell biological activity assay method based on detecting STAT phosphorylation levels to evaluate the neutralizing effect of screening antibodies on IL-31. The present invention selects the above-mentioned 1G2, 3G12, 4G4, 5D4, 6F3, 6F5, 7F9, 8A2, 9D8, 12B3, 15A4, 16B6 and 19G11 antibodies to perform phosphorylation inhibition tests on signal pathways. The specific steps are as follows:

[0065] 1. Cell preparation: Canine macrophage-like DH82 cells (Cell Bank of the Chinese Academy of Sciences, catalog number TCO3) were cultured at 37°C and 5% CO2 until they were in good condition according to the instructions;

[0066] 2. Canine IFN-γ induction: The above cells were plated at 10w / well in a 96-well plate and cultured with MEM complete medium (10% FBS, 2mmol / L GlutaMax, 1mmol / L sodium pyruvate), and 10ng / mL canine IFN-γ was added for induction for 24 hours;

[0067] 3. Starvation treatment: After induction, starve the cells with MEM basal medium for 2 h;

[0068] 4. IL-31 treatment: remove the above culture medium, divide into different antibody groups, add mixed culture fluids of different antibodies (different hybridoma cell supernatants + 1 μg / mL IL-31 recombinant protein), and continue culturing for 1 hour;

[0069] 5. Cell lysis: Remove the culture medium, wash the wells twice with PBS, and lyse the cells with cell lysis buffer.

[0070] 6. Detect the lysis samples according to the STAT3 phosphorylation detection kit (PathScan (R) Phospho-Stat3 (Tyr705) Sandwich ELISA Kit, CST) and collect the detection data.

[0071] The results are as follows Figure 2 As shown, it can be seen that 3G12 antibody has an inhibitory effect on STAT3 phosphorylation in DH82 cells, while other antibodies have almost no inhibitory effect. Based on this, the present invention continues to use 3G12 antibody as the subsequent experimental antibody.

[0072] Example 5. Validation of the Animal Model for Screening Monoclonal Antibody 3G12

[0073] 1. Establishment of the Itch Model

[0074] This model was established with reference to the literature "IL-31-induced pruritus in dogs: a novel experimental model to evaluate anti-pruritic effects of canine therapeutics" (Veterinary Dermatology, 2015). All dogs were fed under the same conditions. All dogs were weighed and divided into 4 groups. The average weight difference between different treatment groups was controlled within 20%. The groups are as follows:

[0075] Table 1: Experimental groups

[0076] Group quantity Recombinant protein IL-31 injection dosage Experimental Group 1 4 3 μg / kg Experimental Group 2 4 6 μg / kg Experimental Group 3 4 9 μg / kg Control group 4 PBS

[0077] The fur of the forelimb vein was shaved with a razor, and the dogs were placed in the video room 1 hour before the start of the observation to start the adaptation. The prepared recombinant protein IL-31 injection solution was injected intravenously 20 minutes before the start of the observation, and the injection dose was given according to Table 1 above; 20 minutes after the injection, continuous video recording began for 2 hours; the camera observed the dogs vertically below. After the end of the video recording, the dogs in different groups were fed normally under the same conditions until the next experiment; if necessary, the experiment could be repeated 1 month later. According to the literature, the observer selected discrete time periods (1 minute) for observation. The observer judged "yes" or "no" according to the above time according to the itch behavior (licking / biting paws, flanks, anal area, scratching flanks or neck, digging the ground, shaking head, rubbing buttocks on the cage floor) and recorded it in the table. The cumulative number of "yes" made during each observation time provided the itch score PSI.

[0078] The present invention uses recombinant IL-31 for modeling. During the modeling process, individual non-responsive dogs are eliminated, and at least 4 dogs are counted in each group. Figure 3 Data show that at a dose of 3 μg / kg, the itch scores of different dogs in the group varied relatively greatly; at a dose of 6 μg / kg, the itch scores of different dogs in the group varied relatively little, and when the dose was further increased to 9 μg / kg, the itch scores did not increase significantly. Therefore, the present invention uses a dose of 6 μg / kg to establish an itch model and conduct efficacy studies.

[0079] Example 6. In vivo efficacy study of mouse 3G12

[0080] 1. The 2 mg / kg injection dose of ceramide was used as the positive control group (4 statistical experimental dogs), PBS was used as the negative control group (4 statistical experimental dogs), and different injection doses of mouse 3G12 antibody (0.125 mg / kg, 0.5 mg / kg and 2 mg / kg) were used as the experimental group (4 statistical experimental dogs). The above pruritus model was used for in vivo pharmacodynamics study. On D0, different concentrations of antibody 3G12 antibody were subcutaneously injected into the dogs (the negative control group was injected with an equal volume of PBS); on D1 and D7, recombinant protein IL-31 was injected intravenously into the forelimbs. The baseline score of pruritus was recorded 30 minutes before injection, and the pruritus score was recorded for 2 hours after injection. According to the pruritus score, the data of different groups were processed with Prism 8.0 software. If P < 0.05, it indicates that there is a significant difference between the antibody injection group and the PBS injection group. If P > 0.05, it indicates that there is no significant difference between the antibody injection group and the PBS injection group.

[0081] Since the 3G12 antibody in this example is a mouse antibody, the efficacy of the mouse 3G12 antibody has begun to disappear on D7, probably due to the dog's autoimmune reaction and enzyme degradation. Therefore, this example only counts the experimental results of the first 7 days. Figure 4 The results showed that on D1 day, a dose of 0.125 mg / kg of 3G12 antibody was already able to produce a sufficiently good effect, indicating that the 3G12 antibody has a sufficiently good ability to neutralize IL-31.

[0082] Example 7. Gene sequencing

[0083] The purified 3G12 hybridoma cell line was amplified and 3×10 6 After cell lysis, mRNA was extracted and reverse transcribed into cDNA. After antibody gene amplification, it was cloned into pcDNA3.4 vector. After Sanger sequencing, the variable region nucleic acid sequence of the antibody was obtained. After bioinformatics analysis, the amino acid sequence of 3G12 antibody as shown in Table 2 was obtained:

[0084] Table 2: 3G12 antibody amino acid sequence

[0085]

[0086]

[0087] Example 8. Caninization of Antibodies

[0088] After bioinformatics software analysis and IMGT analysis to predict antibody functional regions, as well as laboratory expression, canine IgGA (accession number: AAL35301.1) was finally selected as the canine heavy chain framework structure. After NCBI retrieval and IMGT analysis, canine Igκ (accession number: DBA44934.1) was selected as the canine light chain constant region splicing framework structure. The above framework was used for variable region replacement or splicing operations. According to Table 3, the underlined part in the sequence SEQ ID NO: 15 is the canine heavy chain variable region sequence, and the region sequence can be replaced with the VH amino acid sequence of the 3G12 antibody; the sequence SEQ ID NO: 17 is the canine light chain constant region sequence, and the sequence is spliced ​​with the VL amino acid of the 3G12 antibody to form a new chimeric antibody light chain and heavy chain combination, and the caninized 3G12 antibody is obtained. The caninized 3G12 antibody gene sequence was inserted into the pcDNA3.4 vector through the restriction site by gene recombination. After the vector was successfully constructed after verification, it was transiently transformed into CHO cells for expression. The culture supernatant was collected and the antibodies were purified for subsequent validation experiments.

[0089] Table 3: Constant region sequences of canine antibody heavy and light chains and caninized 3G12 antibody sequences

[0090]

[0091]

[0092] Example 9. Affinity comparison test

[0093] The affinity test uses the indirect ELISA method, and the specific process is as follows:

[0094] 1. Setting: The control group was the ceramide group, the experimental group was the caninized 3G12 group of the present invention, and the antibody concentration gradient was set to: 4000 ng / mL, 800 ng / mL, 160 ng / mL, 32 ng / mL, 6.4 ng / mL, 1.28 ng / mL, 0.256 ng / mL and 0.051 ng / mL.

[0095] 2. Indirect ELISA operation: 50 ng of canine IL-31 fusion protein was coated on the plate at each well. The primary antibody was caninized 3G12 antibody. The enzyme-labeled secondary antibody was Dog IgG antibody (HRP) (GeneTex). Other operations were the same as the above indirect ELISA operation. The test values ​​were read.

[0096] 3. Data processing: Prism8.0 was used to process the above Elisa test values, with the above certoin and 3G12 antibody concentration gradient values ​​as the horizontal axis and the OD value as the vertical axis, to fit the curve function equation and take the EC50 value. The results are as follows Figure 5 As shown in Table 4:

[0097] Table 4: EC50 values ​​of the ceramide group and the experimental group

[0098] Group EC50 value Cetirizine group 16.4 ng / mL Caninized 3G12 group 3.6 ng / mL

[0099] From Table 4, Figure 5 It can be seen that the affinity of 3G12 antibody to canine IL-31 antigen is improved after caninization, and its affinity is even higher than that of ceramide. It can be seen that the effect of the caninized 3G12 antibody constructed by the present invention is better than that of ceramide.

[0100] Example 10. In vivo efficacy study of caninized 3G12 antibody

[0101] The 2 mg / kg injection dose of ceramide was used as the positive control group (4 statistical experimental dogs), PBS was used as the negative control group (4 statistical experimental dogs), and different injection doses of caninized 3G12 antibody (0.125 mg / kg, 0.5 mg / kg and 2 mg / kg) were used as the experimental group (4 statistical experimental dogs). The above pruritus model was used for in vivo pharmacodynamics study. On D0 day, different antibodies were injected subcutaneously into the dogs (the negative control group was injected with an equal volume of PBS); on D1, D7, D14 and D28, recombinant protein IL-31 was injected intravenously into the forelimbs. The baseline score of pruritus was recorded for 30 minutes before injection. After 20 minutes of injection, the pruritus score was recorded for 2 hours. According to the pruritus score, the data of different groups were processed with Prism 8.0 software. If P<0.05, it indicates that there is a significant difference between the antibody injection group and the PBS injection group. If P>0.05, it indicates that there is no significant difference between the antibody injection group and the PBS injection group.

[0102] like Figure 6 As shown, under the same conditions, the caninized 3G12 antibody has an inhibitory effect on IL-31-mediated pruritus that is no less than that of chloramphenicol, and there is no significant difference between 0.5 mg / kg of caninized 3G12 antibody and 2 mg / kg of chloramphenicol.

[0103] Example 11. Verification of the effect of caninized 3G12 antibody mutation sequence

[0104] 1. Mutated antibodies:

[0105] In order to further improve the performance of 3G12 antibody, such as the signal pathway blocking effect, half-life and affinity of the antibody, the inventors mutated some sites in the CDR region sequence of the antibody and tested the effect of the mutated antibody. Based on informatics analysis and protein spatial structure simulation, the current mature molecular biology methods for antibody modification were used to mutate several sites in the caninized 3G12 sequence. We selected the following sites for mutation, as shown in Table 5:

[0106] amino acid D at position 6 in the heavy chain CDR1 sequence GYNFTDNS;

[0107] amino acids E and E at positions 4 and 7 in the heavy chain CDR2 sequence INTETGEP;

[0108] amino acids D and E at positions 4 and 6 in the heavy chain CDR3 sequence AKGDYEY;

[0109] amino acid E at position 4 in the light chain CDR1 sequence ESVENYGISF;

[0110] amino acid E at position 5 in the light chain CDR3 sequence QQSKEVPWT;

[0111] Table 5: Caninized 3G12 antibody mutation information

[0112]

[0113] 2. Effect verification

[0114] The above-mentioned mutated antibodies were verified for their effects, specifically: the above-mentioned antibodies were transiently expressed by CHO cells, and the supernatant was collected for purification. The above purified samples (mutated antibodies), unmutated caninized antibodies 3G12 and ceramide were subjected to phosphorylation inhibition tests and affinity tests of the IL-31 signaling pathway. The affinity experimental steps refer to Example 9; different antibody concentrations (15.6ng / mL, 62.5ng / mL, 250ng / mL, 1000ng / mL, 4000ng / mL, 16000ng / mL and 64000ng / mL) were set for the phosphorylation inhibition experiment, and the specific steps refer to Example 4.

[0115] The results of affinity test and phosphorylation inhibition experiment are shown in Table 6 and Figure 7 , 8 As shown, the antibody with the strongest affinity and phosphorylation inhibition ability is 3G12-m4, followed by 3G12-m6.

[0116] Table 6: Affinity and phosphorylation inhibition results of different antibodies

[0117] Antibody EC50(ng / mL) IC50(ng / mL) Cetirizine 17.13 1235 3G12 4.14 706 3G12-m1 9.28 1070 3G12-m2 7.23 1111 3G12-m3 6.50 1066 3G12-m4 1.69 406 3G12-m5 9.04 1167 3G12-m6 2.91 497 3G12-m7 15.51 1191 3G12-m8 13.89 1239 3G12-m9 27.56 1832 3G12-m10 34.49 1943 3G12-m11 45.57 2417 3G12-m12 28.36 1964 3G12-m13 76.56 2973 3G12-m14 93.67 3526 3G12-m15 27.10 1990 3G12-m16 13.11 1197 3G12-m17 37.02 2051

[0118] From the above results, it can be seen that the present invention has carried out mutation modification on the caninized 3G12 antibody, and some of the mutated antibodies have improved the affinity for IL-31 protein and the ability to inhibit STAT phosphorylation. Although the ability of some of the mutated antibodies has been reduced, they still retain the affinity for canine IL-31 antigen and the ability to inhibit STAT phosphorylation, so that they retain the function of anti-canine IL-31 antibodies and can still be used as anti-canine IL-31 antibodies.

[0119] The partially mutated antibody of the present invention is to exchange aspartic acid (D) with glutamic acid (E). This is because the molecular structures and properties of aspartic acid (D) and glutamic acid (E) are similar, and the replaced protein or polypeptide can maintain the structure and properties of the original protein or polypeptide. Therefore, the 3G12 mutant antibody in which part of aspartic acid (D) and glutamic acid (E) are exchanged in the present invention has the function of the original antibody. At the same time, the performance of this mutated antibody is also verified by the above experiment, and it can still be used as an antibody against canine IL-31. In addition, in addition to the exchange of amino acids with similar properties, in order to improve the performance of the antibody, some other mutations have been tried, and the antibodies after these mutations still retain the performance of the unmutated antibody to varying degrees.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An anti-canine interleukin-31 antibody or an antigen-binding portion thereof, characterized in that The amino acid sequence of the heavy chain CDR1 of the antibody or its antigen-binding portion is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 3; the amino acid sequence of the light chain CDR1 of the antibody is shown in SEQ ID NO: 4, the amino acid sequence of the light chain CDR2 is AAS, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:

5.

2. The antibody or antigen-binding portion thereof according to claim 1, wherein The nucleotide sequence of the heavy chain CDR1 of the antibody is shown in SEQ ID NO: 8, the nucleotide sequence of the heavy chain CDR2 is shown in SEQ ID NO: 9, and the nucleotide sequence of the heavy chain CDR3 is shown in SEQ ID NO: 10; the nucleotide sequence of the light chain CDR1 of the antibody is shown in SEQ ID NO: 11, the nucleotide sequence of the light chain CDR2 is GCTGCATCC, and the nucleotide sequence of the light chain CDR3 is shown in SEQ ID NO:

12.

3. The antibody or antigen-binding portion thereof according to claim 1, wherein The heavy chain amino acid sequence of the antibody is shown in SEQ ID NO: 6; the light chain amino acid sequence of the antibody is shown in SEQ ID NO:

7.

4. The antibody or antigen-binding portion thereof according to claim 1, wherein The heavy chain nucleotide sequence of the antibody is shown in SEQ ID NO: 13; the light chain nucleotide sequence of the antibody is shown in SEQ ID NO:

14.

5. The antibody or antigen-binding portion thereof according to any one of claims 1 to 4, characterized in that Also included are canine IgGA heavy chain frameworks, and canine Igκ light chain constant region frameworks.

6. The antibody or antigen-binding portion thereof according to claim 5, wherein The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 19; the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:

20.

7. The antibody or antigen-binding portion thereof according to claim 1, wherein The antibody or antigen-binding portion thereof is a mutant of the interleukin-31 antibody according to claim 1, wherein the mutant is any one of the following or a combination of any one of the following heavy chains and light chains: 1) mutating the amino acid D at position 6 in the heavy chain CDR1 sequence GYNFTDNS as shown in SEQ ID NO: 1 to amino acid E, amino acid N or amino acid V; 2) mutating the amino acid E at position 4 in the heavy chain CDR2 sequence INTETGEP as shown in SEQ ID NO: 2 to amino acid D; 3) mutating the amino acid E at position 7 in the heavy chain CDR2 sequence INTETGEP as shown in SEQ ID NO: 2 to amino acid D, amino acid Q, amino acid L or amino acid T; 4) mutating the amino acid D at position 4 in the heavy chain CDR3 sequence AKGDYEY as shown in SEQ ID NO: 3 to amino acid E, amino acid K or amino acid I; 5) mutating the amino acid E at position 6 in the heavy chain CDR3 sequence AKGDYEY as shown in SEQ ID NO: 3 to amino acid D, amino acid N or amino acid Q; 6) mutating the amino acid E at position 4 in the light chain CDR1 sequence ESVENYGISF as shown in SEQ ID NO: 4 to amino acid D; 7) The amino acid E at position 5 in the light chain CDR3 sequence QQSKEVPWT as shown in SEQ ID NO: 5 is mutated to amino acid D.

8. A kit for detecting canine interleukin-31, characterized in that: The kit comprises the anti-canine interleukin-31 antibody or antigen-binding portion thereof according to any one of claims 1 to 7.

9. Use of the anti-canine interleukin-31 antibody or antigen-binding portion thereof according to any one of claims 1 to 7 or the kit for detecting canine interleukin-31 according to claim 8 in the preparation of a pharmaceutical composition for treating, preventing and / or alleviating diseases related to IL-31.

10. The use according to claim 9, characterized in that The IL-31-related disease is selected from pruritus, allergy, nasal polyps or asthma; Preferably, the pruritus is selected from atopic dermatitis, eczema, psoriasis, scleroderma and pruritus; Preferably, the allergy is selected from allergic rhinitis, allergic dermatitis, summer eczema, urticaria, airway inflammatory diseases, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease and autoimmune induced inflammatory processes.

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