Anti-canine PD-L1 monoclonal antibody and application thereof in preparation of anti-cancer drugs

Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to an anti-canine PD-L1 monoclonal antibody and application thereof in preparation of anti-cancer drugs. The invention provides an anti-canine PD-L1 monoclonal antibody, the amino acid sequences of complementarity determining regions of a light chain of the anti-canine PD-L1 monoclonal antibody are respectively QISVSHNGYTN, KVS and FQGPWTSHV, and the amino acid sequences of complementarity determining regions of a heavy chain of the anti-canine PD-L1 monoclonal antibody are respectively TDYLGYAF, GGSSGIHP and PIAYARND. The antibody has good affinity to PD-L1 on the surfaces of canine tumor cells, can target tumors and effectively inhibit tumor growth, and can be used for treating canine cancers and infectious diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biopharmacy, and particularly relates to an anti-canine PD-L1 monoclonal antibody and application thereof in preparation of anti-cancer drugs. BACKGROUND

[0002] Programmed Death Ligand 1 (PD-L1), also known as Cluster of Differentiation 274 (CD274) or B7 Homolog Protein 1 (B7-H1), is a type I transmembrane protein with a molecular weight of about 40 kDa. The protein has a wide tissue expression characteristic, and its basic expression can be found in activated T cells, B cells, monocytes, dendritic cells, macrophages and other immune cell subgroups, and also presents a constitutive expression characteristic in a variety of non-hematopoietic cells.

[0003] In the tumor immune microenvironment, the abnormal expression mechanism of PD-L1 has important pathological significance. When the PD-L1 overexpressed on the surface of tumor cells specifically binds to the Programmed Death-1 (PD-1) receptor, the PD-1 / PD-L1 immune checkpoint pathway can be activated through cascade signal transduction. The immune suppression effect mediated by the pathway specifically manifests as follows: by lowering the activation threshold of effector T cells, inhibiting the proliferation and differentiation of cytotoxic T lymphocytes, and reducing the secretion level of tumor-killing cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), the tumor cells ultimately escape from immune surveillance and clearance. It is worth noting that the expression regulation of PD-L1 has a dual mechanism, which can maintain the basic expression level in white blood cells and non-hematopoietic cells, and can also be significantly up-regulated by inflammatory factors (such as IFN-γ and IL-6) or oncogenic signaling pathways (such as PI3K / AKT / mTOR pathway). Clinical pathological analysis confirms that the overexpression of PD-L1 in a variety of solid tumor tissues is significantly positively correlated with the increase in the number of tumor infiltrating lymphocytes (TILs) and poor prognosis, suggesting that it plays a key regulatory role in tumor occurrence, invasion, metastasis and immune escape. This finding lays a theoretical foundation for the development of immune checkpoint inhibitors targeting the PD-1 / PD-L1 pathway.

[0004] Based on the above mechanism, the PD-L1 antibody as a typical immune checkpoint inhibitor has a multi-target regulation mechanism. The PD-L1 antibody can competitively block the PD-1 / PD-L1 signal pathway by high-affinity binding to the extracellular domain of the PD-L1 protein; it can also specifically interfere with the co-inhibitory interaction of B7.1 and PD-L1, thereby comprehensively restoring the immune activation function of T cells and promoting Th1-type cytokine secretion. The PD-L1 antibody can induce mitochondria-dependent apoptosis of tumor cells, and the pro-apoptotic effect has a significant dose-dependent relationship with caspase-3 / 7 activation. More notably, in a combination therapy regimen, the PD-L1 antibody exhibits a unique synergistic effect: when combined with cytotoxic chemotherapy drugs (such as cisplatin, paclitaxel) or molecular targeted drugs (such as EGFR inhibitors), it can significantly enhance the cytotoxic effect of other treatment methods by down-regulating the expression level of anti-apoptotic proteins (such as Bcl-2, Survivin) in tumor cells, and this phenomenon has been fully verified in preclinical animal models.

[0005] Therefore, the development of anti-PD-L1 monoclonal antibodies with high affinity has become a key technical direction for immunotherapy. The successful development of such new antibodies not only provides an innovative solution for the immunotherapy of canine tumors, but also has the potential to expand its application to clinical intervention of immune regulation abnormality related diseases such as autoimmune diseases and chronic viral infections. SUMMARY

[0006] The purpose of the present application is to provide a monoclonal antibody capable of effectively inhibiting canine tumors and its application in the preparation of anti-cancer drugs.

[0007] The present application provides an anti-canine PD-L1 monoclonal antibody, wherein the amino acid sequences of the complementarity determining regions of the light chain are QISVSHNGYTN, KVS and FQGPWTSHV, respectively, and the amino acid sequences of the complementarity determining regions of the heavy chain are TDYLGYAF, GGSSGIHP and PIAYARND, respectively.

[0008] In some embodiments, the amino acid sequence of the light chain is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2.

[0009] In some embodiments, the anti-canine PD-L1 monoclonal antibody is secreted by the hybridoma cell with the accession number CGMCC No. 46336.

[0010] The present application also provides a hybridoma cell secreting an anti-canine PD-L1 monoclonal antibody, which has the accession number CGMCC No. 46336.

[0011] The application also provides an anticancer drug comprising any of the anti-canine PD-L1 monoclonal antibodies.

[0012] In some embodiments, the anticancer drug further comprises a pharmaceutically acceptable carrier or excipient.

[0013] The carrier includes liposomes and nanoparticles. The excipient includes binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavorings, fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants.

[0014] In some embodiments, the anticancer drug is an injection or an oral preparation.

[0015] The injection includes solutions, suspensions, emulsions, and powder injections. The oral preparation includes tablets, capsules, oral liquids, granules.

[0016] The anticancer drug can effectively inhibit malignant tumors of canine animals.

[0017] The malignant tumors of canine animals include canine ductal adenocarcinoma, canine renal carcinoma, canine uterine smooth muscle carcinoma, canine skin melanoma, canine oral carcinoma, and canine breast carcinoma.

[0018] The use of the anti-canine PD-L1 monoclonal antibody in the preparation of an anticancer drug also belongs to the scope of the application.

[0019] In some embodiments, the anticancer drug is a drug for inhibiting canine ductal adenocarcinoma, canine renal carcinoma, canine uterine smooth muscle carcinoma, canine skin melanoma, canine oral carcinoma, and canine breast carcinoma.

[0020] The use of the anti-canine PD-L1 monoclonal antibody in the treatment of canine tumor diseases also belongs to the scope of the application.

[0021] The application also provides a preparation method of an anticancer drug, which comprises: culturing the hybridoma cells with the preservation number of CGMCC No. 46336, isolating and purifying the monoclonal antibody secreted by the hybridoma cells, and taking the monoclonal antibody as the pharmaceutical efficacy component of the anticancer drug.

[0022] In some embodiments of the method, the hybridoma cells are cultured, the supernatant of the cell culture solution is collected, and the monoclonal antibody in the supernatant of the cell culture solution is purified.

[0023] In other embodiments of the method, the hybridoma cells are inoculated into the abdominal cavity of a mouse, the ascites is collected after the abdominal swelling of the mouse, and the monoclonal antibody in the ascites is purified.

[0024] The application provides an anti-canine PD-L1 monoclonal antibody, which is named mAb-CPDL5. Cell immunofluorescence experiments prove that the mAb-CPDL5 can specifically bind to PD-L1 proteins on the surfaces of canine tubular adenocarcinoma cells (CMT-1026), canine kidney epithelial cells (MDCK) and canine breast cancer cells (U27) Figure 3 ). Immunohistochemical experiments prove that the mAb-CPDL5 can specifically bind to positive cells of canine uterine smooth muscle tumors, canine skin melanoma tumors, canine oral tumors and canine breast tumors Figure 5 ). Animal in vivo experiments prove that the tumor volume and tumor mass of breast cancer tumor-bearing mice treated by the mAb-CPDL5 are significantly reduced Figure 4 . Therefore, the mAb-CPDL5 has good affinity to PD-L1 on the surface of canine tumor cells, can target tumors and effectively inhibit tumor growth. The anti-canine PD-L1 monoclonal antibody can be used as an immune checkpoint inhibitor for treating cancers and infectious diseases of canine animals. The application provides a precise immunotherapy for treating canine tumor diseases and fills the blank of tumor immunotherapy in the field of veterinary medicine.

[0025] The preservation information of the hybridoma cell secreting the anti-canine PD-L1 monoclonal antibody is as follows:

[0026] Biological material: CPDL5

[0027] Preservation number: CGMCC No. 46336

[0028] Classification name: mouse hybridoma cell

[0029] Preservation date: March 28, 2025

[0030] Preservation unit: China General Microbiological Culture Collection Center

[0031] Address: No. 3, Yikhina, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a statistical curve of the detection results of the mouse serum antibody titer. The abscissa represents the dilution ratio of the serum of the immunized mouse. Negative indicates a negative control (mouse negative serum), and blank indicates a blank control (1xPBS). The ordinate represents the OD 450nm value. The numbers 1, 2, 3 and 4 in the legend are the numbers of the immunized mice.

[0033] Figure 2The results of Western Blot detection of anti-canine PD-L1 monoclonal antibody mAb-CPDL5; the left lane is the protein molecular weight marker, and the right lane is the whole protein of canine breast cancer cells (U27).

[0034] Figure 3 The results of cell immunofluorescence experiments of anti-canine PD-L1 monoclonal antibody mAb-CPDL5 with different types of canine cancer cells; Merge represents the merging of DAPI and FITC photos.

[0035] Figure 4 The results of treating breast cancer tumor-bearing mice with anti-canine PD-L1 monoclonal antibody mAb-CPDL5; A: the weight change curve of the mice, the horizontal coordinate represents the number of days after the mice were inoculated with breast cancer cells (4T1); B: the tumor volume change curve of the mice, the horizontal coordinate represents the number of days after the mice were inoculated with breast cancer cells (4T1); C: the tumor weight of the mice at the end of the experiment; D: the tumor dissection photos of the mice at the end of the experiment; Con represents the control group (injected with normal saline), and PD-L1 represents the treatment group (injected with monoclonal antibody mAb-CPDL5); * indicates P<0.05 (significant difference).

[0036] Figure 5 The results of immunohistochemical experiments of anti-canine PD-L1 monoclonal antibody mAb-CPDL5 with different types of canine malignant tumor tissues.

[0037] SEQUENCE DESCRIPTION

[0038] In the attached sequence listing, the amino acid sequences follow the standard convention of starting at the amino terminus and proceeding toward the carboxy terminus.

[0039] SEQ ID NO: 1 Amino acid sequence of the light chain of monoclonal antibody mAb-CPDL5;

[0040] SEQ ID NO: 2 Amino acid sequence of the heavy chain of monoclonal antibody mAb-CPDL5;

[0041] SEQ ID NO: 3 Amino acid sequence of the complementarity determining region LCDR1 of the light chain of monoclonal antibody mAb-CPDL5;

[0042] SEQ ID NO: 4 Amino acid sequence of the complementarity determining region LCDR3 of the light chain of monoclonal antibody mAb-CPDL5;

[0043] SEQ ID NO: 5 Amino acid sequence of the complementarity determining region HCDR1 of the heavy chain of monoclonal antibody mAb-CPDL5;

[0044] SEQ ID NO: 6 amino acid sequence of the complementarity determining region HCDR2 of the heavy chain of the monoclonal antibody mAb-CPDL5;

[0045] SEQ ID NO: 7 amino acid sequence of the complementarity determining region HCDR3 of the heavy chain of the monoclonal antibody mAb-CPDL5;

[0046] SEQ ID NO: 8 amino acid sequence of the canine PD-L1 protein. DETAILED DESCRIPTION

[0047] The following examples are provided:

[0048] 1. An anti-canine PD-L1 monoclonal antibody, the amino acid sequences of the complementarity determining regions of the light chain thereof are QISVSHNGYTN, KVS and FQGPWTSHV respectively, and the amino acid sequences of the complementarity determining regions of the heavy chain thereof are TDYLGYAF, GGSSGIHP and PIAYARND respectively.

[0049] 2. The anti-canine PD-L1 monoclonal antibody of Example 1, wherein the amino acid sequence of the light chain is shown as SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO: 2.

[0050] 3. The anti-canine PD-L1 monoclonal antibody of Example 2, wherein the antibody is secreted by the hybridoma cell with the accession number of CGMCC No. 46336.

[0051] 4. A hybridoma cell secreting the anti-canine PD-L1 monoclonal antibody, with the accession number of CGMCC No. 46336.

[0052] 5. An anti-cancer drug comprising the anti-canine PD-L1 monoclonal antibody of any one of Examples 1-3.

[0053] 6. The anti-cancer drug of Example 5, wherein the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0054] 7. The anti-cancer drug of Example 6, wherein the drug is an injection or an oral preparation.

[0055] 8. The anti-cancer drug of any one of Examples 5-7, wherein the drug is capable of effectively inhibiting a malignant tumor of a canine.

[0056] 9. Use of the anti-canine PD-L1 monoclonal antibody of any one of Examples 1-3 in the preparation of an anti-cancer drug.

[0057] 10. A method for preparing an anticancer drug, comprising: culturing the hybridoma cell with the accession number CGMCC No. 46336, isolating and purifying the monoclonal antibody secreted by the hybridoma cell, and taking the monoclonal antibody as the pharmaceutical effective component of the anticancer drug.

[0058] The application will be further described in detail below with reference to examples. It should be understood that the following examples are only used to explain and illustrate the application, and do not limit the scope of the application in any way.

[0059] The BALB / c mice used in the following examples were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0060] The mouse myeloma cells (sp2 / 0), canine kidney epithelial cells (MDCK), canine breast cancer cells (U27) and mouse breast cancer cells (4T1) used in the following examples were purchased from the American Type Culture Collection (ATCC), wherein the sp2 / 0 cells were numbered as ATCC CRL-1581, the MDCK cells were numbered as ATCC CCL-34, the U27 cells were numbered as ATCC CRL-3456, and the 4T1 cells were numbered as ATCC CRL-2539.

[0061] The canine tubular adenocarcinoma cells (CMT-1026) used in the following examples were isolated from the breast cancer tissue of a clinical canine, and the cells were deposited in the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC No. 22349.

[0062] The reagents not specifically described in the following examples are all conventional reagents in the art, which can be commercially available or prepared according to conventional methods in the art. The experimental methods not specifically described in the following examples are all conventional experimental methods in the art, which can be referred to relevant experimental manuals, known documents or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0063] Example 1

[0064] Obtaining of the anti-canine PD-L1 monoclonal antibody

[0065] The amino acid sequence of canine programmed death receptor ligand 1 (PD-L1) (Genbank accession number: NP_001278901) was searched in the database NCBI (National Center for Biotechnology Information). The canine PD-L1 was expressed in prokaryotes using the prokaryotic expression vector pET-28a and E. coli BL21, and the canine PD-L1 protein (SEQ ID NO: 8) was obtained by purification. The purified canine PD-L1 protein (SEQ ID NO: 8) was used as an antigen to immunize mice, and hybridoma cell strains were screened to prepare an anti-canine PD-L1 monoclonal antibody.

[0066] 1. Immunization of mice

[0067] Four 8-week-old BALB / c mice of SPF level were selected, numbered 1, 2, 3, and 4. The four mice were initially injected intraperitoneally with 60 μg of purified canine PD-L1 protein, and were boosted every 14 days with 30 μg of purified canine PD-L1 protein. After three boosts, the mice were bled from the orbit, and the serum antibody titer was measured by ELISA. The details are as follows:

[0068] (1) The purified canine PD-L1 protein was diluted to 2 μg / mL and coated in a 96-well enzyme-labeled plate at 100 μL per well, and incubated at 4°C for 12 hours.

[0069] (2) The canine PD-L1 protein in the enzyme-labeled plate was tapped off, and the plate was washed three times with PBST for 30 seconds each time, and the PBST was shaken off.

[0070] (3) 2% BSA blocking solution (prepared with PBS) was added at 100 μL per well, and incubated at 37°C for 1 hour.

[0071] (4) The blocking solution in the enzyme-labeled plate was tapped off, and washed three times with PBST for 30 seconds each time, and the PBST was shaken off.

[0072] (5) The immune mouse serum was diluted with 1×PBS at a ratio of 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, and 1:102400, respectively, and then the diluted solution was added to the wells at 100 μL per well; blank control was added with 1×PBS at 100 μL per well; negative control was added with 1:200 diluted mouse negative serum at 100 μL per well. Incubate at 37°C for 2 hours.

[0073] (6) The liquid in the enzyme-labeled plate was tapped off, and washed three times with PBST for 30 seconds each time, and the PBST was shaken off.

[0074] (7) Add 100 μL of HRP-labeled goat anti-mouse secondary antibody (Jackson ImmunoResearch, 115-035-003 Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L); diluted 1:5000) diluted in 1× PBS to each well and incubate at 37°C for 1 hour.

[0075] (8) Tap off the liquid in the ELISA plate, wash it five times with PBST, 30 seconds each time, and spin dry the PBST.

[0076] (9) Add TMB single-component colorimetric solution for color development, 100 μL per well; after the desired color development is achieved, add 1 M HCL to terminate the color development, 100 μL per well.

[0077] (10) Read OD 450nm , the mouse serum antibody titer is greater than the maximum OD 450nm Minimum OD of / 2 450nm The dilution to which the reading corresponds.

[0078] The results are as follows Figure 1 As shown, the serum antibody titer of mouse No. 3 was the highest, reaching 1:51200. Mouse No. 3 was intraperitoneally injected with 50 μg of purified canine PD-L1 protein for pulse immunization.

[0079] 2. Cell fusion

[0080] Take the 3rd mouse 3 days after impact immunization, take the eyeball to take blood, then pull the neck to death, soak in 75% alcohol for 5 min. Pour a small amount of serum-free IMDM medium into the dish, put the cell screen and syringe inner core into the dish. Take off the mouse's spleen with scissors and tweezers and put it on the cell screen. Gently crush the spleen with the syringe inner core, and suck the obtained spleen cells into a centrifuge tube containing sp2 / 0 cells (mouse myeloma cells), centrifuge at 1500 rpm for 5 min. (At the same time, take off the mouse's thymus with scissors and tweezers and crush it, and transfer the obtained thymus cells to a 15 mL centrifuge tube, add 2 mL of HAT medium and 1 mL of HT, and place it in the incubator for standby. ) After centrifugation, pour off the supernatant, and gently blow the spleen cells and sp2 / 0 cells with serum-free IMDM medium, centrifuge at 1500 rpm for 5 min. Try to pour off the supernatant after centrifugation, and tap the tube bottom to suspend the cells. Put the centrifuge tube into warm water at 37°C, slowly add 1 mL of PEG (polyethylene glycol) within 1 min, and then stand in warm water for 1 min. Slowly add 2 mL of serum-free IMDM medium within 2 min, then slowly add 8 mL of serum-free IMDM medium within 2 min. Centrifuge at 1000 rpm for 5 min. Pour off the supernatant, add 10 mL of newborn calf serum, and carefully blow the cells, then pour into the thymus cells prepared in advance. Add sterilized semi-solid medium to 50 mL, mix well, and pour into 30 cell culture dishes. Put the cell culture dishes into a humid box, then put them into the incubator for culture.

[0081] 3. Screening of hybridoma cells

[0082] After cell fusion, single cell clones are selected and cultured in 96-well cell culture plates (previously plated with thymus cells, 100 μL / well). Discard all the supernatant of the single clone cells in the 96-well cell culture plate, add IMDM medium containing 20% newborn calf serum (containing HT), 200 μL / well, for the first screening.

[0083] The purified canine PD-L1 protein was used to coat the enzyme-labeled plate, and the first screening of the monoclonal cells was subjected to a second screening by ELISA. The method was as follows: canine PD-L1 protein was diluted with coating solution to a final concentration of 2 μg / mL, and 100 μL / well was added to the enzyme-labeled plate, which was incubated at 4°C overnight; the liquid in the enzyme-labeled plate was tapped off, and PBST was washed 3 times for 30 s each time, and the PBST was shaken dry; blocking solution (2% skim milk powder) was added, 200 μL / well, and incubated at 37°C for 2 h; the liquid in the enzyme-labeled plate was tapped off, and PBST was washed 3 times for 30 s each time, and the PBST was shaken dry; monoclonal cell culture supernatant (primary antibody), sp2 / 0 cell culture supernatant (negative control), PBS (blank control), and mouse positive serum diluted 1000 times with PBS (positive control) were added to each well, 100 μL / well, and incubated at 37°C for 1 h; the liquid in the enzyme-labeled plate was tapped off, and PBST was washed 3 times for 30 s each time, and the PBST was shaken dry; secondary antibody (Jackson ImmunoResearch, 115-035-003 Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L), diluted 20000 times with PBS) was added, 100 μL / well, and incubated at 37°C for 1 h; the liquid in the enzyme-labeled plate was tapped off, and PBST was washed 3 times for 30 s each time, and the PBST was shaken dry; color developing solution was added, 100 μL / well, and color developed for about 10 min; termination solution was added to terminate the reaction, 50 μL / well; the absorbance was measured at dual wavelengths (450 nm, 630 nm), and the data was recorded and saved. After the second screening, 40 hybridoma cell strains were obtained.

[0084] The enzyme-labeled plate was coated with canine PD-L1 protein and His-tag protein, respectively, and the 40 hybridoma cell strains obtained in the second screening were subjected to a third screening by ELISA, and the method was the same as the second screening. After the third screening, 1 positive hybridoma cell strain was obtained, which was named CPDL5.

[0085] 4. Monoclonal antibody subclass identification

[0086] The monoclonal antibody secreted by the positive hybridoma cell strain CPDL5 was identified by a monoclonal antibody subclass identification kit (subclass coating antibody: 1010-01; Goat anti-mouse IgM-HRP: 1020-05; Goat Anti-Mouse IgG1, Human ads-HRP: 1070-05; Goat Anti-Mouse IgG2a, Human ads-HRP: 1080-05; Goat Anti-Mouse IgG2b, Human ads-HRP: 1090-05; Goat Anti-Mouse IgG3, Human ads-HRP: 1100-05; Goat Anti-Mouse IgA-HRP: 1040-05; Goat Anti-Mouse Kappa-HRP: 1090-05; Goat Anti-Mouse Lambda-HRP: 1060-05) of Beijing Xingboshiping Biotechnology Co., Ltd. according to the kit instructions.

[0087] The method is as follows: dilute the "subclass coating antibody" with coating solution to a final concentration of 2 μg / mL, add to the enzyme-labeled plate, 100 μL / well, 4°C overnight; tap off the liquid in the enzyme-labeled plate, wash with PBST for 3 times, 30 s each time, and spin dry PBST; add blocking solution (2% skimmed milk powder), 200 μL / well, 37°C for 2 h; tap off the liquid in the enzyme-labeled plate, wash with PBST for 3 times, 30 s each time, and spin dry PBST; add CPDL5 cell culture supernatant (primary antibody) and sp2 / 0 cell culture supernatant (negative control) to each well, 100 μL / well, 37°C for 1 h; tap off the liquid in the enzyme-labeled plate, wash with PBST for 3 times, 30 s each time, and spin dry PBST; dilute each type of subclass secondary antibody with PBS, add to the appropriate wells, 100 μL / well, 37°C for 1 h; tap off the liquid in the enzyme-labeled plate, wash with PBST for 3 times, 30 s each time, and spin dry PBST; add color developing solution, 100 μL / well, color develop for about 10 min; add stop solution to stop the reaction, 50 μL / well; measure the absorbance at 450 nm and 630 nm, record and save the data.

[0088] The monoclonal antibody secreted by the hybridoma cell strain CPDL5 was identified as IgG1 by subclass identification. The monoclonal antibody was named mAb-CPDL5.

[0089] 5. Purification of monoclonal antibody

[0090] The hybridoma cell CPDL5 was inoculated into the abdominal cavity of a mouse, and ascites was collected and purified by the ammonium caprylate sulfate method. The specific steps are as follows: 2 parts of 0.06 mol / L, pH 5.0 acetic acid buffer was added to 1 part of pretreated ascites, and the pH was adjusted to 4.5 with 0.1 mol / L HCL; slowly add caprylic acid drop by drop under stirring at room temperature, add 33 μL of caprylic acid per milliliter of pre-diluted ascites, stand at 4°C for 2h, then centrifuge at 15000 rpm for 30 min, discard the precipitate; after filtering the supernatant, add 1 / 10 volume of 0.1 mol / L PBS, pH 7.4, 8.5% NaCl, and adjust the pH to 7.4 with 1 mol / L NaOH; add 0.277 g of ammonium sulfate per milliliter of the mixture, and add it within 30 min under 4°C ice bath, stand for more than 1 h; centrifuge at 4°C 12000 rpm for 30 min, discard the supernatant; dissolve the precipitate in appropriate amount of PBS (pH 7.4) containing 137 mmol / L NaCl, 2.6 mmol / L KCl, 0.2 mmol / L EDTA, and dialyze 50-100 times the above PBS at 4°C overnight; centrifuge at 4°C 12000 rpm for 30 min to remove insoluble residues; measure the IgG content by spectrophotometer, the formula is as follows: IgG content = (1.45 OD 280 -0.74 OD 260 ) × sample dilution factor.

[0091] After purification by the ammonium caprylate sulfate method, a monoclonal antibody mAb-CPDL5 with a concentration of 2.75 mg / mL was obtained.

[0092] 6. Western blot detection of monoclonal antibody

[0093] Prepare a polyacrylamide gel. Take an appropriate amount of canine breast cancer cells (U27), extract the total protein and dilute to 40 μL, add 5x denaturing non-reducing buffer at a volume ratio of 4:1, boil in a 100°C metal bath for 5 minutes, centrifuge at 12,000g for 5 minutes, and obtain the protein sample. Add 20 μL of protein sample per well, and perform polyacrylamide gel electrophoresis at a constant voltage of 80V until the indicator reaches the bottom of the gel plate. After electrophoresis, cut the gel at the desired protein size, cut 6 pieces of filter paper slightly larger than the gel, and cut a piece of PVDF membrane the same size as the gel. Soak the gel and filter paper in the electrotransfer buffer. Soak the PVDF membrane in methanol for 30 seconds with tweezers, then take it out, and activate the PVDF membrane's ability to absorb protein. According to the order of negative electrode, 3 pieces of filter paper, protein gel, PVDF membrane, 3 pieces of filter paper, and positive electrode, layer by layer, slowly squeeze out the air bubbles in the middle with tweezers to prevent air bubbles from blocking protein transfer. Perform electrotransfer in an ice bath to ensure low temperature throughout to prevent overheating from affecting the transfer effect. Set the electrophoresis instrument to 300mA constant current for 1 hour.

[0094] After the end of the electrotransfer, the PVDF membrane was taken out with tweezers to prevent the PVDF membrane from sticking to other proteins, and the membrane was washed 3 times with PBST buffer. Blocking: 5% skimmed milk powder was prepared with PBS buffer as blocking solution, and the PVDF membrane was blocked at room temperature for 2 hours. The PVDF membrane was taken out and washed 3 times with PBST buffer. First antibody incubation: the purified monoclonal antibody mAb-CPDL5 with a concentration of 2.75 mg / mL was diluted 1:1000 with blocking solution and added to the protein binding surface of the PVDF membrane, and incubated on a shaker at room temperature for 2 hours. The PVDF membrane was taken out and washed 3 times with PBST buffer. Second antibody incubation: the HRP-labeled goat anti-mouse secondary antibody (Jackson ImmunoResearch, 115-035-003 Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L)) was diluted 1:5000 with blocking solution and added to the protein binding surface of the PVDF membrane, and incubated on a shaker at room temperature for 2 hours. The PVDF membrane was taken out and washed 3 times with PBST buffer. ECL development: the luminescent agent and stabilizer in the ECL luminescent kit (MCE, Cat. No.: HY-K2005) were mixed in a volume ratio of 1:1 to make them uniform, and then added dropwise to the PVDF membrane. The PVDF membrane was placed in the gel imaging system for exposure and photography.

[0095] The results are shown in Figure 2 The monoclonal antibody mAb-CPDL5 can specifically bind to the components of U27 canine breast cancer cells.

[0096] 7. Sequence analysis of monoclonal antibody

[0097] The obtained monoclonal antibody mAb-CPDL5 was sequenced, and the light chain sequence and heavy chain sequence of the antibody were analyzed using geneious software, and the complementarity determining region (complementarity determining region, CDR) was determined, and the results are as follows.

[0098] Light chain sequence of mAb-CPDL5:

[0099] GDILMTQSPLSLPVSLGDQASISCRSSQISVSHNGYTNVEWYLQKAGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGPWTSHVFGGGTKLEIKRADAAPTGSIF (SEQ ID NO: 1)

[0100] Heavy chain sequence of mAb-CPDL5:

[0101] EFQVKLQESGPELVRPGTSVKVSCKASTDYLGYAFIEWVKQRPGQGLEWIGVGGSSGIHPKYNEKFKGKATLTSDKSSSTAYMQLSSLTSDDSAVYFCPIAYARNDWGQGTLVTVSAAKTTPPSVYPLAPVCGDKL (SEQ ID NO: 2)

[0102] The complementarity determining regions of the light chain are denoted LCDR1, LCDR2 and LCDR3, respectively, and have the following amino acid sequences:

[0103] LCDR1: QISVSHNGYTN (SEQ ID NO: 3)

[0104] LCDR2: KVS

[0105] LCDR3: FQGPWTSHV (SEQ ID NO: 4)

[0106] The complementarity determining regions of the heavy chain are denoted HCDR1, HCDR2 and HCDR3, respectively, and have the following amino acid sequences:

[0107] HCDR1: TDYLGYAF (SEQ ID NO: 5)

[0108] HCDR2: GGSSGIHP (SEQ ID NO: 6)

[0109] HCDR3: PIAYARND (SEQ ID NO: 7)

[0110] 8. Preservation of hybridoma cells

[0111] The above hybridoma cell CPDL5 was sent to the China General Microbiological Culture Collection Center for preservation, with the preservation name CPDL5, the preservation number CGMCC No. 46336, and the preservation date March 28, 2025.

[0112] Example 2

[0113] Cellular immunofluorescence experiment of anti-canine PD-L1 monoclonal antibody with different types of cancer cells

[0114] The binding of the monoclonal antibody mAb-CPDL5 obtained in Example 1 to the cell surface PD-L1 protein of different types of cancer cells was analyzed by cellular immunofluorescence experiment. The different types of cancer cells used in this experiment were canine ductal adenocarcinoma cells (CMT-1026), canine kidney epithelial cells (MDCK) and canine breast cancer cells (U27), respectively.

[0115] The experimental method is as follows:

[0116] 1) Take out the different kinds of cancer cell cell crawl sheet that grows 48 hours, move into a clean six hole board, soak in cold PBS no more than 3 minutes.

[0117] 2) Each glass drops 4% of polyethylene glycol, fixes on ice for 14 minutes. Note, to be horizontal, cover evenly. (Prepare to block serum, dilute according to 1:10 with PBS-Triton, shake mix well).

[0118] 3) Wash the cell crawl sheet with pre-cooled PBS for 5 minutes, repeat 2 times, shake bed 150 rpm, wash gently.

[0119] 4) Wipe the edge of the crawl sheet, draw a circle around the edge of the crawl sheet with a DOCK pen (to prevent the reagent added on the crawl sheet from losing tension and causing dry sheet).

[0120] 5) Put the crawl sheet into the corresponding hole of the lower row and the upper row mark of the six hole board respectively (the blocking serum is added, the board is dry at this time, the next board is dry).

[0121] 6) Add 10% normal blocking serum to each crawl sheet with a micropipette, block for 30 min in a humid box at room temperature. (Prepare primary antibody: use monoclonal antibody mAb-CPDL5 as primary antibody, dilute the antibody with PBS-Triton according to 1:100, shake mix well).

[0122] 7) Primary antibody incubation: carefully add the already diluted primary antibody with a micropipette (remove the blocking liquid from each sheet, wipe dry the outside of the circle, add the primary antibody, note: do not touch the cells), place in a humid box, incubate overnight in a 4℃ refrigerator, reserve the remaining primary antibody.

[0123] 8) The next day, gently remove the cover, move to room temperature, incubate for 1 hour in a humid box. Note not to dry the sheet.

[0124] 9) Wash with PBS-Tween 3 times, 5 min each time.

[0125] 10) Wipe dry the four corners of the crawl sheet, then put it back into the original hole of the first row of the six hole board respectively. Add FITC labeled goat anti-mouse secondary antibody (FITC goat anti-mouse IgG (Abeam, ab6785), dilute according to 1:500 with PBS-Triton) evenly in the dark, place flat, incubate for 1 hour at room temperature (as the reagent will volatilize, check and add the secondary antibody appropriately to avoid dry sheet).

[0126] 11) After the crawl sheet is dried, add a drop of blue fluorescent DNA dye DAPI, incubate for 4 min at room temperature, wash with PBS-Tween 3 times, shake by hand.

[0127] 12) After the slide has dried in a dark place, add a drop of mounting medium to the slide, place the slide upside down on the mounting medium, and seal the slide with nail polish (to prevent the mounting medium from spilling out and to secure the slide). As fluorescence intensity decreases with light stimulation, take a picture as soon as possible.

[0128] The results are as follows Figure 3 As shown, the surfaces of canine ductal adenocarcinoma cells (CMT-1026), canine renal epithelial cells (MDCK), and canine breast cancer cells (U27) all emit green fluorescence, indicating that the monoclonal antibody mAb-CPDL5 can specifically bind to the PD-L1 protein on the surface of these cells.

[0129] Example 3

[0130] Treatment of breast cancer-bearing mice with anti-canine PD-L1 monoclonal antibodies

[0131] The inhibitory effect of the monoclonal antibody mAb-CPDL5 obtained in Example 1 on tumor growth was detected by in vivo experiments in breast cancer tumor-bearing mice.

[0132] Ten female BALB / c mice, 4 weeks old, with an average weight of 13±2 g were selected. Mouse breast cancer cells (4T1) were subcutaneously injected into the fourth pair of mammary pads on the right breast of the mice. Each mouse was inoculated with 2.5×10 6 cells. On the 7th day after inoculation of cancer cells, 10 mice were randomly divided into two groups, with 5 mice in each group, which were set as the control group and the treatment group respectively. The control group was given 0.9% normal saline at a dose of 200 μL / mouse. The treatment group was given the monoclonal antibody mAb-CPDL5 according to the weight of the mice, at a dose of 10 mg / kg body weight. An intraperitoneal injection was performed on the 7th and 10th days after inoculation of cancer cells, for a total of 2 doses. The weight of the mice was measured before each administration to determine the dosage of mAb-CPDL5, and the maximum and minimum diameters of the tumor were measured with a ruler. The experiment ended on the 13th day after inoculation of cancer cells. All nude mice were killed by cervical dislocation, and the in situ tumors in the body were taken, weighed, photographed, and the maximum and minimum diameters of the tumors were measured (unit: mm). The formula for calculating tumor volume is: V (mm 3 ) = maximum diameter × minimum diameter 2 / 2.

[0133] The results are as follows Figure 4 As shown, there was no significant difference in the body weight of mice between the control group and the treatment group ( Figure 4 A). Tumor volume of breast cancer-bearing mice treated with mAb-CPDL5 compared with the control group ( Figure 4 B) and tumor mass ( Figure 4 C) were significantly decreased, indicating that mAb-CPDL5 can effectively inhibit tumor growth.

[0134] Example 4

[0135] Immunohistochemistry experiment of anti-canine PD-L1 monoclonal antibody with different types of canine cancer tissues

[0136] The binding of the monoclonal antibody mAb-CPDL5 obtained in Example 1 to cells expressing PD-L1 in different types of canine cancer tissues was analyzed by immunohistochemistry experiment. The canine cancer tissues used in the experiment were from canine uterine smooth muscle malignant tumor, canine skin melanoma malignant tumor, canine oral malignant tumor and canine breast malignant tumor, taken from the diseased dogs diagnosed in animal hospitals in Beijing area.

[0137] The experimental method is as follows:

[0138] 1) Different types of canine cancer tissues (canine uterine smooth muscle malignant tumor, canine skin melanoma malignant tumor, canine oral malignant tumor and canine breast malignant tumor) were taken respectively to make paraffin sections.

[0139] 2) The antibody was tested by immunohistochemical staining using an automatic staining machine. The deparaffinization and hydration conditions were used, and the specific steps were as follows: incubation at 60°C for 30 min, and washing with deparaffinization solution for 3 times.

[0140] 3) 150 μL of endogenous peroxidase blocking solution was added to the tissue section, and incubated at room temperature for 5 min. The section was washed with PBS for 3 times.

[0141] 4) The antigen repair used citrate antigen repair solution. The repair solution was preheated in a microwave oven, high fire for 2 min, soaking in repair solution for 2 min, then low fire for 3 min, avoiding boiling, room temperature balance for 2 min, repeated heating for a total of 4 times, and low fire for 5 min repeated twice, natural cooling for 1 h.

[0142] 5) The blocking solution (5% BSA) was added to the tissue section, and incubated at room temperature for 10 min. The excess liquid was shaken off.

[0143] 6) The purified monoclonal antibody mAb-CPDL5 in Example 1 was used as the primary antibody. The mAb-CPDL5 was diluted to a final concentration of 1 μg / mL using antibody diluent. 150 μL of the primary antibody diluent was added to the tissue section, and incubated at room temperature for 30 min. The section was washed with PBS for 3 times.

[0144] 7) The secondary antibody (Jackson ImmunoResearch, 115-035-003 Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L)) was diluted according to 1:200 using antibody diluent. 150 μL of the secondary antibody diluent was added to the tissue section, and incubated at room temperature for 8 min. The section was washed with PBS for 3 times.

[0145] 8) Add 150 μL DAB chromogenic solution drop on the tissue section, incubate at room temperature for 10 min. Rinse the section with tap water, counterstain with hematoxylin, incubate at room temperature for 5 min.

[0146] 9) Rinse with deionized water for 1 min, 95% ethanol for 1 min, 100% ethanol twice, xylene once, neutral balsam mounting, and microscopic examination.

[0147] The results are shown in Figure 5 The monoclonal antibody mAb-CPDL5 can specifically bind to canine uterine smooth muscle malignant tumor, canine skin melanin malignant tumor, canine oral malignant tumor and canine breast malignant tumor cells, and the PD-L1 positive cells are brown.

Claims

1. An anti-canine PD-L1 monoclonal antibody, wherein the amino acid sequences of the complementary determining regions of its light chain are QISVSHNGYTN, KVS, and FQGPWTSHV, respectively, and the amino acid sequences of the complementary determining regions of its heavy chain are TDYLGYAF, GGSSGIHP, and PIAYARND, respectively.

2. The anti-canine PD-L1 monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO:

2.

3. The anti-canine PD-L1 monoclonal antibody according to claim 2, characterized in that The antibody is secreted by the hybridoma cell with a deposit number of CGMCC No.46336.

4. A hybridoma cell secreting anti-canine PD-L1 monoclonal antibody, whose deposit number is CGMCC No.46336.

5. An anticancer drug comprising the anti-canine PD-L1 monoclonal antibody according to any one of claims 1 to 3.

6. The anticancer drug according to claim 5, characterized in that The drug further comprises a pharmaceutically acceptable carrier or excipient.

7. The anticancer drug according to claim 6, characterized in that The medicine is an injection or oral preparation.

8. The anticancer drug according to any one of claims 5 to 7, characterized in that The medicine can effectively inhibit malignant tumors in canines.

9. Use of the anti-canine PD-L1 monoclonal antibody according to any one of claims 1 to 3 in the preparation of anticancer drugs.

10. A method for preparing an anticancer drug, comprising: The hybridoma cells with a preservation number of CGMCC No. 46336 are cultured, the monoclonal antibodies secreted by the hybridoma cells are separated and purified, and the monoclonal antibodies are used as the active ingredients of the anticancer drug.

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