Antibodies against canine il-31 and uses thereof

Abstract

The application relates to an antibody or an antigen binding part thereof against canine IL-31 and application thereof, and belongs to the technical field of antibody preparation. The application relates to an anti-canine interleukin-31 antibody or an antigen binding part thereof, an amino acid coding the antibody or the antigen binding part thereof, a polynucleotide, a vector containing the polynucleotide, a host cell containing the polynucleotide or the vector, 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 can be specifically combined with a canine IL-31 antigen, and can effectively relieve symptoms such as pruritus, allergy, nasal polyps or asthma. The mutated anti-canine IL-31 antibody also has the corresponding antigen canine IL-31 combination function, and can also treat IL-31 related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibody preparation, and particularly relates to an antibody against canine IL-31 and application thereof. BACKGROUND

[0002] Atopic dermatitis (AD) is an inflammatory and pruritic allergic skin disease with heredity. The severe pruritus brings more inconvenience to the life of pets and owners, especially the secondary symptoms such as alopecia and erythema, which particularly arouse the concern of pet owners. Globally, about 450 million dogs suffer from chronic or lifelong such 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 skin disease pruritus. 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 has been cloned by scientists, and there have been relatively many studies on IL-31. According to research, IL-31 combines with the complex receptor composed of IL-31RA (interleukin 31 receptor A, IL31RA) and OSMR (Recombinant Oncostatin M Receptor, OSMR), and after the activation of the receptor, the STAT (signal transducer and activator of transcription, STAT) phosphorylation of the JAK (janus kinase) receptor is caused, resulting in the up-regulation of target genes and causing pruritus behavior.

[0004] More and more researchers have been involved in the research of treating atopic dermatitis by blocking the IL-31 pathway. Lokivetmab (Cytopoint, Zoetis) is the only canine-derived monoclonal antibody currently approved for the treatment of related dermatitis in dogs, and its target is canine IL-31, which is injected once every four to eight weeks. According to clinical trials, Lokivetmab can significantly reduce the pruritus symptoms of dogs and significantly improve the skin condition of dogs, which shows that the IL-31 monoclonal antibody is effective in treating atopic dermatitis. Although some products in the prior art show certain effects in treating animal atopic dermatitis, the high cost of these products limits the treatment of animal atopic dermatitis. Therefore, developing more canine IL-31 antibodies has become a problem to be solved by those skilled in the art. SUMMARY

[0005] The present application aims to overcome the deficiencies of the prior art and provide an anti-dog interleukin-31 antibody and application thereof. The IL-31 monoclonal antibody developed by the present application can be used for treating atopic dermatitis, and can also be used for treating diseases such as allergy, nasal polyps and asthma.

[0006] To achieve the above-mentioned object, the technical scheme adopted by the present application is as follows:

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

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

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

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

[0011] As a preferred embodiment of the first aspect, it further comprises a dog IgGA dog heavy chain framework structure and a dog Ig kappa 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 in SEQ ID NO: 19; and the amino acid sequence of the light chain of the antibody is shown in 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 anti-IL-31 antibody, which is any one of the following or a combination of the heavy chain and the light chain of the following:

[0014] 1) mutating the 6th amino acid D in the heavy chain CDR1 sequence GYNFTDNS having the amino acid sequence of SEQ ID NO: 1 into an amino acid E, an amino acid N or an amino acid V;

[0015] 2) mutating the 4th amino acid E in the heavy chain CDR2 sequence INTETGEP having the amino acid sequence of SEQ ID NO: 2 into an amino acid D;

[0016] 3) mutating the 7th amino acid E in the heavy chain CDR2 sequence INTETGEP having the amino acid sequence of SEQ ID NO: 2 into an amino acid D, an amino acid Q, an amino acid L or an amino acid T;

[0017] 4) mutating the 4th amino acid D in the heavy chain CDR3 sequence AKGDYEY having the amino acid sequence of SEQ ID NO: 3 into an amino acid E, an amino acid K or an amino acid I;

[0018] 5) mutating the 6th amino acid E in the heavy chain CDR3 sequence AKGDYEY having the amino acid sequence of SEQ ID NO: 3 into an amino acid D, an amino acid N or an amino acid Q;

[0019] 6) mutating the 4th amino acid E in the light chain CDR1 sequence ESVENYGISF having the amino acid sequence of SEQ ID NO: 4 into an amino acid D;

[0020] 7) mutating the 5th amino acid E in the light chain CDR3 sequence QQSKEVPWT having the amino acid sequence of SEQ ID NO: 5 into an amino acid D.

[0021] In a second aspect, the present application also provides a kit for detecting canine IL-31, which comprises the anti-canine IL-31 antibody or antigen binding portion thereof according to the first aspect.

[0022] In a third aspect, the present application also provides use of the anti-canine IL-31 antibody or antigen binding portion thereof according to the first aspect or the kit for detecting canine IL-31 according to the second aspect in the preparation of a pharmaceutical composition for treating, preventing and / or alleviating a disease associated with IL-31.

[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 disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease and autoimmune-induced inflammatory processes.

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

[0025] The anti-canine IL-31 antibody disclosed in the present application can specifically bind to canine IL-31 and has an affinity not weaker than that of the product Selumetinib. It has been verified that it has good biological activity in cells and animals and can effectively relieve the related symptoms of the model animals in a canine pruritus model. These related diseases include pruritus, allergy, nasal polyps or asthma; pruritus includes atopic dermatitis, eczema, psoriasis, scleroderma and pruritus; allergy includes allergic dermatitis, summer eczema, urticaria, airway inflammatory disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease and autoimmune-induced inflammatory processes. Meanwhile, the antibody has no obvious immunogenicity after canineization modification. Meanwhile, the mutant anti-canine IL-31 antibody also has the corresponding binding function to the antigen canine IL-31 and can also treat IL-31 related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Electrophoretogram of recombinant protein canine IL-31 antigen;

[0027] Figure 2 Schematic diagram of the results of the IL-31 signal pathway phosphorylation inhibition test of different antibodies in Example 4;

[0028] Figure 3 Schematic diagram of the results of the injection of IL-31 protein concentration and pruritus score in the establishment of a pruritus model in Example 5;

[0029] Figure 4 Schematic diagram of the results of the pharmacodynamic detection of murine 3G12 antibody in experimental dogs in Example 6;

[0030] Figure 5 Schematic diagram of the results of the affinity comparison of Selumetinib and canineized 3G12 antibody in Example 9;

[0031] Figure 6 Schematic diagram of the results of the pharmacodynamic detection of canineized 3G12 antibody in experimental dogs in Example 10;

[0032] Figure 7 Schematic diagram of the results of the affinity test of the mutant canineized 3G12 antibody in Example 11;

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

[0034] In order to better illustrate the purposes, technical solutions and advantages of the present application, the present application will be further described below in combination with specific examples.

[0035] Example 1. Preparation of antigen

[0036] The canine IL-31 amino acid sequence (NP_001159386.1) retrieved from NCBI was deduced and a gene sequence was synthesized, a prokaryotic expression system pET30a(+) was used to construct a vector, and a recombinant protein canine IL-31 was expressed. After the recombinant protein was expressed and purified, it was verified that the molecular size (predicted 17.79 kDa, see Figure 1 ) and other information were correct, and then it was used for mouse immunization.

[0037] Example 2. Immunization of mice

[0038] The immunized mice were Bal b / c (purchased from: Southern Medical University Experimental Animal Management Center), and the mouse age was 6 weeks. The above-mentioned purified recombinant canine IL-31 was used as an immunogen to immunize mice, and 20 mice were immunized in one batch, and 3 immunizations were performed, and the immunization process was as follows:

[0039] 1. The first immunization was performed according to 50 μg of immunogen per mouse;

[0040] 2. The second immunization was performed after three weeks, and the second immunization was performed according to 25 μg of immunogen per mouse;

[0041] 3. The third immunization was performed after two weeks, and the third immunization was performed according to 25 μg of immunogen per mouse;

[0042] 4. One week after the third immunization, blood was collected from the eyelids, and the titer of the immunized mice was detected 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 sampling: under sterile conditions, the spleen was taken out, the membranous tissue was peeled off, and the like, and was placed in incomplete culture medium. The incomplete culture medium was injected into the spleen by a syringe to separate the spleen cells. The remaining spleen tissue was ground by a cell screen, and the cells were collected and counted.

[0046] 1.2 SP2 / 0 cell collection: the SP2 / 0 cells were round and transparent, and the cells were collected in a 50 ml centrifuge tube and counted.

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

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

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

[0050] 2. Indirect ELisa screening positive wells

[0051] 2.1 Plate coating: dilute the dog IL-31 fusion protein to 2ug / ml coating solution with 1x coating buffer solution, add the coating solution to each well of the detection plate, 50ul per well, and react for at least 12 hours at 2-8°C.

[0052] 2.2 Blocking: wash the coated plate with TBST 4 times, after removing the remaining liquid, add blocking solution, 100ul per well, and incubate at 37°C for 2 hours; wash the plate with TBST 4 times and reserve.

[0053] 2.3 Sample incubation: take the supernatant of the cell culture plate after fusion for detection, use the serum of the immunized mouse as positive control, use cell culture medium as negative control, and use PBS as blank control, 100ul per well, incubate at 37°C for 1h.

[0054] 2.4 Secondary antibody incubation: wash the detection plate with TBST 4 times, dilute the goat anti-mouse secondary antibody (Servi, HRP*Goat Anti-Mouse IgG(H+L)) with blocking solution 1:10000, 100ul per well, incubate at 37°C for 1h.

[0055] 2.5 Color development and termination: wash the detection plate with TBST 4 times, after removing the remaining liquid, add color developing solution, 100ul per well, and stand at room temperature for 10min; add 50ul of termination solution to all wells of the ELISA reaction plate.

[0056] 2.6 Reading: read the absorbance value at 450nm with a microplate reader.

[0057] 2.7 Select the well with higher detection value as suspected positive well, and repeat the detection next day. The well with still higher positive value is regarded as positive well. After the above detection, 13 positive wells are screened, including 1G2, 3G12, 4G4, 5D4, 6F3, 6F5, 7F9, 8A2, 9D8, 12B3, 15A4, 16B6 and 19G11.

[0058] 3. Monoclonalization

[0059] 3.1 First round of monoclonalization: collect the cells in the above positive wells, dilute the cells to single cell / 200 μL using monoclonal cell culture solution, and add the cell suspension to a 96-well cell culture plate. After 8 days of culture, detect according to the above indirect ELisa method.

[0060] 3.2 Second round of monoclonalization: analyze the ELisa detection results of the first round of subcloning. If the positive well rate is 100%, it is determined that the cell purity is sufficient, otherwise, select the positive well for the next round of monoclonalization. The operation of the second round of monoclonalization is the same as that of the first round of monoclonalization.

[0061] 3.3 After the above positive well cells are subjected to multiple rounds of monoclonalization, the hybridoma cell purity reaches 100%, and the monoclonalization is terminated.

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

[0063] Example 4. Inhibition test of IL-31 signal pathway phosphorylation

[0064] IL-31 regulates the expression of related genes through a signal pathway, thereby affecting the itching behavior of dogs, and neutralizing IL-31 can block the itching behavior. It is known that the complex receptor composed of IL-31 receptor A (IL-31RA) and oncostatin M receptor (OSMR) mediates the Janus kinase phosphorylation and activates the STAT signal cascade after binding with IL-31. Therefore, the present application establishes a cell biology activity determination method based on the detection of the STAT phosphorylation level to evaluate the neutralization effect of the screened antibody on IL-31. The above 1G2, 3G12, 4G4, 5D4, 6F3, 6F5, 7F9, 8A2, 9D8, 12B3, 15A4, 16B6 and 19G11 antibodies are selected for the phosphorylation inhibition test of the signal pathway. The specific steps are as follows:

[0065] 1. Cell preparation: Dog macrophage-like DH82 cells (Chinese Academy of Sciences Cell Bank, Catalog No. TCO3) were cultured to good condition at 37℃, 5% CO2 according to the instructions;

[0066] 2. Dog IFN-γ induction: The above cells were inoculated into a 96-well plate at 10w / well, and were cultured with MEM complete medium (10% FBS, 2mmol / L GlutaMax, 1mmol / L sodium pyruvate) while adding 10ng / mL of dog IFN-γ for 24 hours;

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

[0068] 4. IL-31 treatment: The above medium was removed, and different antibody groups were added with mixed culture solution (different hybridoma cell supernatant + 1μg / mL IL-31 recombinant protein) respectively, and were cultured for 1h;

[0069] 5. Cell lysis: The medium was removed, the wells were washed twice with PBS, and the cells were lysed with cell lysis solution.

[0070] 6. The lysed sample was detected according to the STAT3 phosphorylation detection kit (PathScan(R) Phospho-Stat3 (Tyr705) Sandwich ELISA Kit, CST), and the detection data were collected.

[0071] The results are shown in Figure 2 It can be seen that the 3G12 antibody has an inhibitory effect on the STAT3 phosphorylation of DH82 cells, and the other antibodies have little inhibitory effect. Based on this, the 3G12 antibody is used as the subsequent experimental antibody.

[0072] Example 5. Verification of screening of monoclonal antibody 3G12 by animal model

[0073] 1. Establishment of an itch model

[0074] The model is established according to the reference "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 were divided into 4 groups, and the average weight difference between different treatment groups was controlled within 20%, and the groups were as follows:

[0075] Table 1: Experimental grouping

[0076] Group Number Injection dose of recombinant protein IL-31 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 shaver removes the fur at the forelimb vein, and enters the video room 1 h before the observation starts, and starts to adapt. 20 min before the start of observation, start intravenous injection of prepared recombinant protein IL-31 injection, the injection dose is carried out according to the above table 1; 20 min after injection, start continuous video recording for 2 h; the camera vertically observes the dog below. After the video recording is finished, the different groups of dogs are normally fed under the same conditions, and are ready for the next experiment; if necessary, the experiment can be repeated after 1 month. According to the literature, the observer selects a discrete time period (1 min) for observation, and the observer judges "yes" or "no" according to the itching behavior (licking / biting the paw, lateral abdomen, anal region, scratching the lateral abdomen or neck, digging, shaking the head, and scratching the buttocks on the cage floor) according to the above time, and records it in the table. The cumulative number of "yes" in each observation time provides the itching score PSI.

[0078] In the modeling process of the present application, individual non-responding dogs are removed, and at least 4 dogs are counted in each group. According to the data, at a dose of 3 μg / kg, the itching score changes of different dogs in the group are relatively large; at a dose of 6 μg / kg, the itching score changes of different dogs in the group are relatively small, and the itching score is not significantly improved when the dose is further increased to 9 μg / kg. Therefore, the present application uses a dose of 6 μg / kg to establish an itching model for efficacy study. Figure 3

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

[0080] 1. The positive control group (4 experimental dogs) is injected with a dose of 2 mg / kg of seratrodast, the negative control group (4 experimental dogs) is injected with PBS, and the experimental group (4 experimental dogs) is injected with different doses of mouse 3G12 antibody (0.125 mg / kg, 0.5 mg / kg and 2 mg / kg). The above itching model is used for in vivo pharmacodynamic study. On D0, different concentrations of antibody 3G12 are injected subcutaneously into the dogs (the negative control group is injected with the same volume of PBS); on D1 and D7, recombinant protein IL-31 is injected into the forelimb vein, 30 min before injection, the video is recorded to count the baseline score of itching, and 20 min after injection, the video is recorded for 2 h to count the itching score. According to the itching score, Prism 8.0 software is used to process the data of different groups, 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 embodiment is a murine antibody, at D7, the efficacy of the murine 3G12 antibody has begun to disappear due to the immune response of the dog and the degradation of the enzyme, so this embodiment only counts the experimental results of the first 7 days. Figure 4 The results show that at D1, the 3G12 antibody at a dose of 0.125 mg / kg has already achieved a good enough effect, which indicates that the 3G12 antibody has a good enough ability to neutralize IL-31.

[0082] Example 7. Gene sequencing

[0083] The purified 3G12 hybridoma cell strain was expanded, and 3x10 6 cells were collected, the mRNA was extracted after cell lysis and reverse transcribed into cDNA, the antibody gene was amplified and cloned into the pcDNA3.4 vector, and the variable region nucleic acid sequence of the antibody was obtained after Sanger sequencing, and the 3G12 antibody amino acid sequence shown in Table 2 was obtained through bioinformatics analysis:

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

[0085]

[0086]

[0087] Example 8. Caninization of antibody

[0088] After bioinformatics software analysis and IMGT analysis to predict the functional regions of the antibody and laboratory expression, etc., canine IgGA (accession number: AAL35301.1) was finally selected as the canine heavy chain framework structure. Through 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 replaced or spliced with the variable region. According to Table 3, the part of the sequence SEQ ID NO: 15 with a horizontal line is the canine heavy chain variable region sequence, which 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, which is spliced with the VL amino acid of the 3G12 antibody, so as to form a new combination of chimeric antibody light chain and heavy chain, and obtain the canine 3G12 antibody. The canine 3G12 antibody gene sequence is inserted into the pcDNA3.4 vector through the enzyme cutting site by gene recombination, and after the vector is verified to be successfully constructed, it is transiently transfected into CHO cells for expression. The culture supernatant is collected, the antibody is purified, and is used for subsequent verification test.

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

[0090]

[0091]

[0092] Example 9. Affinity comparison test

[0093] The affinity test adopts an indirect ELisa method, and the specific procedure is as follows:

[0094] 1. Settings: the control group is the cetirizine group, the experimental group is the dog-sourced 3G12 group of the application, and the antibody concentration gradient is set as: 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: dog IL-31 fusion protein 50 ng / well is coated, the primary antibody is dog-sourced 3G12 antibody, the enzyme-labeled secondary antibody is Dog IgG antibody (HRP) (GeneTex), and other operations are the same as the above indirect ELisa operation, and the detection value is read.

[0096] 3. Data processing: Prism 8.0 processes the above ELisa detection value, the above cetirizine and 3G12 antibody concentration gradient values are taken as the abscissa, the OD value is taken as the ordinate, the curve function equation is fitted, the EC50 value is taken, and the results are shown in Figure 5 and Table 4:

[0097] Table 4: EC50 value results of the cetirizine group and the experimental group

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

[0099] It can be known from Table 4, Figure 5 that the affinity of the 3G12 antibody to the dog IL-31 antigen is improved after the dog-sourcing modification, and the affinity is also higher than that of cetirizine, so the effect of the dog-sourced 3G12 antibody constructed in the application is better than that of cetirizine.

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

[0101] The in vivo pharmacodynamics study was performed using the above-mentioned itch model. The positive control group (4 statistical experimental dogs) was injected with cetirizine at a dose of 2 mg / kg, the negative control group (4 statistical experimental dogs) was injected with PBS, and the experimental group (4 statistical experimental dogs) was injected with canineized 3G12 antibody at different doses (0.125 mg / kg, 0.5 mg / kg and 2 mg / kg). On D0, different antibodies were injected subcutaneously into the dogs (the negative control group was injected with PBS of the same volume); on D1, D7, D14 and D28, recombinant protein IL-31 was injected intravenously into the forelimbs. The baseline score of itch was recorded 30 min before injection, and the score of itch was recorded 2 h after injection. According to the score of itch, Prism 8.0 software was used to process the data of different groups. If P<0.05, it indicated that there was a significant difference between the antibody injection group and the PBS injection group. If P>0.05, it indicated that there was no significant difference between the antibody injection group and the PBS injection group.

[0102] As shown in Figure 6 , under the same conditions, the canineized 3G12 antibody had an inhibitory effect on IL-31-mediated itch that was not inferior to cetirizine. There was no significant difference between 0.5 mg / kg canineized 3G12 antibody and 2 mg / kg cetirizine.

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

[0104] 1. Mutant antibody:

[0105] In order to further improve the performance of 3G12 antibody, such as the signal pathway blocking effect of the antibody, the half-life and the affinity, the inventors mutated some sites in the CDR region sequence of the antibody and detected the effect of the mutated antibody. According to information analysis and protein space structure simulation, using the mature antibody modification molecular biology means at present, some sites in the canineized 3G12 sequence were mutated. We selected the following sites for mutation, as shown in Table 5:

[0106] The 6th amino acid D in the heavy chain CDR1 sequence GYNFTDNS;

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

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

[0109] The 4th amino acid E in the light chain CDR1 sequence ESVENYGISF;

[0110] The 5th amino acid E in the light chain CDR3 sequence QQSKEVPWT;

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

[0112]

[0113] 2. Effect verification

[0114] The above mutated antibodies were subjected to effect verification, specifically: the above antibodies were expressed by CHO cells, and the supernatant was collected and purified. The above purified samples (mutated antibodies), the unmutated caninized antibody 3G12 and cetirizine were subjected to IL-31 signal pathway phosphorylation inhibition test and affinity test. The affinity experiment step was referred to Example 9; the phosphorylation inhibition experiment was set to different antibody concentrations (15.6 ng / mL, 62.5 ng / mL, 250 ng / mL, 1000 ng / mL, 4000 ng / mL, 16000 ng / mL and 64000 ng / mL), and the specific steps were referred to Example 4.

[0115] The affinity test and phosphorylation inhibition experiment results are shown in Table 6 and Figure 7 、 8 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) Setocin 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 caninized 3G12 antibody is subjected to mutation modification in the application, and the affinity of some of the mutated antibodies to IL-31 protein and the STAT phosphorylation inhibition ability are improved, the ability of some of the mutated antibodies is reduced, but the affinity and STAT phosphorylation inhibition ability to canine IL-31 antigen are still retained, so that the function of the anti-canine IL-31 antibody is retained, and the antibody can still be used as an anti-canine IL-31 antibody.

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

[0120] Finally, it should be noted that the above examples are merely intended to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

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

5.

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

12.

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

7.

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

14.

5. The antibody of any one of claims 1-4, wherein The antibody further comprises a canine IgGA canine heavy chain framework structure, and a canine Ig kappa light chain constant region framework structure.

6. The antibody of claim 5, wherein The amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO: 19; the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO:

20.

7. A kit for detecting canine interleukin-31, characterized by, The kit comprises the anti-canine interleukin-31 antibody of any one of claims 1-6.

8. Use of the anti-canine interleukin-31 antibody of claim 1 in the manufacture of a pharmaceutical composition for treating canine pruritus; the canine pruritus is selected from atopic dermatitis, eczema, psoriasis, or scleroderma.

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