A monoclonal antibody against canine IL-31 and its application

Abstract

The present invention discloses a monoclonal antibody against canine IL-31 and its application, belonging to the field of biotechnology. The monoclonal antibody against canine IL-31 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises complementary determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementary determining regions CDR-L1, CDR-L2, and CDR-L3; wherein, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are respectively shown in SEQ ID NO:10-12; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are respectively shown in SEQ ID NO:17-19. The monoclonal antibody against canine IL-31 of the present invention has good blocking activity and can inhibit itching symptoms. Therefore, it has good application prospects in the preparation of drugs for preventing and / or treating animal pruritus.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a monoclonal antibody against canine IL-31 and application thereof. Background Art

[0002] Interleukin-31 (IL-31), also known as IL-31, is an inflammatory cytokine discovered in 2004. It plays a key role in skin itching in humans and animals. IL-31 belongs to the IL-6 family, has four antiparallel α helices, and is mainly produced by helper T cells type 2 (Th2). IL-31 molecules bind to heterodimeric receptors composed of IL-31 receptor A (IL31RA) and oncostatin M receptor β (OSMRβ), activating downstream JAK-STAT, MAPK, PI3K and other signal transduction pathways, transmitting itch signals to the central nervous system, activating adjacent nerve branches through axon reflex mechanisms, and releasing neuropeptides into the skin, causing inflammation and itching. Studies have shown that IL-31 can stimulate canine dorsal root ganglia, causing a strong itchy response, and the circulating concentration of IL-31 in the blood of dogs with atopic dermatitis is significantly increased compared with healthy dogs. In addition, IL-31 molecules also play an important role in diseases such as lung inflammation, inflammatory bowel disease, allergic rhinitis and mastocytosis.

[0003] At present, the number of pets raised is increasing, and the demand for pet disease research is also increasing. However, domestic research in the pet field is relatively small and backward, and cannot meet the many problems faced by the pet industry. Itching is one of the common symptoms of canine skin diseases and is a typical clinical symptom of atopic dermatitis and allergic dermatitis. In view of the fact that canine IL-31 molecules are involved in promoting symptoms such as skin itching and allergies, it endangers the health of pet skin. However, there are few studies on canine IL-31 in China and related therapeutic products are scarce. In addition, monoclonal antibody drugs have the characteristics of strong specificity and sensitive response. They are increasingly used in clinical practice and can specifically target a certain target.

[0004] Therefore, it is of great significance to develop a monoclonal antibody with good blocking activity that can block the subsequent signaling pathway of canine IL-31 molecules and achieve the purpose of inhibiting itching. Summary of the invention

[0005] The object of the present invention is to provide a monoclonal antibody against canine IL-31 and its application. The monoclonal antibody against canine IL-31 in the present invention has good blocking activity. The monoclonal antibody specifically binds to canine IL-31 and blocks its binding to canine IL-31 receptor A. Therefore, it has good application prospects in the preparation of drugs for preventing and / or treating animal pruritus.

[0006] In the first aspect, the present invention provides an anti-canine IL-31 monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementary determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprising complementary determining regions CDR-L1, CDR-L2, and CDR-L3; wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are respectively as shown in SEQ ID NOs: 10-12; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are respectively as shown in SEQ ID NOs: 17-19.

[0007] In some embodiments, the heavy chain variable region also includes framework regions HC-FR1, HC-FR2, HC-FR3, and HC-FR4, and the light chain variable region also includes framework regions LC-FR1, LC-FR2, LC-FR3, and LC-FR4; wherein the amino acid sequences of HC-FR1, HC-FR2, HC-FR3, and HC-FR4 are shown in SEQ ID NOs: 13-16, respectively; the amino acid sequences of LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are shown in SEQ ID NOs: 20-23, respectively.

[0008] In some embodiments, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 26, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 27.

[0009] In the present invention, the inventors further studied and found that after the monoclonal antibody or its antigen-binding fragment against canine IL-31 is caninized, the caninized monoclonal antibody obtained has better blocking activity.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding any one of the above-mentioned anti-canine IL-31 monoclonal antibodies or antigen-binding fragments thereof.

[0011] In some preferred embodiments, the nucleic acid molecules include a nucleic acid molecule encoding a heavy chain variable region whose nucleotide sequence is shown in SEQ ID NO: 24 and a nucleic acid molecule encoding a light chain variable region whose nucleotide sequence is shown in SEQ ID NO: 25.

[0012] The nucleic acid molecules provided by the present invention can usually be obtained by PCR amplification or artificial synthesis.

[0013] In a third aspect, the present invention provides a recombinant vector comprising the above nucleic acid molecule.

[0014] The recombinant vectors in the present invention include cloning vectors and expression vectors. The cloning vectors are used to replicate related sequences, and the expression vectors are used to express related genes.

[0015] In a fourth aspect, the present invention provides a recombinant cell comprising the above nucleic acid molecule or the above recombinant vector.

[0016] In some embodiments, the method for preparing a recombinant cell comprises the step of transforming the above-mentioned recombinant vector into an expression host cell.

[0017] In a fifth aspect, the present invention provides a method for detecting the blocking activity of any of the above-mentioned anti-canine IL-31 monoclonal antibodies or their antigen-binding fragments, comprising the following steps: S1, constructing a pSTAT-luc vector and a pcDNA3.1-cILRA vector respectively; S2, transfecting the pSTAT-luc vector and the pcDNA3.1-cILRA vector into HeLa cells; S3, mixing the anti-canine IL-31 monoclonal antibody or its antigen-binding fragment with the IL-31 protein for incubation, then adding them to HeLa cells for incubation, and finally detecting fluorescein to obtain the blocking activity of the monoclonal antibody or its antigen-binding fragment.

[0018] In some embodiments, the pSTAT-luc vector is obtained by inserting the STAT nucleotide sequence (SEQ ID NO: 3) into the multiple cloning site region of the reporter gene vector pGL3-promotor; the pcDNA3.1-cILRA vector is obtained by replacing the sequence between the Hind Ⅲ and Xho Ⅰ sites in pcDNA3.1 with the IL-31RA nucleotide sequence (SEQ ID NO: 4).

[0019] In a sixth aspect, the present invention provides use of any of the above-mentioned anti-canine IL-31 monoclonal antibodies or antigen-binding fragments thereof, the above-mentioned nucleic acid molecules, the above-mentioned recombinant vectors or the above-mentioned recombinant cells in the preparation of a drug for preventing and / or treating animal pruritus.

[0020] In some embodiments, the animal comprises a dog.

[0021] In the present invention, the animal may be any common animal in the art, such as a dog, a cat, or a dog. In the present invention, the animal is preferably a dog.

[0022] In a seventh aspect, the present invention provides a pharmaceutical composition comprising any one of the above-mentioned anti-canine IL-31 monoclonal antibodies or antigen-binding fragments thereof.

[0023] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0024] The beneficial effect of the present invention is that, different from the prior art, the present invention provides a monoclonal antibody against canine IL-31 with good blocking activity, which specifically binds to canine IL-31 and blocks its binding to canine IL-31 receptor A, thereby blocking the canine IL-31 downstream signaling pathway and inhibiting itching symptoms. Therefore, the present invention has good application prospects in the preparation of drugs for preventing and / or treating animal pruritus. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The nucleic acid electrophoresis diagram of the canine IL-31 gene in Example 1, wherein M: Maker; 1: negative control; 2: target fragment;

[0026] Figure 2 The SDS-PAGE and Western blot results of the canine IL-31 recombinant protein in Example 1 are shown, wherein M: Marker; 1: empty control; 2: 16°C, 0.5mmol / L IPTG supernatant; 3: 16°C, 1.0mmol / L IPTG supernatant; 4: 37°C, 0.5mmol / L IPTG supernatant; 5: 37°C, 1.0mmol / L IPTG supernatant; 6: 16°C, 0.5mmol / L IPTG precipitate; 7: 16°C, 1.0mmol / L IPTG precipitate; 8: 37°C, 0.5mmol / L IPTG precipitate; 9: 37°C, 1.0mmol / L IPTG precipitate;

[0027] Figure 3 The SDS-PAGE and Western blot results of the optimal expression of canine IL-31 recombinant protein in Example 1, wherein M: protein marker; 1: empty control; 2: unpurified control; 3-6: flow-through; 7-18: purified samples;

[0028] Figure 4 This is a graph showing the results of the detection of the activation effect of the canine IL-31 recombinant protein on the STAT luciferase reporter system in Example 1, wherein ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001;

[0029] Figure 5A This is a graph showing the change in serum titer of immunized mice detected by indirect ELISA on the 14th day in Example 2;

[0030] Figure 5B This is a graph showing the change in serum titer of immunized mice detected by indirect ELISA on day 28 in Example 2;

[0031] Figure 5CThis is a graph showing the change in serum titer of immunized mice detected by indirect ELISA on the 35th day in Example 2;

[0032] Figure 5D This is a graph showing the changes in serum titer of immunized mice detected by indirect ELISA on days 0-35 in Example 2;

[0033] Figure 6 This is a graph showing the titer of monoclonal antibody 3F9 in ascites fluid detected by indirect ELISA in Example 5;

[0034] Figure 7 This is a graph showing the titer of monoclonal antibody 3F9 in ascites fluid detected by indirect immunofluorescence in Example 5;

[0035] Figure 8 This is the SDS-PAGE result of the purified monoclonal antibody 3F9 in Example 6;

[0036] Fig. 9 The Western blot results of the binding of ascites monoclonal antibody 3F9 to canine interleukin 31 and feline interleukin 31 in Example 7 are shown;

[0037] Fig.10 This is a graph showing the blocking activity of monoclonal antibody 3F9 detected using the STAT luciferase reporter system in Example 8;

[0038] Fig.11 The Western blot results of different binding regions (N1, N2, N3) of monoclonal antibody 3F9 and canine IL-31 recombinant protein in Example 9 are shown;

[0039] Fig.12 This is a Western blot result diagram of the binding of caninized recombinant monoclonal antibody c3F9 to canine IL-31 recombinant protein in Example 12;

[0040] Fig.13 This is a graph showing the blocking activity of caninized recombinant monoclonal antibody c3F9 detected using the STAT luciferase reporter system in Example 12. DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] The experimental methods without specific conditions in the examples are usually carried out according to conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by MR Green, Molecular Biology by Robert F Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial channels unless otherwise specified.

[0043] Example 1 Preparation of canine IL-31 recombinant protein and detection of biological activity

[0044] 1.1 Expression and purification of canine IL-31 recombinant protein

[0045] Peripheral blood mononuclear cells (PBMCs) were isolated from canine peripheral blood, and total RNA of canine PBMCs was extracted and reverse transcribed to synthesize cDNA. According to the nucleotide sequence of canine IL-31 registered on NCBI (accession number: NM_001165914.1), primers were designed and PCR amplification was performed using the above cDNA as a template. The electrophoresis of the amplified product is shown in Figure 1 shown.

[0046] After homologous recombination of the amplified target fragment with the linearized pET-30a vector, it was transferred into DH5α competent cells, and single colonies were picked for PCR identification and sequencing verification. The nucleotide sequence of the canine IL-31 recombinant protein is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The correctly sequenced pET-30a-cIL-31 recombinant plasmid was transformed into BL21 (DE3) competent cells for expression. During the induction expression process, the expression conditions of 16°C, 37°C and 0.5mmol / L and 1.0mmol / L IPTG were set respectively. After induction expression, the bacteria were collected and broken by ultrasonic disruptor. The supernatant and precipitate after breaking were subjected to SDS-PAGE and Western-blot analysis respectively. The results are shown as follows Figure 2 shown.

[0047] according to Figure 2 The expression conditions of 16°C, 140rpm, and 1.0mmol / L IPTG were selected. After 24h of induction, a large number of expressed bacteria were collected, resuspended, and crushed by pressure. The precipitate was collected and dissolved with inclusion body solution and filtered through a 0.22μm filter. Canine IL-31 was affinity purified using a Ni-NTA column. The purified protein was analyzed by SDS-PAGE and Western-blot to verify the purification effect. The results are shown in Figure 3The protein was dialyzed for refolding and concentrated by ultrafiltration.

[0048] 1.2 Establishment of STAT luciferase reporter system and detection of canine IL-31 recombinant protein activity

[0049] Canine IL-31 recombinant protein binds to its receptor and can activate the downstream JAK / STAT pathway, thereby activating STAT molecules. By constructing the pSTAT-luc plasmid, the biological activity of IL-31 can be detected. Since the activated STAT can upregulate the expression of p21WAF1 / CIP1, IRF-1, c-fos, and VEGF genes, the fragments of the promoter regions of these four genes that can bind to STAT1 or STAT3 are selected, and the specific STAT binding element sequence is shown in SEQ ID NO: 3. The above STAT binding element sequence is inserted into the multiple cloning site region of the reporter gene vector pGL3-promotor to form a plasmid pSTAT-luc with a STAT binding element, a promoter, and a luciferase structure, which is synthesized and constructed by Sangon Biotech Co., Ltd.

[0050] At the same time, a plasmid of full-length canine interleukin 31 receptor A (IL-31RA) was constructed. The nucleotide sequence of canine IL-31RA (SEQ ID NO: 4) was amplified from the cDNA of canine PBMC, and its amino acid sequence is shown in SEQ ID NO: 5. The IL-31RA nucleotide sequence was substituted for the sequence between the Hind Ⅲ and Xho Ⅰ sites in pcDNA3.1 to obtain the pcDNA3.1-cILRA plasmid.

[0051] The biological activity of canine IL-31 recombinant protein was detected using the STAT dual luciferase reporter system. The specific steps are as follows:

[0052] (1) Cell plating: When HeLa cells are cultured to the logarithmic growth phase, they are digested with trypsin, centrifuged at 1000 rpm for 5 min, resuspended in DMEM medium containing 10% FBS, inoculated into a 48-well cell culture plate, and cultured in a cell culture incubator at 37°C with 5% CO2;

[0053] (2) Transfection: When the cells grow to 60%-70% density in the well, transfection can be performed. 2 h before transfection, the cell culture medium is replaced with fresh complete culture medium, and the pSTAT-luc plasmid, internal control pTK plasmid, and pcDNA3.1-cILRA plasmid are co-transfected into HeLa cells;

[0054] (3) Adding protein: 12 hours after transfection, pre-dilute canine IL-31 recombinant protein to 1000 ng / mL, then perform a 1:10-fold serial dilution, set up 4 dilutions in total, make 3 replicates for each dilution, and add the protein to the transfected cell wells; use the standard protein sample BSA as a negative control, and incubate the cells in an incubator at 37°C, 5% CO2 for 20 hours;

[0055] (4) Detection and data analysis: Remove the culture medium, wash the cells once with PBS, add 50 μL 1×PLB to each well to lyse the cells, shake at room temperature for 15 minutes, add 100 μL LAR II to each well in a light-proof plate and then add 20 μL lysate supernatant, read the relative light units (RLU) on the cell culture plate using a SpectraMax M5 microplate reader as 1, then add 100 μL Stop Buffer and read the relative light units as 2. The ratio of the two is the relative expression level. If there is a difference, it means that the STAT reporter system is activated by the canine IL-31 recombinant protein, which proves the biological activity of the protein. The results are as follows Figure 4 shown.

[0056] from Figure 4 It can be seen that canine IL-31 recombinant protein can significantly activate STAT molecules, its relative light unit is higher than that of the cell control group, and the degree of activation is positively correlated with the concentration of canine IL-31.

[0057] Example 2 Immunization of mice with canine IL-31 recombinant protein and detection of mouse serum antibody titer

[0058] 2.1 Immunization of mice

[0059] Four 6-week-old SPF-grade BALB / c female mice were selected, and the canine IL-31 recombinant protein prepared in Example 1 was mixed with Freund's adjuvant in a ratio of 1:1 and emulsified, and the canine IL-31 recombinant protein emulsified with adjuvant was subcutaneously injected into the back of the neck of the mice, 100 μg per mouse, and booster immunization was performed once every two weeks. Before each immunization and 7 days after the third immunization, the mice were bled from the eye sockets, and the serum was separated and stored for later use.

[0060] 2.2 Establishment of indirect ELISA detection method

[0061] The optimal coating concentration of the antigen and the optimal dilution of the serum were determined by the square array titration method. The canine IL-31 recombinant protein was diluted from 4μg / mL to 0.125μg / mL with ELISA coating solution, and 6 dilution concentrations of the protein were set. It was coated vertically on a 96-well ELISA plate, 100μL per well, and placed at 4℃ overnight for coating; after washing with PBST, 100μL of blocking solution was added to each well, and blocked at 37℃ for 1h. After blocking, wash 3 times with PBST; dilute the positive serum from 1:1000 to 1:1024000, and set a total of 11 dilutions. Serum with different dilutions was added horizontally to the ELISA plate in turn, 100μL per well, and negative mouse serum was set as a negative control. The dilution method was the same as the positive serum, and the plate was incubated at 37℃ for 1h. After incubation, wash with PBST for 3 times; dilute HRP-labeled goat anti-mouse IgG antibody 1:5000, add to the wells, and incubate at 37°C for 30 minutes. After incubation, wash with PBST for 3 times, 5 minutes each time; add ELISA colorimetric solution, stand at room temperature in the dark for 10 minutes to develop color, then add stop solution to terminate the color development reaction, and measure the OD value with an enzyme marker and record the data.

[0062] Result determination: Calculate the P / N value (“P” represents the absorbance value OD of the sample to be tested 630 Value, "N" negative control OD 630 In general, the serum dilution with the positive serum OD630 closest to 1, the dilution with a higher P / N value and a smaller protein concentration was selected. Finally, it was determined that the optimal coating concentration of canine IL-3 recombinant protein 1 was 0.5 μg / mL.

[0063] 2.3 Detection of serum antibody levels in immunized mice

[0064] After diluting the canine IL-31 recombinant protein at a protein coating concentration of 0.5 μg / mL, coat the ELISA plate at 100 μL / well and place it at 4°C overnight. Dilute the mouse serum 2-fold from 1:500 to 1:64000, with a total of 8 dilutions, and use negative serum as a control. Each serum needs to be replicated for 3 times. Add the diluted serum as the primary antibody to the well, then incubate with HRP-labeled goat anti-mouse IgG, and finally use a colorimetric solution for color development. Detect OD using an enzyme reader 630 The value was used to determine the mouse with the highest serum antibody titer. The results were as follows Figures 5A-5D shown.

[0065] from Figures 5A-5D It can be seen that after three immunizations, the serum antibody level of mice showed a rising trend and reached the highest point. At a dilution of 1:64000, OD 630The value was still higher than 2. Among them, the serum antibody level of H5 mice was the highest, with an OD of 1:64000. 630 If the value is still higher than 3, the mouse is selected for subsequent cell fusion.

[0066] Example 3 Preparation of hybridoma cells

[0067] 3.1 Preparation of SP2 / 0 myeloma cells

[0068] SP2 / 0 myeloma cells frozen in the laboratory were revived and cultured in 1640 complete medium containing 20% ​​FBS. When the cells grew well and the cell count reached 1×10 7 About 10 to 14 days, collect the cells. Inject the cells subcutaneously at multiple points on the back of the neck into 6-8 week-old BALB / c mice. After about 10-14 days, solid tumors can be seen growing on the back of the mice. After the mice are killed by dislocating the neck, they are soaked in 75% alcohol for 5 minutes. The mice are fixed on the clean bench, and the solid tumors are removed by sterile operation. They are placed in a homogenizer and fully ground with 1640 basal medium. The upper cell suspension is gently aspirated through a 40μm cell sieve and placed in a sterile centrifuge tube for use. The cell suspension in the centrifuge tube is centrifuged at 1000g for 10 minutes, and the supernatant is discarded. The cell pellet is resuspended with 15mL 1640 basal medium. Add 15mL of lymphocyte separation solution to another 50mL sterile centrifuge tube, gently add the resuspended myeloma cell suspension to the separation solution along the tube wall, and centrifuge at 1000g for 10 minutes. Carefully pipette the dense white cell layer at the boundary of the two interfaces into another centrifuge tube, wash it twice with 1640 basal medium, resuspend it with 10 mL 1640 basal medium, count and freeze it for later use.

[0069] 3.2 Preparation of feeder cells

[0070] Take a blank BALB / c mouse, remove the eyeball and take blood, then kill it by cervical dislocation, and soak the mouse in 75% alcohol for 5 minutes; put the mouse in a clean bench, fix it in supine position, and use forceps and surgical scissors to cut the skin along the midline of the abdomen to fully expose the peritoneum; replace a set of sterile scissors and forceps, cut the peritoneum along the midline of the abdomen, expose the abdominal organs, and find the spleen in the left upper abdomen of the mouse; put the spleen in a homogenizer, add 1640 basal culture medium for grinding; transfer the cell suspension in the homogenizer to a 50mL centrifuge tube through a 70μm cell strainer; transfer the cell suspension in the centrifuge tube to another 50mL centrifuge tube through a 40μm cell strainer to obtain dispersed single cells, centrifuge at 1000r / min for 10min, and collect spleen cells. After counting the cells, 3×10 cells should be plated per well of a 96-well cell culture plate. 4Calculate the required amount of feeder cells based on the amount of cells per well. Resuspend the cells in 1640 complete medium containing HAT, add the cell suspension to a 96-well plate at 100 μL / well, and culture in a cell culture incubator at 37°C with 5% CO2.

[0071] 3.3 Preparation of immune spleen cells

[0072] According to the results of mouse serum antibody titer in Example 2, the mice with the highest titer were selected. Four days before cell fusion, the mice with higher antibody titer were subjected to intraperitoneal impaction, and the antigen without adjuvant and emulsification was injected into the mouse peritoneal cavity, and 100 μg was injected into each mouse. Immune splenocytes were taken for cell fusion 3d after impact immunization. BALB / c mice after intraperitoneal impact were taken, and the orbital bleeding was killed, and the blood was collected and the serum was separated, which was the positive serum. The mice were soaked in 75% alcohol for 5 min. According to the steps of preparing feeder layer cells, immune splenocytes were prepared, and the cells were counted and used for standby.

[0073] 3.4 Cell fusion

[0074] SP2 / 0 myeloma cells were fused with immune spleen cells to form hybridoma cells.

[0075] 3.5 Screening of hybridoma cells

[0076] On the 5th day after cell fusion, add 50μL HAT medium to each well. Starting from the 7th day, discard half of the medium every other day and add HT medium. At the same time, observe the size of the cell colony. When the hybridoma cells in the cell wells reach 1 / 4 of the bottom area of ​​the wells, the hybridoma cells can be screened. Before each test, aspirate the medium in the wells and place it in a 96-well plate at 4°C for standby use. Choose indirect ELISA and indirect immunofluorescence for detection:

[0077] (1) Indirect ELISA test:

[0078] The protein was diluted to the optimal coating concentration with ELISA coating solution and then coated on the ELISA plate for subsequent detection. The hybridoma supernatant was used as the primary antibody, and the negative serum and positive serum were used as negative and positive controls, respectively. 630 The holes with higher values ​​are marked.

[0079] (2) Indirect immunofluorescence detection:

[0080] The hybridoma supernatant was used as the primary antibody, and the negative serum and FLAG monoclonal antibody were used as the negative control and positive control, respectively. After observation under an inverted fluorescence microscope, photos were taken and saved, and the wells with specific fluorescence were marked.

[0081] Example 4 Subcloning of canine IL-31-positive hybridoma cells

[0082] Hybridoma cells that are positive in both ELISA and IFA tests are selected for subcloning. Feeder cells are prepared the day before subcloning. During subcloning, hybridoma cells are collected and transferred to sterile 1.5mL EP tubes, and the cells are counted under a microscope. According to the counting results, the hybridoma cells are gradually diluted to 10 cells per milliliter, and the cell suspension at this concentration is added to a 96-well plate with feeder cells, 100μL per well, that is, theoretically there is 1 hybridoma cell in each well. The hybridoma cells of an original well generally need to be subcloned into a 96-well plate, and the cell plate is placed in a cell culture incubator for culture. Observe the growth state of the hybridoma cells. When they grow to 1 / 5-1 / 3 of the culture well, they need to be tested by indirect ELISA and IFA methods. Select the wells that are positive in both detection methods for the next subcloning. After repeating 3 times, when the positive rate of each hybridoma cell well reaches 100%, a single cloned hybridoma cell line is obtained, and finally the m3F9 strain is selected to establish a hybridoma cell line.

[0083] Example 5 Large-scale preparation and verification of canine IL-31 monoclonal antibody

[0084] 5.1 Large-scale preparation of canine IL-31 monoclonal antibody

[0085] Take 10-14 week old SPF BALB / c mice and inject 0.5 mL of Freund's incomplete adjuvant into the peritoneum of each mouse. After 7 days, take the m3F9 hybridoma cells in good condition and in the logarithmic growth phase, resuspend them in 200 μL 1640 basal medium, and inject the hybridoma cells into the peritoneum of the pre-sensitized mice. Each mouse is injected with 10 6 Observe the condition of the mouse and the ascites. When the mouse abdomen becomes obviously swollen, has difficulty in moving, and has shortness of breath, the ascites can be collected.

[0086] After disinfecting the mouse's abdominal fur with alcohol cotton balls, pierce the mouse's abdominal cavity with the needle of a 20mL syringe in a clean bench until light yellow liquid flows out. Collect the ascites in a 15mL centrifuge tube. Centrifuge at 2000r / min for 10min. The supernatant is the monoclonal antibody ascites, and the precipitate is the hybridoma cells, which can be frozen. The mice can continue to be raised, and the ascites can be collected again after 2-3 days until the mice have no ascites.

[0087] 5.2 Verification of the effect of canine IL-31 ascites monoclonal antibody

[0088] (1) Indirect ELISA:

[0089] The ascites monoclonal antibody was detected using the established indirect ELISA detection method. The ascites monoclonal antibody was diluted from 1:2000 and doubled to 1:4096000. The diluted ascites monoclonal antibody was added to the ELISA plate as the primary antibody. After the secondary antibody was added and the color development was completed, the OD was detected and recorded. 630 The result is as follows. Figure 6 shown.

[0090] from Figure 6 The data in Figure 2 show that the ascites monoclonal antibody has a high titer. At a dilution of 1:1024000, OD 630 The value is still greater than 1.

[0091] (2) Indirect immunofluorescence:

[0092] The ascites monoclonal antibody was detected using the established indirect immunofluorescence detection method. The ascites monoclonal antibody was diluted from 1:500 to 1:10000, and the diluted monoclonal antibody of different concentrations was added to the cell wells as the primary antibody, followed by incubation with AF488-goat anti-mouse IgG (H+L) and color development. The experimental results are shown in Figure 2. Figure 7 shown.

[0093] from Figure 7 When the ascites monoclonal antibody was diluted from 1:500 to 1:10000, the monoclonal antibody was able to bind to the cells expressing canine IL-31 recombinant protein, and specific green fluorescence appeared.

[0094] Example 6 Purification of canine IL-31 monoclonal antibody

[0095] The collected m3F9 ascites monoclonal antibody was purified by protein A affinity chromatography, and the collected eluate was tested for its purification effect by SDS-PAGE. Figure 8 shown.

[0096] from Figure 8 It can be seen that the purified monoclonal antibody shows two clear and clean bands on the electrophoresis diagram, located at 25kDa and 50kDa respectively, which is consistent with the size of the antibody light chain and heavy chain, proving that the monoclonal antibody was successfully purified and has a high purity.

[0097] Example 7 Western blot analysis of canine IL-31 monoclonal antibody and cross-reaction with feline IL-31 recombinant protein

[0098] Peripheral blood mononuclear cells (PBMCs) were isolated from cat peripheral blood, and total RNA of cat PBMCs was extracted and reverse transcribed to synthesize cDNA. According to the nucleotide sequence of cat IL-31 registered on NCBI (accession number: XM_045042055.1), primers were designed and PCR amplification was performed using the above cDNA as a template. After homologous recombination of the amplified product with the pET-30a vector, prokaryotic expression was performed, and the expression product was affinity purified by Ni-NTA column to obtain cat IL-31 protein, whose amino acid sequence is shown in SEQ ID NO: 6.

[0099] The canine IL-31 recombinant protein and the cat IL-31 protein were subjected to SDS-PAGE gel electrophoresis and transferred to a PVDF membrane by wet transfer. After blocking, canine IL-31 monoclonal antibody was added for incubation. The results are as follows: Fig. 9 shown.

[0100] from Fig. 9 It can be seen that the monoclonal antibody can react with canine IL-31 recombinant protein and cat IL-31 protein, and specific bands can be observed at the positions of 23kDa and 35kDa, respectively, proving that the monoclonal antibody can not only specifically bind to canine IL-31 recombinant protein, but also cross-react with cat IL-31 protein.

[0101] Example 8 Blocking Activity Detection of Canine IL-31 Monoclonal Antibody

[0102] The blocking activity of canine IL-31 monoclonal antibody was detected using the STAT luciferase reporter system established in Example 1. The concentration of the purified monoclonal antibody was measured, and it was diluted to 50 μg / mL, 10 μg / mL, 5 μg / mL, and 1 μg / mL according to the set concentration gradient. The diluted monoclonal antibody was co-incubated with canine IL-31 recombinant protein, and added to the cell wells that had been transfected with pSTAT-luc plasmid, internal control pTK plasmid, and pcDNA3.1-cILRA plasmid. In addition, four groups of controls were set up: SP2 / 0 myeloma cell culture medium supernatant was co-incubated with canine IL-31 recombinant protein as a negative control; the monoclonal antibody was co-incubated with DMEM as a monoclonal antibody control; a cell control was set; and the canine IL-31 recombinant protein was set as a negative control. 20 hours after the addition of the protein, the luciferin in each well was detected using a dual luciferase kit, and the results are as follows. Fig.10 shown.

[0103] from Fig.10 It can be seen that the monoclonal antibody has the ability to significantly inhibit the activation of canine IL-31 downstream STAT molecules, and the inhibitory effect is proportional to the concentration of the monoclonal antibody, proving that the monoclonal antibody has blocking activity.

[0104] Example 9 Detection of the binding region between canine IL-31 monoclonal antibody and antigen

[0105] The B cell epitopes and spatial structure of the canine IL-31 recombinant protein were predicted. According to the properties of the protein, canine IL-31 was divided into three segments and named N1, N2, and N3, respectively. The amino acid sequences of the N1, N2, and N3 proteins are shown in SEQ ID NOs: 7-9, respectively. The three proteins were expressed using the method in Example 1 and purified using a nickel affinity column. The purified proteins were analyzed using SDS-PAGE to verify the purification effect. Subsequently, the N1, N2, and N3 proteins were transferred to a PVDF membrane. After blocking, canine IL-31 monoclonal antibodies were incubated, and His antibodies were set as positive controls, and no primary antibody was added as negative controls. Then, HRP-labeled sheep anti-mouse IgG secondary antibodies were incubated, and finally ECL color developing solution was used to collect images in a chemiluminescence imaging system, as shown in FIG. Fig.11 shown.

[0106] from Fig.11 It can be seen that after incubation with the m3F9 monoclonal antibody, a specific band appeared at the N1 protein, indicating that the monoclonal antibody bound to the N1 protein (H25-P80) region.

[0107] Example 10 Sequencing of the variable region sequence of canine IL-31 monoclonal antibody

[0108] Take the hybridoma cells in Example 4, blow them up and transfer them to a centrifuge tube, centrifuge them at 1000r / min for 5min, discard the supernatant, lyse the cell pellet with Trizol reagent, and extract total RNA from it. Then use reverse transcription reagent to synthesize its cDNA. Design upstream and downstream primer sets for mouse heavy chain (VH) and light chain (VL) (the specific sequences are shown in Table 1 below), and use the cDNA of the above hybridoma cells as a template to amplify the nucleotide sequence of the variable region. After connecting the sequence to the pMD19-T vector, send it for sequencing. Comparison of sequencing results, the sequence of the complementary determining region and the skeleton region of the variable region of the antibody is obtained, as follows:

[0109] Table 1 Sequence list of upstream and downstream primer sets

[0110]

[0111] The amino acid sequence of the heavy chain variable region CDR-H1 of the anti-canine IL-31 monoclonal antibody (GDSITSGY (SEQ ID NO: 10)), the amino acid sequence of CDR-H2 (ISYSGTT (SEQ ID NO: 11)), the amino acid sequence of CDR-H3 (ARYPTYRYAMDY (SEQ ID NO: 12)), the amino acid sequence of HC-FR1 (DVQLKESGPSLVKPSQTLSLTCSVT (SEQ ID NO: 13)), the amino acid sequence of HC-FR2 (WNWIRKFPGNKLEYMGF (SEQ ID NO: 14)), the amino acid sequence of HC-FR3 (GYNPSLKSRISITRDTSKNQYYLHLNSVTTEDTATYYC (SEQ ID NO: 15)), and the amino acid sequence of HC-FR4 (WGQGTSVTVSS (SEQ ID NO: 16));

[0112] The amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody against canine IL-31 (ESVDSYGNGF (SEQ ID NO: 17)), the amino acid sequence of CDR-L2 (RAS (SEQ ID NO: 18)), the amino acid sequence of CDR-L3 (HQSYEDPWT (SEQ ID NO: 19)), the amino acid sequence of LC-FR1 (EILLTQFPASLAVSLGQRATISCRAS (SEQ ID NO: 20)), the amino acid sequence of LC-FR2 (MHWYQQKPGQSPKLLIY (SEQ ID NO: 21)), the amino acid sequence of LC-FR3 (NLESGIPARFSGSGSRTDFTLTINPVEADDVATYYC (SEQ ID NO: 22)), and the amino acid sequence of LC-FR4 (FGGGTKLEIK (SEQ ID NO: 23)).

[0113] Example 11 Caninization and purification of canine IL-31 monoclonal antibody

[0114] According to the amino acid sequence of the canine IL-31 monoclonal antibody variable region in Example 10, the nucleotide sequence of the canine IL-31 monoclonal antibody variable region was synthesized, as follows:

[0115] Nucleotide sequence of the heavy chain variable region of the monoclonal antibody against canine IL-31 (SEQ ID NO: 24)GACGTCCAGCTGAAGGAGTCAGGACCTAGCCTCGTGAAAACCTTCTCAGACTCTGTCCCTCACCTGTTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAATTCCCAGGAAATAAACTTGAGTACATGGGGTTCATAAGCTACAGTGGTACCACTGGCT ACAATCCATCTCTCAAAAGTCGAATCTCCATCACTCGAGACACATCCAAGAACCAGTACTACCTACATTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGATATCCTACCTACCGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0116] The nucleotide sequence of the light chain variable region of the monoclonal antibody against canine IL-31 (SEQ ID NO: 25) GAAATTTTGCTGACCCAGTTTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAATGGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGTCACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCACCAAAGTTATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0117] The gene fragments of the heavy chain variable region and light chain variable region of the above-synthesized monoclonal antibody were constructed into the plasmid pFUSE-CHIg-dG2 containing the canine heavy chain constant region and the plasmid pFUSE2-CLIg-dK containing the light chain constant region, respectively, to obtain caninized heavy chain and light chain recombinant plasmids. In the above recombinant plasmids, the amino acid sequences of the heavy chain and light chain of the monoclonal antibody against canine IL-31 are as follows:

[0118] Amino acid sequence of the heavy chain of the monoclonal antibody against canine IL-31 (SEQ ID NO: 26)

[0119] MYRMQLLSCIALSLALVTNSASDVQLKESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGFISYSGTTGYNPSLKSRISITRDTSKNQYYLHLNSVTTEDTATYYCARYPTYRYAMDYWGQGTSVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQKSLSHSPGK

[0120] Amino acid sequence of the light chain of the monoclonal antibody against canine IL-31 (SEQ ID NO: 27)

[0121] MYRMQLLSCIALSLALVTNSLEEILLTQFPASLAVSLGQRATISCRASESVDSYGNGFMHWYQQKPGQSPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCHQSYEDPWTFGGGTKLEIKLERTDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0122] The obtained caninized heavy chain and light chain recombinant plasmids were transfected into HEK-293T cells at a mass ratio of heavy chain: light chain = 3:2. After 48 hours of culture, the secreted monoclonal antibody was collected from the supernatant of the cell culture medium, which was the caninized IL-31 monoclonal antibody and named c3F9. The obtained monoclonal antibody was purified by Protein A affinity chromatography to remove the interference of foreign proteins.

[0123] Example 12 Validation of caninized IL-31 monoclonal antibody

[0124] 12.1 Western blot analysis of caninized IL-31 monoclonal antibody

[0125] The canine IL-31 recombinant protein was subjected to SDS-PAGE gel electrophoresis and transferred to a PVDF membrane by wet transfer. After blocking, canine IL-31 monoclonal antibody was added as the primary antibody for incubation, and HRP-labeled sheep anti-canine IgG was used as the secondary antibody. The binding of canine recombinant antibody to canine IL-31 protein was detected by Western blot. The results are shown in Fig.12 shown.

[0126] from Fig.12 It can be seen that the caninized monoclonal antibody can specifically bind to canine recombinant IL-31 protein.

[0127] 12.2 Validation of the blocking activity of caninized IL-31 monoclonal antibody

[0128] The blocking activity of the caninized recombinant monoclonal antibody was detected using the STAT luciferase reporter system established in Example 1. The purified caninized recombinant monoclonal antibody was incubated with canine IL-31 protein at concentrations of 200 μg / mL, 100 μg / mL, and 50 μg / mL, respectively. At the same time, other monoclonal antibodies were incubated with canine IL-31 protein as a control. The blocking activity of the caninized recombinant monoclonal antibody was detected according to the protein biological activity detection method in Example 1. The results are as follows: Fig.13 shown.

[0129] from Fig.13 It can be seen that the caninized recombinant antibody c3F9 has a better ability to block the activation of canine IL-31 downstream STAT molecules, and shows a dose-dependent effect.

[0130] In summary, the anti-canine IL-31 monoclonal antibody of the present invention has good blocking activity. The monoclonal antibody specifically binds to canine IL-31 and blocks its binding to canine IL-31 receptor A. Furthermore, the monoclonal antibody is caninized, and the obtained caninized monoclonal antibody has better blocking activity.

[0131] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0132] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against canine IL-31, characterized in that: It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementary determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementary determining regions CDR-L1, CDR-L2, and CDR-L3; Among them, the amino acid sequences of the CDR-H1, CDR-H2, and CDR-H3 are shown as SEQ ID NOs: 10-12, respectively; the amino acid sequences of the CDR-L1 and CDR-L3 are shown as SEQ ID NOs: 17 and 19, respectively, and the amino acid sequence of the CDR-L2 is RAS.

2. The anti-canine IL-31 monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain variable region also includes framework regions HC-FR1, HC-FR2, HC-FR3, and HC-FR4, and the light chain variable region also includes framework regions LC-FR1, LC-FR2, LC-FR3, and LC-FR4; Among them, the amino acid sequences of HC-FR1, HC-FR2, HC-FR3, and HC-FR4 are shown in SEQ ID NOs: 13-16, respectively; the amino acid sequences of LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are shown in SEQ ID NOs: 20-23, respectively.

3. The anti-canine IL-31 monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain is shown in SEQ ID NO:

27.

4. A nucleic acid molecule encoding the anti-canine IL-31 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that: Comprising the nucleic acid molecule of claim 4.

6. A recombinant cell, characterized in that Comprising the nucleic acid molecule according to claim 4 or the recombinant vector according to claim 5.

7. A method for detecting the blocking activity of the anti-canine IL-31 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The steps include: S1, construct pSTAT-luc vector and pcDNA3.1-cILRA vector respectively; S2, transfecting the pSTAT-luc vector and the pcDNA3.1-cILRA vector into HeLa cells; S3, mixing the anti-canine IL-31 monoclonal antibody or its antigen-binding fragment with the IL-31 protein, and then adding it to the HeLa cells for incubation, and finally detecting fluorescein to obtain the blocking activity of the monoclonal antibody or its antigen-binding fragment; The pcDNA3.1-cILRA vector is obtained by replacing the sequence between the Hind Ⅲ and Xho Ⅰ sites in pcDNA3.1 with the IL-31RA nucleotide sequence shown in SEQ ID NO:

4.

8. Use of the anti-canine IL-31 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5 or the recombinant cell according to claim 6 in the preparation of a medicament for preventing and / or treating animal pruritus.

9. A pharmaceutical composition, characterized in that The invention comprises the anti-canine IL-31 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

10. The composition according to claim 9, characterized in that Also included are pharmaceutically acceptable carriers.

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