Chimeric monoclonal antibodies targeting canine nerve growth factor and uses thereof

Abstract

The application discloses chimeric monoclonal antibodies targeting canine nerve growth factor and application thereof, and belongs to the technical field of biological medicine. The application provides canine-mouse chimeric monoclonal antibodies c1D4 and c3E7, wherein the amino acid sequence of the heavy chain variable region VH of c1D4 is shown as SEQ ID NO. 6, the amino acid sequence of the light chain variable region VL is shown as SEQ ID NO. 13; the amino acid sequence of the heavy chain variable region VH of c3E7 is shown as SEQ ID NO. 20, the amino acid sequence of the light chain variable region VL is shown as SEQ ID NO. 25, the amino acid sequence of the heavy chain constant region CH is shown as SEQ ID NO. 11, and the amino acid sequence of the light chain constant region CL is shown as SEQ ID NO. 18. It is verified that c1D4 and c3E7 have therapeutic activity in a mouse osteoarthritis pain model induced by MIA and a canine osteoarthritis pain model induced by LPS.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to chimeric monoclonal antibodies targeting canine nerve growth factor and their applications. Background Technology

[0002] Osteoarthritis (OA) is a chronic, progressive joint disease characterized by degeneration of articular cartilage, synovitis, subchondral bone remodeling, and changes in periarticular tissues. Pain is one of the most prominent clinical manifestations of OA, leading to decreased mobility and limited joint function in affected dogs, thus impacting their quality of life. OA and its related clinical symptoms have been reported to have a high incidence in dogs, becoming a significant disease affecting the health and welfare of companion animals. Currently, clinical treatment primarily aims to relieve pain, improve joint function, and enhance the animal's quality of life. Non-steroidal anti-inflammatory drugs (NSAIDs) remain commonly used for pain management in canine OA; however, some dogs may experience gastrointestinal, renal, and hepatic adverse reactions during long-term use, and individual tolerance to these drugs varies. Therefore, it is necessary to develop therapeutic drugs with different mechanisms of action that can effectively relieve OA-related pain in dogs.

[0003] Nerve growth factor (NGF) is an important neurotrophic factor involved in the regulation of sensory nerve function. NGF participates in the sensitization of peripheral nociceptive neurons by binding to tyrosine receptor kinase A (TrkA) and activating downstream signaling pathways, and is closely related to the occurrence and maintenance of osteoarthritis (OA)-related pain. Anti-NGF antibodies can specifically bind to NGF and block the NGF / TrkA signaling pathway, thereby reducing peripheral pain sensitization and alleviating OA-related pain. Therefore, NGF has become one of the important targets for the treatment of OA-related pain.

[0004] Although existing technologies have been used to treat canine OA pain targeting NGF, it remains necessary to obtain antibodies with different antigen-binding sequences that can specifically bind to canine NGF and efficiently inhibit its biological activity. This would provide new antibody drug candidates for the treatment of canine OA and related pain, and also potentially provide a new technological foundation for the independent research and development of canine OA biotherapeutic drugs in my country. Summary of the Invention

[0005] The purpose of this invention is to provide chimeric monoclonal antibodies targeting canine nerve growth factor and their applications, thereby addressing the problems existing in the prior art. The canine-mouse chimeric monoclonal antibodies c1D4 and c3E7 provided by this invention possess high affinity, high blocking activity, and good safety. Verification has shown that c1D4 and c3E7 exhibit therapeutic activity in both MIA-induced mouse osteoarthritis pain models and LPS-induced canine osteoarthritis pain models. This invention provides new ideas and methods for the biotherapy of canine osteoarthritis and lays a technological foundation for the independent research and development of drugs for canine osteoarthritis.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a canine-mouse chimeric monoclonal antibody c1D4 that specifically binds to canine nerve growth factor. The canine-mouse chimeric monoclonal antibody c1D4 includes a murine heavy chain variable region VH, a murine light chain variable region VL, a canine heavy chain constant region CH, and a canine light chain constant region CL. The mouse-derived heavy chain variable region VH includes heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO.7, heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO.8, and heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO.9; The mouse-derived light chain variable region VL includes light chain CDR1 with the amino acid sequence shown in SEQ ID NO.14, light chain CDR2 with the amino acid sequence shown in SEQ ID NO.15, and light chain CDR3 with the amino acid sequence shown in SEQ ID NO.16; The amino acid sequence of the constant region CH of the canine heavy chain is shown in SEQ ID NO.11; The amino acid sequence of the constant region CL of the canine light chain is shown in SEQ ID NO.18.

[0007] Optionally, the amino acid sequence of the variable region VH of the mouse heavy chain is shown in SEQ ID NO.6; The amino acid sequence of the variable region VL of the mouse light chain is shown in SEQ ID NO.13.

[0008] Optionally, the nucleotide sequence of the gene encoding the variable region VH of the mouse heavy chain is shown in SEQ ID NO.5; The nucleotide sequence of the gene encoding the variable region VL of the mouse light chain is shown in SEQ ID NO.12.

[0009] The present invention also provides the use of the canine-mouse chimeric monoclonal antibody c1D4 in the preparation of a medicament for treating osteoarthritis and / or osteoarthritis-related pain.

[0010] The present invention also provides a medicament for treating osteoarthritis and / or osteoarthritis-related pain, wherein the medicament uses the canine-mouse chimeric monoclonal antibody c1D4 as the active ingredient.

[0011] The present invention also provides a canine-mouse chimeric monoclonal antibody c3E7 that specifically binds to canine nerve growth factor, wherein the canine-mouse chimeric monoclonal antibody c3E7 comprises a mouse heavy chain variable region VH, a mouse light chain variable region VL, a canine heavy chain constant region CH, and a canine light chain constant region CL. The mouse-derived heavy chain variable region VH includes heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO.21, heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO.22, and heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO.23; The mouse-derived light chain variable region VL includes light chain CDR1 with the amino acid sequence shown in SEQ ID NO.26, light chain CDR2 with the amino acid sequence shown in SEQ ID NO.27, and light chain CDR3 with the amino acid sequence shown in SEQ ID NO.28; The amino acid sequence of the constant region CH of the canine heavy chain is shown in SEQ ID NO.11; The amino acid sequence of the constant region CL of the canine light chain is shown in SEQ ID NO.18.

[0012] Optionally, the amino acid sequence of the variable region VH of the mouse heavy chain is shown in SEQ ID NO.20; The amino acid sequence of the variable region VL of the mouse light chain is shown in SEQ ID NO.25.

[0013] Optionally, the nucleotide sequence of the gene encoding the variable region VH of the mouse heavy chain is shown in SEQ ID NO.19; The nucleotide sequence of the gene encoding the variable region VL of the mouse light chain is shown in SEQ ID NO.24.

[0014] The present invention also provides the use of the canine-mouse chimeric monoclonal antibody c3E7 in the preparation of a medicament for treating osteoarthritis and / or osteoarthritis-related pain.

[0015] The present invention also provides a medicament for treating osteoarthritis and / or osteoarthritis-related pain, wherein the medicament uses the canine-mouse chimeric monoclonal antibody c3E7 as the active ingredient.

[0016] The present invention discloses the following technical effects: This invention provides two chimeric monoclonal antibodies, c1D4 and c3E7, with different antigen-binding sequences. The amino acid sequence of the heavy chain variable region VH (mouse-derived) of c1D4 is shown in SEQ ID NO. 6, the amino acid sequence of the light chain variable region VL (mouse-derived) is shown in SEQ ID NO. 13, the amino acid sequence of the heavy chain constant region CH (canine-derived) is shown in SEQ ID NO. 11, and the amino acid sequence of the light chain constant region CL (canine-derived) is shown in SEQ ID NO. 18. Similarly, the amino acid sequence of the heavy chain variable region VH (mouse-derived) of c3E7 is shown in SEQ ID NO. 20, the amino acid sequence of the light chain variable region VL (mouse-derived) is shown in SEQ ID NO. 25, the amino acid sequence of the heavy chain constant region CH is shown in SEQ ID NO. 11, and the amino acid sequence of the light chain constant region CL is shown in SEQ ID NO. 18. Experimental results show that c1D4 and c3E7 can specifically bind to canine NGF, exhibiting high affinity, high blocking activity, and good safety. Verification showed that c1D4 and c3E7 both exhibited therapeutic activity in MIA-induced and LPS-induced osteoarthritis-related pain models in mice. This invention provides new ideas and methods for the biological treatment of canine osteoarthritis and lays a technological foundation for the independent research and development of drugs for canine osteoarthritis and related pain. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The plasmid map of the recombinant expression plasmid pCMV-cNGF-Fc; Figure 2 The plasmid map of the recombinant expression plasmid pCMV-cNGF-GST; Figure 3 The plasmid map of the recombinant expression plasmid pCMV-cTrkA-Fc; Figure 4 The results of indirect ELISA detection of serum anti-canine NGF antibody titers in two immunized mice; Figure 5 The results of indirect ELISA detection of anti-canine NGF antibody titer in hybridoma cell culture supernatant; Figure 6 The results of indirect ELISA detection of anti-canine NGF antibody titer in hybridoma cell ascites fluid; Figure 7The results are from an indirect ELISA assay to detect the binding activity of 10 murine monoclonal antibodies to canine NGF. Figure 8 The results of competitive ELISA detection for blocking canine NGF-TrkA with 10 murine monoclonal antibodies; Figure 9 The results show the detection of the inhibition of canine NGF-induced TF-1 cell proliferation by 10 murine monoclonal antibodies. Figure 10 The results of detecting the inhibition of canine NGF-induced PC-12 cell differentiation by 10 murine monoclonal antibodies; Figure 11 The results are from an indirect ELISA assay to detect the binding activity of six canine-mouse chimeric monoclonal antibodies to canine NGF. Figure 12 The results of competitive ELISA detection for blocking canine NGF-TrkA with 6 chimeric canine-mouse antibodies; Figure 13 The results of detecting the inhibition of canine NGF-induced TF-1 cell proliferation by six chimeric canine and mouse antibodies; Figure 14 The results show the detection of the inhibition of canine NGF-induced PC-12 cell differentiation by six chimeric canine and mouse antibodies. Figure 15 The results show the changes in body weight of mice in each group in a MIA-induced mouse osteoarthritis model. Figure 16 The results of electronic Von Frey assays in each group of mice in a MIA-induced mouse osteoarthritis model; Figure 17 The results of the bipedal balance test in each group of mice in the MIA-induced mouse osteoarthritis model; Figure 18 The standing scores of dogs in each group on day 6 in the LPS-induced canine osteoarthritis pain model. Figure 19 The results of single hind limb weight-bearing scores of dogs in each group on day 6 in the LPS-induced canine osteoarthritis pain model; Figure 20 The results of dynamic scores of dogs in each group on day 6 in the LPS-induced canine osteoarthritis pain model; Figure 21 The results of the comprehensive scores of each group of dogs on day 6 in the LPS-induced canine osteoarthritis pain model; Figure 22 The standing scores of dogs in each group on day 28 in the LPS-induced canine osteoarthritis pain model. Figure 23 The results of single hind limb weight-bearing scores of dogs in each group on day 28 in the LPS-induced canine osteoarthritis pain model; Figure 24 The results of dynamic scores of dogs in each group on day 28 in the LPS-induced canine osteoarthritis pain model; Figure 25 The results show the comprehensive scores of dogs in each group on day 28 in the LPS-induced canine osteoarthritis pain model. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1 1. Experimental Materials 1.1 Biomaterials PC-12 cells, TF-1 cells, and SP2 / 0 cells were purchased from ATCC; HEK-293F cells were purchased from Zhuhai Kairui Biotechnology Co., Ltd. SPF-grade BALB / c female mice aged 6-8 weeks and 8-10 weeks, and SPF-grade C57BL / 6 male mice aged 8-10 weeks were purchased from Hubei Provincial Center for Disease Control and Prevention. Healthy Beagle dogs aged 1-1.5 years were purchased from Jinan Xilingjiao Breeding Center.

[0025] 1.2 Main Instruments and Equipment The refrigerated centrifuge was purchased from Eppendorf (Germany); the constant voltage and constant current electrophoresis apparatus was purchased from Beijing Liuyi Instrument Factory; the vertical electrophoresis tank and matching SDS-PAGE gel preparation device were purchased from Wuhan Boster Biological Engineering Co., Ltd.; the Syngene protein gel imaging system (USA) was purchased from LABCONCO (USA); the CO2 cell culture incubator was purchased from Sanyo (Japan); the fluorescence inverted microscope was purchased from OLYMPUS (Japan); the ultrapure water system was purchased from Milpore (USA); the ultra-high speed refrigerated centrifuge was purchased from Beckman Coulter (USA); the rapid shaker was purchased from Haimen Qilin Bell Instrument Manufacturing Co., Ltd.; the multi-functional microplate reader was purchased from BMG LABTECH (Germany); and the AKTA Pure 25 protein purification system was purchased from GE Healthcare (USA).

[0026] 1.3 Routine Biochemical Reagents Freund's complete adjuvant, Freund's incomplete adjuvant, HAT medium additive (50×), HT medium additive (50×), polyethylene glycol (PEG) cell fusion reagent, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; penicillin-streptomycin solution (100×) was purchased from Beijing Beyotime Biotechnology Co., Ltd.; DNA gel extraction kit, HiScript II Reverse Transcriptase, and Phanta Flash Super-Fidelity DNA Polymerase were purchased from Nanjing Novizan Biotechnology Co., Ltd.; HEK-293F serum-free suspension medium, KPM, TA-293, KE-293, and KT-Feed were purchased from Zhuhai Kairui Biotechnology Co., Ltd.; RPMI-1640 medium was purchased from Gibco; fetal bovine serum (FBS) and horse serum (HS) were purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; HRP-goat anti-mouse IgG antibody and HRP-goat anti-canine IgG were purchased from Wuhan Boster Biological Products Co., Ltd.; EZ-Link The Sulfo-NHS-LC-Biotin kit and Alexa Fluor 488-labeled anti-rabbit IgG fluorescent secondary antibody were purchased from Thermo Fisher Scientific; skim milk powder was purchased from Yili; the whole genome extraction kit and removable polystyrene microplate were purchased from Wuhan Keqian Biotechnology Co., Ltd.; filter paper and TMB chromogenic solution were purchased from Fiyobio Biotechnology Co., Ltd.; and the mouse monoclonal antibody subtype identification kit (catalog number SEK003) was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. All other chemical reagents were of analytical grade unless otherwise specified.

[0027] 1.4 Preparation of cell culture medium 1×Phosphate-buffered saline (PBS): Weigh out 8.0 g of NaCl, 0.2 g of KCl, 1.42 g of Na2HPO4 and 0.27 g of KH2PO4, respectively, and dissolve them thoroughly in 800 mL of ultrapure water. Adjust the pH to 7.4 using NaOH or HCl solution, add ultrapure water to a final volume of 1 L, sterilize, and store at room temperature.

[0028] Hybridoma cell culture medium: RPMI-1640 medium with 20% (v / v) FBS and 1% (v / v) penicillin-streptomycin solution added.

[0029] HAT cell culture medium: RPMI-1640 medium supplemented with 2% (v / v) HAT medium additive (50×), 20% (v / v) FBS and 1% (v / v) penicillin-streptomycin solution.

[0030] HT cell culture medium: RPMI-1640 medium with 2% (v / v) HT medium additive (50×), 20% (v / v) FBS and 1% (v / v) penicillin-streptomycin solution added.

[0031] PC-12 cell culture medium: RPMI-1640 medium supplemented with 15% (v / v) horse serum, 5% (v / v) FBS and 1% (v / v) penicillin-streptomycin solution.

[0032] TF-1 cell culture medium: RPMI-1640 medium with 10% (v / v) FBS, 1% (v / v) penicillin-streptomycin solution and a final concentration of 2 ng / mL recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF).

[0033] SP2 / 0 cell culture medium: RPMI-1640 medium with 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin solution added.

[0034] 0.25% trypsin solution: Weigh 2.5 g of trypsin, add 1 L of 1×PBS (pH 7.4) and dissolve thoroughly. Filter through a 0.22 µm sterile filter membrane for sterilization, aliquot, and store at -20℃ for later use.

[0035] Cell cryopreservation solution: Prepared by mixing the corresponding basal culture medium, FBS and DMSO in a volume ratio of 8:1:1 according to the basal culture medium used for different cells, i.e., containing 80% (v / v) basal culture medium, 10% (v / v) FBS and 10% (v / v) DMSO. Prepare fresh before use.

[0036] 1.5 Preparation of purification reagents for ascites fluid and FC-tagged fusion protein 2× Equilibrium Buffer: Weigh 4.846 g of Tris-base and 292.5 g of NaCl, add 800 mL of ultrapure water and dissolve thoroughly. Adjust the pH to 8.0 using NaOH or HCl solution, add ultrapure water to bring the volume to 1 L, filter through a 0.22 µm filter membrane and store at room temperature.

[0037] 1×Equilibrium Buffer: Weigh 2.429 g of Tris-base and 146.25 g of NaCl, add 800 mL of ultrapure water and dissolve thoroughly. Adjust the pH to 8.0 using NaOH or HCl solution, add ultrapure water to bring the volume to 1 L, filter through a 0.22 µm filter membrane and store at room temperature.

[0038] Washing Buffer: Weigh 146.25 g of NaCl, dissolve it completely in 800 mL of 2× PBS, adjust the pH to 8.0 using NaOH or HCl solution, add 2× PBS to bring the volume to 1 L, filter through a 0.22 µm filter membrane, and store at room temperature.

[0039] Elution Buffer (0.1 mol / L Glycine-HCl buffer): Weigh 7.507 g of glycine, add 800 mL of ultrapure water to dissolve it completely, adjust the pH to 2.7 using NaOH or HCl solution, add ultrapure water to make up to 1 L, filter through a 0.22 µm filter membrane and store at room temperature.

[0040] 1.6 Preparation of ELISA-related reagents Carbonate coating buffer (pH 9.6): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, add an appropriate amount of water for injection to dissolve them completely, adjust the pH to 9.6 with NaOH solution or HCl solution, and bring the volume to 1 L with water for injection. Store at room temperature for later use.

[0041] Blocking solution: Weigh 5.00 g of bovine serum albumin (BSA), 8.50 g of NaCl and 0.20 g of sodium thimerosal, add 0.50 mL of Tween-20, dissolve thoroughly with water for injection and bring the volume to 1000 mL, store at 4℃ for later use.

[0042] Sample diluent: 1×PBS (pH 7.4).

[0043] Secondary antibody protectant: Weigh out 5.00 g of BSA, 8.00 g of NaCl, 2.90 g of Na2HPO4·12H2O, 0.20 g of KH2PO4, 0.20 g of KCl, 2.00 g of sucrose and 0.20 g of sodium thimerosal, add 0.50 mL of Tween-20, dissolve thoroughly with an appropriate amount of water for injection and bring the volume to 100 mL, store at 4℃ for later use.

[0044] 20× Concentrated Washing Solution: Weigh 170.00 g of NaCl, add 10.00 mL of Tween-20, dissolve thoroughly with an appropriate amount of water for injection, and bring the volume to 1000 mL. Store at room temperature for later use.

[0045] Substrate solution A: Weigh 14.60 g of Na2HPO4·12H2O, 9.33 g of citric acid and 0.52 g of hydrogen peroxide urea, add an appropriate amount of water for injection to dissolve them completely and make up to 1000 mL, adjust the pH to 5.0-5.4, and store at 4℃ for later use.

[0046] Substrate solution B: Weigh 20.00 mg of 3,3',5,5'-tetramethylbenzidine (TMB), dissolve it in 10.00 mL of anhydrous ethanol, and then dilute to 1000 mL with water for injection. Store at 4°C for later use.

[0047] Termination solution: Measure 2.50 mL of hydrofluoric acid (HF) and slowly add it to 900 mL of water for injection. Mix well and then bring the volume up to 1000 mL with water for injection. Store at 4°C for later use.

[0048] 2. Expression and purification of canine NGF protein and canine TrkA protein Construction of the recombinant expression plasmid: pCMV-cNGF-Fc was completed by Beijing Sinocare Technology Co., Ltd. (pCMV-cNGF-Fc plasmid map can be found here). Figure 1 ), pCMV-cNGF-GST (plasmid map see) Figure 2 ) and pCMV-cTrkA-Fc (plasmid map see Figure 3 Synthesis and construction of recombinant plasmids.

[0049] HEK-293F cell culture: HEK-293F cells were cultured at 37℃ and 5% CO2 with shaking at 120 rpm; when passaged, cells were selected at a density of 3-6 × 10⁶ cells / year. 6 Cells with a density of 10 cells / mL and a viability of not less than 95% were adjusted to a cell density of 0.6 × 10⁶ cells / mL using fresh serum-free suspension medium. 6 Cells / mL, passaged every 3 days, and cell viability maintained above 95% during culture.

[0050] Preparation of transfected cells: Before transfection, the density and viability of HEK-293F cells were measured. Cells in the logarithmic growth phase with a density of 2-4 × 10⁻⁴ cells were selected. 6 Cells with a density of 10 cells / mL and a viability greater than 98% were adjusted to a cell density of 2 × 10⁶ cells / mL using fresh serum-free culture medium. 6 Transfection was performed after incubating at 37℃, 5% CO2, and 120 rpm for 10 min with constant temperature shaking.

[0051] Transient transfection (using 100 mL cell suspension as an example): Take two 15 mL sterile centrifuge tubes. Add 5 mL of KPM medium and 100 μg of sterile plasmid to one tube, and add 5 mL of KPM medium and 500 μL of TA-293 transfection reagent to the other tube. Mix them gently. Then add the mixture containing the transfection reagent to the mixture containing the plasmid. Mix gently and let stand at room temperature for 10 min to form a plasmid-transfection reagent complex. Remove the cell culture flask from the constant temperature shaker and add the above plasmid-transfection reagent complex dropwise while shaking. Then put it back into the shaker and continue shaking culture.

[0052] Target protein expression, harvesting, and purification: 24 h after transfection, 600 μL of KE-293 protein expression enhancer and 2 mL of KT-Feed transient transfection nutrient additive were added to increase the expression level of the target protein. On day 6 post-transfection, cell culture medium was collected, centrifuged at 3000 g for 15 min at 4℃, and the culture supernatant was collected. Cell debris was removed by filtration through a 0.22 μm filter to obtain a crude extract containing recombinant target protein. cNGF-Fc and cTrkA-Fc proteins were purified using Protein A affinity chromatography, and cNGF-GST protein was purified using GST affinity chromatography. After purification, protein purity was analyzed by SDS-PAGE, and the target protein was identified by Western blot. Subsequently, dialysis with PBS buffer was performed to remove elution buffer components. Finally, the extract was filtered through a 0.22 μm filter, aliquoted, and stored at -80℃ for later use.

[0053] 3. Biotin labeling Biotin labeling of canine TrkA-Fc recombinant protein was performed using the Thermo EZ-Link Sulfo-NHS-LC-Biotin kit. Before labeling, the Sulfo-NHS-LC-Biotin reagent bottle was removed from the refrigerator and allowed to equilibrate to room temperature. 180 μL of ultrapure water was added to the bottle, and the mixture was gently pipetted to ensure complete dissolution of the Sulfo-NHS-LC-Biotin. The required amount of Sulfo-NHS-LC-Biotin was calculated based on the molar amount and molecular weight of canine TrkA-Fc protein, and labeling was performed at a molar ratio of canine TrkA-Fc protein to Sulfo-NHS-LC-Biotin of 1:20. The calculated amount of Sulfo-NHS-LC-Biotin was added to the canine TrkA-Fc protein solution, mixed thoroughly, and reacted in an ice bath for 2 h. After the reaction, the unbound free Sulfo-NHS-LC-Biotin was removed by ultrafiltration with PBS buffer. After passing the labeling and verification, the canine TrkA-Fc-Biotin (cTrkA-Fc-Biotin) was repackaged and stored at -80℃ for later use.

[0054] 4. Mouse immunization Two 6-8 week old female BALB / c mice were selected for immunization, and the specific immunization program is shown in Table 1. For the first immunization, canine NGF-GST protein was mixed with an equal volume of Freund's complete adjuvant, emulsified thoroughly using an electric emulsifier, and then injected subcutaneously at multiple sites on the back of the mouse. The immunization dose for each mouse was 50 μg. Subsequently, booster immunizations were performed every 14 days according to the immunization program shown in Table 1. For booster immunizations, canine NGF-GST protein was mixed with an equal volume of Freund's incomplete adjuvant, emulsified, and then injected subcutaneously at multiple sites on the back of the mouse. The immunization dose of canine NGF-GST protein for each mouse was 50 μg. Blood was collected from the mice 14 days after the fifth immunization. Blood samples were centrifuged at 4000 rpm for 10 min, and serum was collected and stored at 4℃ for later use.

[0055] Table 1. Immunization schedule for mice 5. Determination of antibody titer in mouse serum The titer of anti-canine NGF antibody in the serum of immunized mice was determined using an indirect enzyme-linked immunosorbent assay (ELISA). The procedure was as follows: Canine NGF-Fc protein was diluted 1:1000 with coating buffer, and 100 μL was added to each well. Coating was performed overnight at 4°C. After coating, the coating buffer was discarded, and 150 μL of blocking buffer was added to each well. The mixture was incubated at 37°C for 2 h. After blocking, the blocking buffer was discarded, and the mice were washed. The immunized mouse serum was pre-diluted to 1:1000, and then serially diluted 2-fold with sample diluent, for a total of 9 serial dilutions. Serum at various dilutions was added to each well of an ELISA plate, 100 μL per well, with four negative control wells included. After incubation at 37°C for 1 h, the plates were washed five times with washing buffer. Then, 100 μL of HRP-labeled goat anti-mouse IgG diluted 1:5000 was added to each well, and the plates were incubated at 37°C for 1 h, followed by five washes with washing buffer. Substrate solution A and substrate solution B were added to each well sequentially, and the plates were incubated at room temperature in the dark for 10 min. Finally, 50 μL of stop solution was added to each well to terminate the reaction, and the absorbance (OD) at 630 nm was measured within 10 min using an ELISA reader. 630 nm ). OD of the serum pore to be tested 630 nm The value was greater than the average OD of the negative control wells. 630 nm A value of 2.1 times was considered positive, and the highest serum dilution factor that indicated a positive result was used as the antibody titer of the mouse serum.

[0056] Serum from A51 and A52 mice was collected on day 14 after the fifth immunization for antibody titer determination. The indirect ELISA results of anti-canine NGF antibody titers in the serum of the two immunized mice are as follows: Figure 1 As shown, the serum antibody titer of A51 mice was 1:64000, and that of A52 mice was 1:32000. Both mice produced high levels of anti-canine NGF serum antibodies, which can be used for subsequent cell fusion.

[0057] 6. Cell fusion (1) Preparation of feeder cells: Eight-week-old female BALB / c mice were euthanized according to the approved animal ethics protocol, and the mice were treated with 75% ethanol for 5 min. Under aseptic conditions, the spleen was removed and washed twice with RPMI-1640 medium to remove the connective tissue attached to the spleen surface. The spleen was transferred to a sterile Petri dish containing RPMI-1640 medium and a 200-mesh cell sieve. The spleen capsule was cut open with ophthalmic scissors, and the spleen cells were gently pressed and ground with a grinding stick to fully release the spleen cells and prepare a spleen cell suspension. The spleen cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 1250 rpm for 10 min, the supernatant was discarded, and the cell pellet was resuspended in an appropriate amount of HAT complete medium. The pellet was then seeded into 96-well cell culture plates at 100 μL per well.

[0058] (2) Preparation of SP2 / 0 myeloma cells: SP2 / 0 myeloma cells were resuscitated and cultured routinely one week before cell fusion. On the day of fusion, SP2 / 0 cells in the logarithmic growth phase and in good growth condition were selected, 10 mL of RPMI-1640 medium was added, and the cells were gently pipetted to detach from the bottom wall of the cell culture flask. The cell suspension was collected and transferred to a 50 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the bottom of the centrifuge tube was gently tapped to loosen the cell pellet, 10 mL of RPMI-1640 medium was added to resuspend the cells, and viable cell counts were performed.

[0059] (3) Preparation of immunosuppressed spleen cells: Mice were selected for cell fusion based on the results of serum antibody titer testing, and a shock immunization was performed 3 days before cell fusion. Three days after the shock immunization, the mice were euthanized according to the approved animal ethics protocol, and their bodies were treated with 75% ethanol for 5 min. The spleen was removed under aseptic conditions and washed twice with RPMI-1640 medium to remove the connective tissue attached to the spleen surface. The spleen was transferred to a sterile Petri dish containing RPMI-1640 medium and a 200-mesh cell sieve. The spleen capsule was cut open with ophthalmic scissors, and the spleen cells were gently pressed and ground with a grinding stick to fully release the spleen cells and prepare a spleen cell suspension. The spleen cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 1250 rpm for 10 min, the supernatant was discarded, and the cell pellet was resuspended in 10 mL of RPMI-1640 medium and viable cell count was performed.

[0060] (4) Cell fusion and selective culture: SP2 / 0 myeloma cells: immune spleen cells = 1:2 were mixed in a 50 mL centrifuge tube and centrifuged at 1000 rpm for 10 min. The supernatant was completely discarded and residual liquid on the tube wall was removed as much as possible. The bottom of the centrifuge tube was gently tapped to loosen the cell pellet. The centrifuge tube containing the mixed cell pellet was placed in a 37°C water bath and 1 mL of 50% (w / v) PEG preheated to 37°C was slowly added dropwise over 1 min. The mixture was gently shaken during the addition. After the PEG addition was completed, the mixture was allowed to stand for 1 min. Then, the fusion was slowly terminated using RPMI-1640 medium preheated to 37°C: 1 mL was added in the first min, 2 mL in the second min, and 3 mL in the third min. The remaining 24 mL of RPMI-1640 medium was then slowly added, and the centrifuge tube was gently inverted once to mix. Centrifuge at 1250 rpm for 10 min, discard the supernatant, gently resuspend the cell pellet in an appropriate amount of HAT complete medium, and seed into 96-well cell culture plates pre-coated with feeder cells. Add 100 μL of cell suspension to each well and incubate the plates at 37°C and 5% CO2. On day 4 after fusion, perform a half-volume medium change, aspirating 100 μL of culture supernatant from each well and adding 100 μL of fresh HAT complete medium. On day 10 after fusion, perform a full-volume medium change, aspirating 200 μL of culture supernatant from each well and adding 250 μL of fresh HAT complete medium. When the fused cell colonies have grown to approximately half the bottom area of ​​the culture wells, collect the culture supernatant for anti-canine NGF antibody detection.

[0061] 7. Screening of positive hybridoma cells (1) Indirect ELISA screening: Indirect ELISA was used to preliminarily screen for antibodies that could bind canine NGF in the supernatant of hybridoma cell culture. Canine NGF-Fc protein was diluted with coating buffer at a ratio of 1:1000, and 100 μL was added to each well. The mixture was incubated overnight at 4°C. After coating, the coating buffer was discarded, and 150 μL of blocking buffer was added to each well. The mixture was incubated at 37°C for 2 h. After blocking, the blocking buffer was discarded and the cells were washed. The supernatant of hybridoma cell culture to be tested was added to the corresponding wells of the ELISA plate, and two negative control wells were set up. The mixture was incubated at 37°C for 1 h and washed 5 times with washing buffer. Then, HRP-labeled goat anti-mouse IgG diluted at a ratio of 1:5000 was added, and the mixture was incubated at 37°C for 1 h. After incubation, the cells were washed 5 times with washing buffer. 50 μL of substrate solution A and substrate solution B were added to each well, and the mixture was developed for 10 min at room temperature in the dark. After color development, add 50 μL of stop solution to each well to terminate the reaction, and measure the absorbance (OD) at 630 nm using a microplate reader within 10 min. 630 nm Select OD 630 nm Cell wells with a diameter >1.0 were used for subsequent competitive ELISA screening.

[0062] (2) Competitive ELISA screening: Hybridoma cell clones capable of blocking canine NGF-TrkA binding were further screened using competitive ELISA. Canine NGF-Fc protein was diluted 1:1000 with coating buffer, and 100 μL was added to each well, incubating overnight at 4°C. After coating, the coating buffer was discarded, and 150 μL of blocking buffer was added to each well, incubating at 37°C for 2 h. After blocking, the blocking buffer was discarded and the cells were washed. In a serum dilution plate, 60 μL of hybridoma cell culture supernatant was thoroughly mixed with an equal volume of 0.5 μg / mL cTrkA-Fc-Biotin working solution. 100 μL of this mixture was then transferred to each well of the pre-coated ELISA plate, with two antibody-free control wells included. The plate was incubated at 37°C for 1 h, followed by washing five times with washing buffer. HRP-labeled streptavidin diluted 1:5000 was then added, and the plate was incubated at 37°C for 1 h, followed by washing five times with washing buffer. 50 μL each of substrate solution A and substrate solution B were added to each well, and the plate was incubated at room temperature in the dark for 10 min. After incubation, 50 μL of stop solution was added to each well to terminate the reaction, and the absorbance (OD) at 630 nm was measured within 10 min using an ELISA reader. 630 nm The blocking rate of hybridoma cell culture supernatant against canine NGF and TrkA binding was calculated using the following formula: Blocking rate (%) = [1 - OD 630 nm (Sample well) / OD 630 nm [(Control wells without antibody)] × 100%. Hybridoma cell clones with strong canine NGF-TrkA competitive blocking activity were screened from high to low based on the blocking rate, and then subcloning was performed.

[0063] 8. Subcloning of positive hybridoma cells Subcloning of positive hybridoma cells obtained through limiting dilution was performed. Hybridoma cells from the positive cell wells were diluted with HT complete medium to adjust the cell concentration to 20 cells / mL. The resulting cell suspension was then seeded into 96-well cell culture plates pre-coated with feeder cells, with an seeding volume of 100 μL per well (theoretically, an average of approximately 2 cells / well). The culture plates were incubated at 37°C and 5% CO2. On days 5-6, cell growth was observed in each well, and the number of cell clones in each well was recorded. When the cell clones grew to approximately 1 / 3-1 / 2 of the bottom area of ​​the culture well, the culture supernatant was collected. The binding activity of the antibody to canine NGF was detected using an indirect ELISA, and the blocking activity of the antibody against canine NGF-TrkA was detected using a competitive ELISA. For cell wells with positive test results, priority was given to selecting cell clones exhibiting monoclonal growth, good cell morphology and growth status, and strong canine NGF binding and competitive blocking activity in the culture supernatant. These clones were then subjected to limiting dilution subcloning again using the method described above. This process was repeated for three rounds to obtain monoclonal hybridoma cell lines with stable culture supernatant test results, capable of binding canine NGF and exhibiting competitive blocking activity. These hybridoma cells were then fixed into specific cell lines. After fixed cell lines, they were transferred to 24-well cell culture plates for expansion. Once the cells were in stable and good growth status, they were transferred to cell culture flasks for further expansion and then cryopreserved.

[0064] 9. Expanded culture of positive hybridoma cells After screening and establishment, positive hybridoma cells were seeded into 6-well culture plates, with culture medium added to each well to a total volume of 2.5 mL. The plates were then incubated statically at 37°C and 5% CO2, and cell growth was observed the following day. Hybridoma cells in good growth condition are typically round or nearly round, with clear cell outlines and good refractive properties. Cells in a state of division and proliferation are visible, and they may form small clusters. Hybridoma cells in good growth condition were further cultured for subsequent antibody preparation and related assays.

[0065] 10. In vivo culture of hybridoma cells and preparation of ascites fluid Eight- to ten-week-old female BALB / c mice were used for in vivo hybridoma cell culture. All animal procedures were performed according to approved animal ethics protocols. Seven days prior to hybridoma cell inoculation, each mouse was pretreated with an intraperitoneal injection of 0.5 mL Freund's incomplete adjuvant. Hybridoma cells in the logarithmic growth phase and in good condition were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in serum-free RPMI-1640 medium to adjust the cell density to 1-2 × 10⁻⁶ cells / mL. 6 The concentration of hybridoma cells was 0.5 mL / mL, and each mouse was injected intraperitoneally with 0.5-1 × 10⁶ cells / mL, which means each mouse was inoculated with 0.5-1 × 10⁶ cells / mL. 6Hybridoma cells were inoculated. The general condition and abdominal changes of the mice were observed daily after inoculation with hybridoma cells. Ascites was collected approximately 7-10 days later, when it had formed and met the collection criteria. The collected ascites was centrifuged at 1200 rpm for 10 min, the upper oil layer was carefully discarded, and the middle layer of pale yellow ascites was collected, aliquoted, and stored at -20℃ for later use.

[0066] 11. Determination of titer of murine monoclonal antibody culture supernatant and ascites fluid The titer of anti-canine NGF monoclonal antibody in hybridoma cell culture supernatant and ascites fluid was determined by indirect ELISA. The procedure was as follows: Canine NGF-Fc protein was diluted 1:1000 with coating buffer, and 100 μL was added to each well. Coating was carried out overnight at 4°C. After coating, the coating buffer was discarded, and 150 μL of blocking buffer was added to each well. The mixture was incubated at 37°C for 2 h. After blocking, the blocking buffer was discarded and the cells were washed. The hybridoma cell culture supernatant was serially diluted 2-fold. 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 2 10 2 11 2 12 2 13 2 14 2 15 2 16 2 17 2 18 2 19 The ascites fluid was serially diluted 10-fold, with each dilution factor being 10. 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8Hybridoma cell culture supernatant and ascites fluid of various dilutions were added to the wells of an ELISA plate, 100 μL per well, with two negative control wells. The plate was incubated at 37°C for 1 h, followed by washing five times with washing buffer. 100 μL of HRP-labeled goat anti-mouse IgG diluted 1:5000 was added to each well, and the plate was incubated at 37°C for 1 h, followed by washing five times with washing buffer. 50 μL each of substrate solution A and substrate solution B were added to each well, and the plate was incubated at room temperature in the dark for 10 min. After incubation, 50 μL of stop solution was added to each well to terminate the reaction, and the absorbance (OD) at 630 nm was measured within 10 min using an ELISA reader. 630 nm ).

[0067] The antibody titers of the culture supernatants and ascites fluid from 10 hybridoma cell lines were determined using the above method. The results showed that the ELISA titers of the culture supernatants from all 10 hybridoma cell lines were higher than 2. 12 ( Figure 2 The ELISA titers of ascites from 10 hybridoma cell lines were all higher than 10. 6 ( Figure 3 ).

[0068] 12. Protein A affinity chromatography for purification of ascites antibodies The murine monoclonal antibody from hybridoma cell ascites fluid was purified using Protein A affinity chromatography. The specific procedures were as follows: The affinity chromatography column was washed with 10 column volumes of PBS, followed by equilibration with 10 column volumes of 1× equilibration buffer; 10 mL of 1× equilibration buffer and 12 mL of 2× equilibration buffer were thoroughly mixed, and 2 mL of thawed ascites fluid was added. After mixing, the mixture was filtered through a 0.45 μm microporous membrane; the filtered sample was slowly added to the affinity chromatography column at a flow rate of 0.5 mL / min; after loading, the column was washed with 10 column volumes of 1× equilibration buffer to remove unbound contaminants; the column was washed with 10 column volumes of washing buffer, followed by equilibration with 10 column volumes of 1× equilibration buffer again; the antibody bound to the column was eluted with 60 mL of elution buffer. 200 μL of pre-chilled 1 M Tris-HCl (pH 9.0) neutralization buffer was added to 2 mL centrifuge tubes beforehand. Each elution fraction was collected separately to ensure timely neutralization of the acidic eluent. After elution, the chromatography column was thoroughly washed with PBS. The elution fraction containing the target antibody was collected, and the antibody was replaced with PBS using ultrafiltration. The purified antibody was analyzed by SDS-PAGE. Protein bands were observed and antibody purity was evaluated by Coomassie Brilliant Blue R-250 staining. The antibody concentration was determined at 280 nm using a micro-ultraviolet spectrophotometer. The purified antibody was aliquoted and stored at -20℃ for later use.

[0069] 13. Subtype identification of murine monoclonal antibodies The subtypes of monoclonal antibodies secreted by 10 hybridoma cell lines were identified using a mouse monoclonal antibody subtype identification kit (SEK003, Beijing Yiqiao Shenzhou Technology Co., Ltd.) via double-antibody sandwich ELISA. The specific procedure was as follows: Rabbit anti-mouse IgG1, rabbit anti-mouse IgG2a, rabbit anti-mouse IgG2b, rabbit anti-mouse IgG3, and rabbit anti-mouse IgM subtype-specific capture antibodies were diluted 1:200 and coated onto an ELISA plate. After coating, blocking buffer was added, and the plate was blocked at 37°C for 2 h. After blocking, the blocking buffer was discarded, and the plate was washed. The purified mouse monoclonal antibodies were diluted to 2 μg / mL with PBS, and 100 μL of the antibody sample was added to each well. One positive control well and one negative control well were also included. The ELISA plate was incubated at 37°C for 1 h, followed by washing 5 times with washing buffer. Then, 100 μL of HRP-labeled rabbit anti-mouse IgG diluted 1:5000 was added to each well, and the plate was incubated at 37°C for 1 h, followed by washing 5 times with washing buffer. 50 μL each of substrate solution A and substrate solution B were added to each well sequentially, and the plate was incubated at room temperature in the dark for 10 min. After incubation, 50 μL of stop solution was added to each well to terminate the reaction. The absorbance (OD) at 630 nm was measured using an ELISA reader within 10 min. 630 nm Based on the colorimetric reaction and OD of the corresponding detection wells for different subtype-specific capture antibodies. 630 nm The value is used to determine the subtype of the monoclonal antibody to be tested.

[0070] The subtypes of monoclonal antibodies secreted by 10 hybridoma cell lines were identified using the above method. The results are shown in Table 2. The results showed that 1D4, 3B10, 4A6, and 4F2 were IgG1 subtypes, while 1E4, 3E7, 4D8, 4G2, 4H5, and 5B11 were IgG2a subtypes.

[0071] Table 2. Subtypes of monoclonal antibodies secreted by 10 hybridoma cell lines 14. Indirect ELISA assay using murine monoclonal antibodies The binding activity of 10 monoclonal antibodies to canine nerve growth factor (NGF) was evaluated using an indirect enzyme-linked immunosorbent assay (ELISA). The specific experimental procedure was as follows: Canine NGF-Fc protein was diluted 1:1000 with coating buffer, and 100 μL was added to each well. Coating was carried out overnight at 4°C. After coating, the coating buffer was discarded, and 150 μL of blocking buffer was added to each well. The mixture was incubated at 37°C for 2 h. After blocking, the blocking buffer was discarded, and the cells were washed. The mouse monoclonal antibody to be tested was diluted to 5 μg / mL with sample diluent, and serially diluted 2-fold using this as the starting concentration. 100 μL of each antibody concentration was added to the corresponding wells of an ELISA plate, with three replicates per antibody concentration. The plates were incubated at 37°C for 1 h. After incubation, the plates were washed five times with washing buffer. Then, 100 μL of HRP-labeled goat anti-mouse IgG diluted 1:5000 was added to each well, and the plates were incubated at 37°C for 1 h. After incubation, the plates were washed five times with washing buffer. 50 μL each of substrate solution A and substrate solution B were added to each well, and the plates were incubated at room temperature in the dark for 10 min. After incubation, 50 μL of stop solution was added to each well to terminate the reaction. The absorbance (OD) at 630 nm was measured using an ELISA reader within 10 min. 630 nm ).

[0072] The binding activity of 10 murine monoclonal antibodies to canine nerve growth factor was detected using the above method, and the results are as follows: Figure 4 As shown in the figure. The results indicate that all 10 murine monoclonal antibodies can specifically bind to canine NGF.

[0073] 15. Competitive ELISA assay using murine monoclonal antibodies The ability of 10 murine monoclonal antibodies to block canine NGF-TrkA was evaluated using a competitive enzyme-linked immunosorbent assay (ELISA). The specific experimental procedure was as follows: Canine NGF-Fc protein was diluted 1:1000 with coating buffer, and 100 μL was added to each well. Coating was carried out overnight at 4°C. After coating, the coating buffer was discarded, and 150 μL of blocking buffer was added to each well. The mixture was incubated at 37°C for 2 h. After blocking, the blocking buffer was discarded and the mixture was washed. The murine monoclonal antibody to be tested was pre-diluted to 200 μg / mL, and serially diluted 2-fold using this as the starting concentration. Each antibody solution was mixed with an equal volume of 0.5 μg / mL cTrkA-Fc-Biotin working solution, so that the highest final concentration of the antibody to be tested in the mixture was 100 μg / mL, and the final concentration of cTrkA-Fc-Biotin was 0.25 μg / mL. After thorough mixing, add 100 μL of the mixture to each well of the pre-coated ELISA plate, with three replicates for each antibody concentration. Include a control well without antibody. Incubate the ELISA plate at 37°C for 1 h, followed by washing five times with washing buffer. Then, add 100 μL of HRP-labeled streptavidin diluted 1:5000 to each well and incubate at 37°C for 1 h, followed by washing five times with washing buffer. Add 50 μL each of substrate solution A and substrate solution B to each well and incubate for 10 min at room temperature in the dark. After incubation, add 50 μL of stop solution to each well to terminate the reaction. Measure the absorbance (OD) at 630 nm using an ELISA reader within 10 min. 630 nm The blocking rate of each concentration of the antibody against the binding of canine NGF to TrkA was calculated using the following formula: Blocking rate (%) = [1 - OD 630 nm (Sample well) / OD 630 nm [(No antibody control wells)] × 100%. The final concentration of the antibody in each well was calculated based on the actual reaction system after equal volumes of antibody solution and cTrkA-Fc-Biotin were mixed. Using the final antibody concentration in each well as the concentration variable and the blocking rate as the response variable, nonlinear regression fitting was employed to calculate the half-maximal inhibitory concentration (IC50) of each murine monoclonal antibody. 50 ).

[0074] The above method was used to evaluate the blocking activity of 10 murine monoclonal antibodies against canine NGF-TrkA binding. The competitive ELISA results are as follows: Figure 5 As shown, the IC50 of 10 murine monoclonal antibodies... 50 The results are shown in Table 3. The results indicate that, except for 4A6, the other nine murine monoclonal antibodies all exhibited highly efficient blocking activity against canine NGF-TrkA binding.

[0075] Table 3. IC50 of 10 murine monoclonal antibodies in competitive ELISA 50 value 16. Experiment on the inhibition of TF-1 cell proliferation by murine monoclonal antibodies The inhibitory effects of 10 murine monoclonal antibodies on canine NGF-mediated biological activity were evaluated using a TF-1 cell proliferation inhibition assay. TF-1 cells were transferred to 50 mL centrifuge tubes, centrifuged at 1200 rpm for 3 min, the supernatant was discarded, and the cell pellet was resuspended in sterile PBS. The cells were then centrifuged again at 1200 rpm for 3 min, and the washing was repeated three times to remove rhGM-CSF from the culture system. After the final wash, the cells were resuspended in complete culture medium free of rhGM-CSF. After cell counting, the cell density was adjusted to 2.5 × 10⁶ cells / mL. 5 Cells were seeded at a concentration of 60 μL / mL into each well of a 96-well cell culture plate. The mouse monoclonal antibody to be tested was pre-diluted to 25 μg / mL, and serially diluted 2-fold using this as the starting concentration. 20 μL of the corresponding antibody solution was added to each well, with four replicates per antibody concentration. The plates were incubated at 37°C for 30 min. After incubation, 20 μL of 1.5 μg / mL canine NGF-Fc working solution was added to each well, bringing the final reaction volume to 100 μL per well. Therefore, the highest final concentration of the antibody in each well was 5 μg / mL, and the final concentration of canine NGF-Fc in each well was 0.30 μg / mL. Five positive control wells and five negative control wells were also included in each cell culture plate. 200 μL of PBS was added to the peripheral wells of the culture plates not used for testing to reduce liquid evaporation during culture. The culture plate was incubated at 37℃ and 5% CO2 for 5 days. After the incubation period, 10 μL of CCK-8 reagent was added to each well, and the plate was incubated at 37℃ for 2 h. After thorough mixing, the absorbance (OD) of each well was measured at 450 nm using a microplate reader. 450 nm The final concentration of the antibody in each well was calculated based on the final reaction volume. Using the final antibody concentration as the concentration variable and the TF-1 cell proliferation inhibition rate as the response variable, a nonlinear regression was used to calculate the half-maximal inhibitory concentration (IC50) of each murine monoclonal antibody. 50 ).

[0076] The inhibitory effects of 10 murine monoclonal antibodies on canine NGF-mediated TF-1 cell proliferation are as follows: Figure 6 As shown, the IC50 of 10 murine monoclonal antibodies... 50 The results are shown in Table 4. The results indicate that all 10 murine monoclonal antibodies were able to inhibit canine NGF-mediated TF-1 cell proliferation to varying degrees, among which 1D4, 3B10, 3E7, 4D8, 4F2 and 5B11 showed strong inhibitory activity.

[0077] Table 4. IC50 values ​​of 10 murine monoclonal antibodies in the TF-1 cell proliferation inhibition assay. 50 value 17. Assay on the inhibition of PC-12 cell differentiation by murine monoclonal antibodies The inhibitory effect of 10 murine monoclonal antibodies on canine NGF-mediated biological activity was evaluated using a PC-12 cell differentiation inhibition assay. The murine monoclonal antibodies were pre-diluted to 50 μg / mL, and serially diluted 2-fold using this as the starting concentration. Each antibody solution was mixed with an equal volume of canine NGF-Fc working solution (2 μg / mL) and incubated at 37°C for 1 h. After incubation, 100 μL of the reaction complex was transferred from each well to a 96-well cell culture plate, with four replicates for each antibody concentration. Subsequently, 100 μL of PC-12 cell suspension was added to each well, along with positive and negative control wells. The culture plates were incubated at 37°C with 5% CO2 for 6 days. After culture, indirect immunofluorescence staining was performed: the culture medium in the wells was discarded, and 4% paraformaldehyde solution was added to each well for fixation at room temperature for 20 min; after fixation, the cells were washed once with PBST, and 50 μL of PBS containing 0.1% Triton X-100 was added to each well for permeation at room temperature for 5 min; after permeation, the cells were washed once with PBST, and 100 μL of PBST containing 1% BSA was added to each well for blocking at room temperature for 1 h; after blocking, 50 μL of rabbit anti-TUBB3 primary antibody diluted 1:5000 was added to each well for incubation at 4℃ overnight; after primary antibody incubation, the cells were washed three times with PBST, and 50 μL of Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody diluted 1:3000 was added to each well for incubation at 37℃ in the dark for 1 h; after secondary antibody incubation, the cells were washed three times with PBST, and finally, the cell nuclei were stained with DAPI. The morphology of PC-12 cells and the fluorescence signal of TUBB3 were observed under a fluorescence microscope. PC-12 cells with neural processes that are twice the length of their cell body's long axis were identified as differentiated cells, and the presence of differentiated cells in each well was counted. The final antibody concentration was calculated based on the final reaction volume of each well, and the half-maximal inhibitory concentration (IC50) of each murine monoclonal antibody was calculated. 50 ).

[0078] The inhibitory effects of 10 murine monoclonal antibodies on canine NGF-induced PC-12 cell differentiation are as follows: Figure 7 As shown, the IC50 of 10 monoclonal antibodies 50 The results are shown in Table 5. The results indicate that all 10 murine monoclonal antibodies were able to inhibit canine NGF-induced PC-12 cell differentiation to varying degrees, with 1D4 and 3E7 showing stronger PC-12 cell differentiation inhibitory activity.

[0079] Table 5. IC50 values ​​of 10 murine monoclonal antibodies in the PC-12 cell differentiation inhibition assay. 50 value 18. Amplification of the variable region sequences of the heavy and light chains of murine monoclonal antibodies (1) Extraction of nucleic acid from hybridoma cells: Select hybridoma cells in good growth condition, transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 10 min, discard the supernatant, add PBS to resuspend the cell pellet, centrifuge at 1000 rpm for 10 min, discard the supernatant, and repeat the above PBS washing step once. After washing, resuspend the cells in 200 μL PBS. Extract nucleic acid from hybridoma cells using a commercial cell genomics extraction kit. Add 10 μL proteinase K to 200 μL of cell suspension, mix thoroughly, add 200 μL of lysis buffer, mix well, and incubate at 55℃ for 10 min to allow the sample to fully lyse and release nucleic acid. Then add 200 μL of binding buffer, mix well, and transfer the resulting mixture to a nucleic acid adsorption column, centrifuge at 12000 rpm for 1 min to allow the nucleic acid to be adsorbed onto the adsorption membrane; discard the flow-through liquid, and repeat the loading until all samples have been adsorbed. Then, 500 μL of wash buffer 1 was added to the adsorption column, and the column was centrifuged at 12000 rpm for 1 min, discarding the flow-through liquid. Next, 500 μL of wash buffer 2 was added, and the column was centrifuged at 12000 rpm for 1 min, discarding the flow-through liquid. After washing, the column was centrifuged at 12000 rpm for 2 min to thoroughly remove residual wash buffer. The adsorption column was transferred to a new nuclease-free centrifuge tube, and 50 μL of RNase-free water was added to the center of the adsorption membrane. After standing at room temperature for 1 min, nucleic acid elution was performed by centrifugation at 12000 rpm for 1 min. The adsorption column was discarded, and the eluent was collected to obtain the DNA / RNA nucleic acid extraction product, which was stored at -20℃ for later use.

[0080] (2) Synthesis of first-strand cDNA: The reaction system is as follows: 12 μL of whole genome and Oligo(dT) 231 μL of VN (50 μM) was added, bringing the total volume to 13 μL. After mixing thoroughly, the mixture was briefly centrifuged to the bottom of the tube and placed in a PCR instrument. The tube was incubated at 65°C for 5 min, followed immediately by an ice bath for 2 min. Then, 4 μL of 5×HiScript II Buffer, 1 μL of dNTP Mix (10 mMeach), 1 μL of HiScript II Reverse Transcriptase (200 U / μL), and 1 μL of RNase inhibitor (40 U / μL) were added to the 13 μL reaction solution, bringing the total reaction volume to 20 μL. After mixing thoroughly, the mixture was briefly centrifuged to the bottom of the tube and placed in a PCR instrument. The reaction program was as follows: 25°C, 5 min; 50°C, 45 min; 85°C, 2 min. The first-strand cDNA was obtained after the reaction and used for subsequent antibody variable region sequence amplification.

[0081] (3) PCR amplification of the variable region sequences of heavy and light chains The primers used for amplifying the variable region of mouse antibodies are as follows: Heavy chain upstream primer HF: CAGGTSCARCTGMAGSAGCC, SEQ ID NO.1; Heavy chain downstream primer HR: GACAGGGATCCAGAGTTCC, SEQ ID NO.2; Light chain upstream primer LF: GGAGACAGRGTCASCATCA, SEQ ID NO.3; Downstream primer for the light chain: CATTCCTGTTGAAGCTCTTGAC, SEQ ID NO.4.

[0082] Note: The degenerate bases in the primer sequences are represented using the IUPAC degenerate base notation, where R=A / G, M=A / C, and S=G / C.

[0083] PCR amplification system: 10 μL 5×SF Buffer (with 10 mM MgSO4), 1 μL dNTP Mix (10 mM each), 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 2 μL cDNA template, 1 μL Phanta Super-Fidelity DNA Polymerase, and 34 μL ddH2O, total volume 50 μL. The amplified PCR products were identified by agarose gel electrophoresis, and the corresponding target bands were excised for further purification.

[0084] (4) Purification and sequencing of PCR amplification products: Transfer the excised gel block to a centrifuge tube, add 600 μL of Buffer GDP to the gel block, and incubate at 55℃ for 10 min to completely dissolve the gel. After dissolving the gel, transfer the resulting solution to a DNA adsorption column and centrifuge at 12000 rpm for 60 s to adsorb DNA onto the membrane. Discard the filtrate in the collection tube, place the adsorption column back into the collection tube, add 300 μL of Buffer GDP, let stand for 1 min, centrifuge at 12000 rpm for 60 s, and discard the filtrate. Then add 700 μL of wash buffer, centrifuge at 12000 rpm for 60 s, and discard the filtrate; repeat the above washing steps once to thoroughly remove salt ions and other impurities. After washing, place the adsorption column in a collection tube and centrifuge at 12000 rpm for 2 min with an empty column to remove residual wash buffer. The adsorption column was transferred to a new sterile 1.5 mL nuclease-free centrifuge tube. 30 μL of elution buffer was added to the center of the adsorption membrane. After standing at room temperature for 2 min, DNA was eluted by centrifugation at 12000 rpm for 1 min. The adsorption column was discarded, and the DNA solution in the centrifuge tube was collected and stored at -20℃ for later use. Finally, the samples were sent to a sequencing company for sequencing. Based on the sequencing results, the coding sequences of the heavy chain variable region and light chain variable region of the monoclonal antibodies secreted by each hybridoma cell were obtained, and the corresponding amino acid sequences were deduced accordingly.

[0085] 19. Construction, expression, and purification of canine-mouse chimeric monoclonal antibodies Based on the aforementioned hybridoma cell screening and functional evaluation results, six murine monoclonal antibodies, namely 1D4, 3B10, 3E7, 4D8, 4F2 and 5B11, were selected for canine-mouse chimerism to construct canine-mouse chimeric monoclonal antibodies c1D4, c3B10, c3E7, c4D8, c4F2 and c5B11, respectively.

[0086] (1) Construction of canine-mouse chimeric antibody expression plasmids: Canine-mouse chimeric antibodies were constructed based on the sequencing results of the variable regions (VH) and (VL) of the heavy chain and light chain of murine monoclonal antibodies. The canine IgG-B heavy chain constant region sequence (IMGT number: AF354265) was obtained from the IMGT database. The VH sequences of each murine monoclonal antibody were ligated with the canine heavy chain constant region sequence to construct the canine-mouse chimeric antibody heavy chain. The canine light chain constant region sequence (IMGT number: IMGT000067) was obtained from the IMGT database. The VL sequences of each murine monoclonal antibody were ligated with the canine light chain constant region sequence to construct the canine-mouse chimeric antibody light chain. The designed canine-mouse chimeric antibody heavy chain and light chain sequences were optimized for expression codons in mammalian cells, and Beijing Yiqiao Shenzhou Technology Co., Ltd. was commissioned to complete gene synthesis and recombinant plasmid construction. The heavy chain and light chain coding sequences were ligated into the pcDNA3.4 eukaryotic expression vector to obtain the corresponding heavy chain expression plasmids and light chain expression plasmids.

[0087] (2) Extraction of recombinant expression plasmids: *E. coli* containing both heavy and light chain expression plasmids were inoculated into LB liquid medium containing ampicillin and cultured overnight at 37°C with shaking. The overnight culture was collected and centrifuged at 5000 rpm for 10 min. The supernatant was discarded and the bacterial pellet was collected. Recombinant plasmids were extracted using a commercial plasmid extraction kit. A large-scale plasmid adsorption column was placed in a collection tube, and 2.5 mL of BL equilibration buffer was added to the column. The column was centrifuged at 8000 rpm for 2 min. 10 mL of P1 solution was added to the bacterial cells to fully resuspend the bacterial suspension. 10 mL of P2 solution was added to the bacterial suspension, and the mixture was inverted 10 times to mix. The suspension was then allowed to stand at room temperature for 5 min for lysis. 10 mL of P4 solution was added to the lysed bacterial suspension, and the mixture was inverted 10 times to terminate the lysis reaction. A white flocculent precipitate was observed at this point. The suspension was centrifuged at 8000 rpm for 20 min. Pour the supernatant from centrifugation into a filter, slowly push the stopper to filter, and collect the filtrate into a new centrifuge tube. Add 0.3 times the volume of isopropanol to the filtrate, then invert to mix. Add the liquid from the previous step to the adsorption column, centrifuge at 8000 rpm for 3 min, discard the bottom waste liquid, and repeat the operation until all liquid has been loaded onto the column. Add 10 mL of PW washing buffer to the adsorption column, centrifuge at 8000 rpm for 3 min, discard the bottom waste liquid, and repeat the washing once. Then add 3 mL of anhydrous ethanol, centrifuge at 8000 rpm for 3 min, and discard the bottom waste liquid. Incubate the adsorption column at 55°C for 5 min to thoroughly remove residual ethanol. Transfer the adsorption column to a new sterile centrifuge tube, add 1 mL of DNase / RNase-free water to the adsorption column, let stand for 5 min, centrifuge at 8000 rpm for 5 min, aliquot the filtrate, and store at -20°C for later use.

[0088] (3) Transient expression of canine-mouse chimeric antibody: Transient expression of canine-mouse chimeric antibody was performed using suspended HEK-293F cells. HEK-293F cells were counted before transfection, and the cell density was adjusted to 2 × 10⁶ cells / year using fresh serum-free suspension medium. 6 Cells / mL. Taking a 100 mL cell suspension system as an example, take two 15 mL centrifuge tubes and add 5 mL of KPM to each. Add 500 μL of transfection reagent (TA-293) to one centrifuge tube; add 35 μg of heavy chain expression plasmid and 65 μg of light chain expression plasmid to the other centrifuge tube. Slowly add the mixture containing the transfection reagent to the mixture containing the expression plasmid, gently pipette to mix, and incubate at room temperature for 10 min to form the transfection complex. Gently shake the HEK-293F cell culture flask, adding the prepared transfection complex while shaking, and then place the cells in a 37°C, 5% CO2 incubator. 2、 Continue oscillating at 120 rpm. 24 h after transfection, add 600 μL of 293 cell protein expression enhancer (KE-293) and 2 mL of transient transfection nutrient supplement (50×KT-Feed). Collect cell culture medium 6 days after transfection. Centrifuge at 3000 g for 15 min at 4℃, collect the culture supernatant, and filter through a 0.22 μm filter to remove cell debris, obtaining the culture supernatant containing canine-mouse chimeric monoclonal antibody.

[0089] (4) Purification of canine-mouse chimeric antibodies: The column equilibration, washing and elution conditions are the same as those in “12. Purification of ascites antibodies by Protein A affinity chromatography”, except that the pretreatment step of the ascites sample is not performed.

[0090] 20. Indirect ELISA assay using chimeric monoclonal antibodies from dogs and mice The binding activity of canine-mouse chimeric monoclonal antibodies c1D4, c3B10, c3E7, c4D8, c4F2, and c5B11 to canine NGF was evaluated using an indirect enzyme-linked immunosorbent assay (ELISA). The assay procedure was the same as in "14. Indirect ELISA assay of mouse monoclonal antibodies," except that HRP-labeled anti-canine IgG diluted 1:5000 was used instead of HRP-labeled goat anti-mouse IgG.

[0091] The indirect ELISA results of 6 canine-mouse chimeric monoclonal antibodies are as follows: Figure 8 As shown in the figure. The results indicate that c1D4, c3B10, c3E7, c4D8, c4F2, and c5B11 can all specifically bind to canine NGF, indicating that canine derivatization of the above six antibodies did not affect their ability to bind to canine NGF.

[0092] 21. Competitive ELISA assay using chimeric monoclonal antibodies from dogs and mice The ability of canine-mouse chimeric monoclonal antibodies c1D4, c3B10, c3E7, c4D8, c4F2, and c5B11 to block canine NGF-TrkA was evaluated using a competitive enzyme-linked immunosorbent assay (ELISA). The assay procedure was the same as in "15. Competitive ELISA assay of mouse monoclonal antibodies," except that the canine-mouse chimeric monoclonal antibodies to be tested were pre-diluted to 100 μg / mL, and serial dilutions were performed in two-fold increments starting at 100 μg / mL.

[0093] The competitive ELISA results of 6 canine and mouse chimeric monoclonal antibodies are as follows: Figure 9 As shown, the IC50 of each antibody 50 The results are shown in Table 6. The results indicate that c1D4, c3B10, c3E7, c4D8, c4F2, and c5B11 can all effectively block the binding of canine NGF-TrkA, indicating that canine derivatization of the above six antibodies did not affect their ability to competitively block canine NGF-TrkA.

[0094] Table 6. IC50 of 6 chimeric monoclonal antibodies from dogs and mice competing with ELISA 50 value 22. Inhibition of TF-1 cell proliferation by canine and mouse chimeric monoclonal antibodies The inhibitory effects of canine-mouse chimeric monoclonal antibodies c1D4, c3B10, c3E7, c4D8, c4F2, and c5B11 on canine NGF-mediated biological activity were evaluated using a TF-1 cell proliferation inhibition assay. The procedure was the same as in "16. Inhibition of TF-1 cell proliferation by mouse monoclonal antibodies".

[0095] The results of the experiment on the inhibition of canine NGF-mediated TF-1 cell proliferation by 6 canine-mouse chimeric monoclonal antibodies are as follows: Figure 10 As shown, the IC50 values ​​of each monoclonal antibody 50 The results are shown in Table 7. The results indicate that c1D4, c3B10, c3E7, c4D8, c4F2 and c5B11 can all inhibit canine NGF-mediated TF-1 cell proliferation, indicating that the canine derivatization of the above antibodies did not affect their ability to competitively block canine NGF-TrkA at the TF-1 cell level.

[0096] Table 7. IC50 values ​​of TF-1 cell proliferation inhibition assay for 6 canine and mouse chimeric monoclonal antibody strains. 50 value 23. Experiment on the inhibition of PC-12 cell differentiation by canine and mouse chimeric monoclonal antibodies The inhibitory effects of canine-mouse chimeric monoclonal antibodies c1D4, c3B10, c3E7, c4D8, c4F2, and c5B11 on canine NGF-mediated biological activity were evaluated using a PC-12 cell differentiation inhibition assay. The procedure was the same as in "17. Assay on PC-12 cell differentiation inhibition by mouse monoclonal antibodies".

[0097] The results of the experiment on the inhibition of differentiation of canine NGF-induced PC-12 cells by six chimeric canine and mouse monoclonal antibodies are as follows. Figure 11 As shown, the IC50 of each antibody 50 The results are shown in Table 8. The results indicate that c1D4, c3B10, c3E7, c4D8, c4F2 and c5B11 can inhibit canine NGF-induced PC-12 cell differentiation. Among them, c1D4 and c3E7 showed strong PC-12 cell differentiation inhibitory activity, indicating that the canine derivatization of the above antibodies did not affect the ability of the monoclonal antibodies to competitively block canine NGF-TrkA at the PC-12 cell level.

[0098] Table 8. IC50 values ​​of six chimeric monoclonal antibody strains of dogs and mice in the PC-12 cell differentiation inhibition assay. 50 value The heavy chain variable region (VH) nucleotide sequence of the c1D4 canine-mouse chimeric monoclonal antibody is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6. The amino acid sequence positions 31-35 are heavy chain CDR1 (SEQ ID NO.7), positions 50-66 are heavy chain CDR2 (SEQ ID NO.8), and positions 99-108 are heavy chain CDR3 (SEQ ID NO.9). The heavy chain constant region (CH) nucleotide sequence is shown in SEQ ID NO.10, and the amino acid sequence is shown in SEQ ID NO.11. The nucleotide sequence of the light chain variable region (VL) is shown in SEQ ID NO.12, and the amino acid sequence is shown in SEQ ID NO.13. The amino acid sequence positions 24-34 are light chain CDR1 (SEQ ID NO.14), positions 50-56 are light chain CDR2 (SEQ ID NO.15), and positions 89-97 are light chain CDR3 (SEQ ID NO.16). The nucleotide sequence of the light chain constant region (CL) is shown in SEQ ID NO.17, and the amino acid sequence is shown in SEQ ID NO.18.

[0099] The heavy chain variable region (VH) nucleotide sequence of the c3E7 canine-mouse chimeric monoclonal antibody is shown in SEQ ID NO.19, and the amino acid sequence is shown in SEQ ID NO.20. The amino acid sequence positions 31-35 are heavy chain CDR1 (SEQ ID NO.21), positions 50-65 are heavy chain CDR2 (SEQ ID NO.22), and positions 98-110 are heavy chain CDR3 (SEQ ID NO.23). The heavy chain constant region (CH) nucleotide sequence is shown in SEQ ID NO.10, and the amino acid sequence is shown in SEQ ID NO.11. The nucleotide sequence of the light chain variable region (VL) is shown in SEQ ID NO.24, and the amino acid sequence is shown in SEQ ID NO.25. The amino acid sequence positions 24-34 are light chain CDR1 (SEQ ID NO.26), positions 50-56 are light chain CDR2 (SEQ ID NO.27), and positions 89-97 are light chain CDR3 (SEQ ID NO.28). The nucleotide sequence of the light chain constant region (CL) is shown in SEQ ID NO.17, and the amino acid sequence is shown in SEQ ID NO.18.

[0100] The specific sequence is shown below: SEQ ID NO.5 (i.e., the murine 1D4 heavy chain variable region nucleotide sequence): CAAGTCCAACTCCAGCAGCCAGGTGCAGAACTGGTGAAACCGGGCGCCAGTGTCAAGATGTCCTGTAAGGCATCTGGATATACGTTCACAAATTACTGGATGCACTGGGTTAAGCAGAGGCCTGGGCAGGGACTCGAATGGATCGGCACAATTGATCCGAGCGATTCCTACACAAGGTA CAACCAGAATTTCAAGGGGAAAGCAACACTCACTGTCGACACAAGTTCATCCACCGCGTACATGCAACTGAGTAGCCTGACCTCCGAGGACTCCGCCGTGTACTACTGTGCCAGGAGAGACGACGGGTACTACTTCGACTACTGGGGGCAAGGAACTACCCTTACTGTCTCCTCT; SEQ ID NO.6 (i.e., the amino acid sequence of the variable region of the mouse 1D4 heavy chain): QVQLQQPGAELVKPGASVKMSCKASGYTFTNYWMHWVKQRPGQGLEWIGTIDPSDSYTRYNQNFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARRDDGYYYFDY WGQGTTLTVSS Note: The underlined sequences are FR1 and FR4 in sequence, FR2 is between CDR1 and CDR2, and FR3 is between CDR2 and CDR3. SEQ ID NO.7: NYWMH; SEQ ID NO.8: TIDPSDSYTRYNQNFKG; SEQ ID NO.9: RDDGYYYFDY; SEQ ID NO.10 (i.e., the nucleotide sequence of the constant region of the canine heavy chain): SEQ ID NO.11 (i.e., the amino acid sequence of the canine heavy chain constant region): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEAAGAPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGK QMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK*; SEQ ID NO.12 (i.e., the nucleotide sequence of the variable region of the mouse 1D4 light chain): GACATCGTAATGACCCAGTCCCACAAATTCATGAGCACATCAGTAGGAGACCGCGTGATCATAACCTGCAAGGCATCCCAGGATGTGTTTACAGCTGTGGCGTGGTTTCAACAGAAGCCCGGGCAATCCCCTAAACTGCTGATCTACTGGGCATCCACTCG GCACACAGGAGTGCCCGACAGGTTCACCGGAAGCGGGTCTGGCACAGACTACACTTTGACGATAAGCTCAATGCAGGCTGAGGACCTGGCCTTGTATTACTGTCAGCAGCACTACAGCTCCCCTTGGACATTCGGCGGGGGCACAAAACTGGAGATCAAA; SEQ ID NO.13 (i.e., the amino acid sequence of the variable region of the mouse 1D4 light chain): DIVMTQSHKFMSTSVGDRVIITC KASQDVFTAVAWFQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSMQAEDLALYYCQQHYSSPWT FGGGTKLEIK Note: The underlined sequences are FR1 and FR4 in sequence, FR2 is between CDR1 and CDR2, and FR3 is between CDR2 and CDR3. SEQ ID NO.14: KASQDVFTAVA; SEQ ID NO.15: WASTRHT; SEQ ID NO.16: QQHYSSPWT; SEQ ID NO.17 (i.e., the nucleotide sequence of the constant region of the canine light chain): CGAAATGACGCCCAGCCTGCTGTGTACCTGTTTCAACCTTCCCCGATCAATTGCACACAGGCTCTGCGAGTGTAGTgTGTCTGCTGAACTCCTTTTATCCCAAGGACATCAACGTGAAGTGGAAGGTCGATGGAGTGATCCAGGATACAGGGATTCAGGAGAGCGT GACTGAGCAGGACAAGGACTCTACGTACTCCCTGTCATCTACGCTGACCATGAGTAGTACAGAATATCTGAGCCACGAGCTTTACTCCTGTGAGATCACCCACAAATCACTGCCGTCCACCCTGATTAAGTCCTTCCAGCGATCAGAGTGCCAGAGGGTTGACTAA; SEQ ID NO.18 (i.e., the amino acid sequence of the canine light chain constant region): RNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD*; SEQ ID NO.19 (i.e., the murine 3E7 heavy chain variable region nucleotide sequence): CAAGTTCAACTGAAAGAATCTGGCCCCGGTCTTGTAGCTCCATCCCAGTCCATGAGTATCACCTGCACAGTCTCTGGCTTTTCCTTGACCGGTTACGGGGTGAATTGGGTTAGGCAGCCTCCCGGGAAGGGGTTGGAGTGGTTGGGCATTATTTGGGGCGATGGAGACACCGACTACAACTC CGCTCTGAAGTCCAGGTTGAGCATCACCAAAGACAACTCTAAGAGCCAGGTCTTTCTGAAGATGAACTCCCTGCAGACAGACGATACTGCTCGCTACTATTGTGCCAGGGACTTCTACTACTACGGGACAAACTTCGCTATGGACTTTTGGGGCCAGGGCACCAGTGTGACGGTCTCCTCC; SEQ ID NO.20 (i.e., the amino acid sequence of the variable region of the mouse 3E7 heavy chain): QVQLKESGPGLVAPSQSMSITCTVSGFSLT GYGVNWVRQPPGKGLEWLGIIWGDGDTDYNSALKSRLSITKDNKSSQVFLKMNSLQTDDTARYYCARDFYYYGTNFAMDF WGQGTSVTVSS Note: The underlined sequences are FR1 and FR4 in sequence, FR2 is between CDR1 and CDR2, and FR3 is between CDR2 and CDR3. SEQ ID NO.21: GYGVN; SEQ ID NO.22:IIWGDGDTDYNSALKS; SEQ ID NO.23: DFYYYGTNFAMDF; SEQ ID NO.24 (i.e., the murine 3E7 light chain variable region nucleotide sequence): GATATTCAAATGACCCAAATCACTTCTTCCTTGTCAGCTTCCCTCGGAGATAGGGTCACCATTTCATGTCGGGCCGGACAGGATTCAAGCAATAACCTGAACTGGTACCAGCAGAAGCCAGACGGCACTGTGAAGCTCCTGATCTATTACACCAGTAGGCT GCATAGTGGCGTGCCCTCAAGGTTTAGCGGCTCAGGAAGTGGAACTGATTACTCTCTGACTATCAGCAACCTCGAGCAAGAGGACATTGCCACATACTTCTGTCAGCAGGGCAAAACTCTTCCATACACATTTGGAGGCGGTACAAAACTCGAGATCAAG; SEQ ID NO.25 (i.e., the amino acid sequence of the variable region of the mouse 3E7 light chain): DIQMTQITSSLSASLGDRVTISC RAGQDSSNNLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTLPYT FGGGTKLEIK Note: The underlined sequences are FR1 and FR4 in sequence, FR2 is between CDR1 and CDR2, and FR3 is between CDR2 and CDR3. SEQ ID NO.26: RAGQDSSNNLN; SEQ ID NO.27: YTSRLHS; SEQ ID NO.28: QQGKTLPYT.

[0101] 24. In vivo efficacy evaluation of canine-mouse chimeric monoclonal antibody in a MIA-induced mouse model of osteoarthritis. To evaluate the in vivo effects of canine and mouse chimeric monoclonal antibodies c1D4 and c3E7 on osteoarthritis-related pain, a mouse osteoarthritis model induced by sodium monoiodide (MIA) was used for efficacy evaluation. Before the experiment, mice were randomly divided into four groups (n=7 per group): a sham treatment group, an isotype control IgG group, a Bedinvetmab group, a c3E7 group, and a c1D4 group. The MIA-induced mouse osteoarthritis model was established on day 0 of the experiment. Anesthesia was induced by inhalation of 2%-3% isoflurane, maintaining the anesthesia concentration at 1.5%-2.0%. Once the mice showed no righting reflex, they were fixed on a sterile operating table. Hair around the right knee joint was removed using a motorized pilaster, and the skin was disinfected sequentially with 75% ethanol and iodine solution. With the right knee joint flexed at approximately 90°, the needle insertion point was gently palpable in the space between the patellar tendons. A 30G insulin injection needle was slowly inserted into the joint cavity below the patellar tendon along the tibial plateau, and 10 μL of MIA solution (0.5 mg / 10 μL / mouse) was slowly injected. The sham treatment group received an equal volume of physiological saline. The entire injection process lasted approximately 10 seconds. After injection, the needle was retained for approximately 5-10 seconds to reduce drug reflux, and then the needle was slowly withdrawn. The puncture site was gently pressed with a sterile cotton swab for a few seconds. After modeling, the mice's recovery was observed. Once they had fully regained spontaneous activity, they were returned to their original cages, and their mental state, diet, water intake, and activity were observed daily.

[0102] On day 8 post-modeling, each treatment group received a single subcutaneous injection. The Bedinvetmab, c3E7, and c1D4 groups received their respective anti-NGF antibodies at a dose of 0.5 mg / kg; the IgG group received an equal dose of isotype control antibody; and the sham treatment group received an equal volume of PBS. All drugs were administered via subcutaneous injection into the back of the neck. During injection, the skin was gently lifted to form a skin fold, and the drug was slowly injected. After injection, any leakage was observed at the injection site.

[0103] To evaluate the ameliorative effect of anti-NGF antibody on MIA-induced osteoarthritis-related pain, dynamic weight bearing (DWB) tests, electronic Von Frey mechanical pain threshold tests, and weight monitoring were performed before and after drug administration. All behavioral experiments were conducted in a quiet, temperature-controlled environment (22±2℃) at fixed times each day (09:00-15:00). Mice were brought into the laboratory 30 minutes before the formal tests to acclimatize to the environment and minimize the influence of environmental factors on stress and behavioral results.

[0104] Bipedal balance and weight-bearing test: The spontaneous weight-bearing capacity of the hind limbs of mice was evaluated using a bipedal balance and weight-bearing tester. Before the formal test, the mice were placed in the instrument for 5 minutes to adapt. During the test, the mice moved freely in the test chamber, and the instrument collected the force data of both hind limbs when they touched the ground in real time. The test ended when the system automatically collected a continuous, stable pressure curve that met the analysis requirements. The test time for each mouse was approximately 3-5 minutes. The instrument software automatically calculated the weight-bearing weight of the left and right hind limbs, and used the percentage of the weight-bearing weight of the affected side (right side) to the total weight-bearing weight of both hind limbs as the evaluation index: Weight-bearing (%) = Weight-bearing weight of the affected side hind limb / (Weight-bearing weight of the affected side hind limb + Weight-bearing weight of the healthy side hind limb) × 100%. A decrease in the percentage of weight-bearing indicated increased pain in the affected joint, while the recovery of weight-bearing indicated pain relief. The bipedal balance and weight-bearing test was conducted 1 day before modeling (-1 day, baseline) and on days 12, 15, 22, 29, and 36 after modeling. On the same day, the weight of each mouse was measured and recorded using an electronic balance to evaluate the overall health status of the animals and the safety of the drugs.

[0105] Electronic Von Frey mechanical pain threshold test: The mechanical pain sensitivity of mice was measured using an electronic Von Frey analgesic device. Before the test, mice were placed in a transparent plexiglass observation box with a metal mesh bottom for 30 minutes to acclimatize. After they remained calm and their limbs were on the ground naturally, the test began. The electronic Von Frey probe was placed vertically under the metal mesh to the center of the affected hind paw sole (avoiding the paw pad and toes), and the pressure was gradually increased at a constant speed. When the mouse exhibited typical escape responses such as rapid paw withdrawal, paw licking, or paw flicking, the pressure was immediately stopped, and the instrument automatically recorded the maximum force (g) applied at that moment as the mechanical paw withdrawal threshold (PWT). Each mouse was measured three times consecutively, with an interval of at least 5 minutes between each test to avoid local sensitization or tissue damage. The average of the three measurements was taken as the final mechanical pain threshold of the animal. Electronic Von Frey tests were performed 2 days before modeling (-2 days, baseline) and on days 11, 14, 21, 28, and 35 after modeling.

[0106] All tests were performed by the same experimenter in an unknown group to reduce human error.

[0107] This invention uses the aforementioned MIA-induced mouse osteoarthritis model to evaluate the in vivo efficacy of the canine-mouse chimeric monoclonal antibodies c1D4 and c3E7. Mouse weight changes are shown below. Figure 12 As shown, the overall body weight of mice in each group remained stable during the experiment, and no significant weight loss was observed after administration of c1D4 and c3E7; the electronic Von Frey measurement results are as follows. Figure 13As shown, compared with the isotype control IgG group, the plantar pain threshold of the affected foot was significantly increased on days 3, 6, 13, 20, and 27 after c1D4 injection (days 11, 14, 21, 28, and 35 after MIA modeling); the plantar pain threshold of the affected foot was also significantly increased on days 3, 13, 20, and 27 after c3E7 injection (days 11, 21, 28, and 35 after MIA modeling); the results of the bipedal balance weight-bearing test are as follows: Figure 14 As shown, compared with the isotype IgG group, the proportion of weight-bearing limbs on the affected side of mice was significantly increased on days 4, 7, 14, 21, and 28 after injection of c1D4 and c3E7, i.e., days 12, 15, 22, 29, and 36 after MIA modeling. In conclusion, this study confirms that both c1D4 and c3E7 have therapeutic activity in the mouse MIA osteoarthritis model.

[0108] 25. In vivo efficacy evaluation of canine-mouse chimeric monoclonal antibody in an LPS-induced canine osteoarthritis model. To evaluate the efficacy of canine-mouse chimeric monoclonal antibodies c1D4 and c3E7 in dogs, a canine knee joint inflammation and pain model was established by intra-articular injection of lipopolysaccharide (LPS). The ameliorative effects of the antibodies on LPS-induced pain-related behaviors and functional changes in the affected hind limb were also evaluated. Before the experiment, based on the weight of the beagle dogs, they were randomly divided into a sham treatment group (Sham), an isotype control IgG group, a positive control Bedinvetmab group, and two canine-mouse chimeric anti-NGF antibody treatment groups (c1D4 and c3E7), with 3 dogs in each group.

[0109] A single subcutaneous injection into the neck and back was administered on day 0. The Bedinvetmab, c3E7, and c1D4 groups were given their respective anti-NGF antibodies at a dose of 0.5 mg / kg; the isotype control IgG group received an equal dose of isotype control antibody; and the blank control group received 2.5 mL of PBS per animal. After injection, the animals were observed for any significant extravasation, local redness, swelling, or other abnormal reactions at the injection site. Daily observations of the animals' mental state, feed intake, water consumption, activity level, and the injection site were maintained after administration.

[0110] To evaluate the in vivo efficacy of different anti-NGF antibodies, acute knee arthritis was induced by LPS on days 6 and 28 after administration. On day 6, the right hind limb knee was used for modeling, and on day 28, the left hind limb knee was used. Animals were appropriately sedated with propofol before LPS modeling, and restrained and aseptically treated after reaching a suitable depth of sedation. The skin around the knee joint on the intended modeling side was prepared and disinfected. With the knee joint slightly flexed at approximately 90°, the needle was inserted into the lateral patellar ligament space. Under aseptic conditions, the needle was slowly inserted into the joint cavity. After confirming the needle tip was within the joint cavity, sterile saline containing 0.25 μg LPS was slowly injected, with a total injection volume of 0.3 mL. After injection, the needle remained in the joint cavity for approximately 10 seconds, then was slowly withdrawn, and the knee joint was gently flexed and extended 5 times to ensure even distribution of LPS within the joint cavity. The sham treatment group received an equal volume of saline.

[0111] To evaluate the ameliorative effect of anti-NGF antibody on LPS-induced acute joint pain, behavioral assessments were conducted at 2, 5, and 7 hours after two LPS modeling events. All behavioral tests were performed in a relatively quiet, temperature-controlled environment by four researchers with unknown animal grouping information. Dogs were transferred to the behavioral testing environment approximately 30 minutes prior to the formal tests to acclimatize and minimize the impact of environmental changes and stress responses on the behavioral results. First, a standing weight-bearing test was used to evaluate the weight-bearing capacity of the affected hind limb in a natural state. Dogs were guided to a flat, non-slip testing area, allowed to stand naturally with their heads facing forward and limbs naturally spread. After the dogs stabilized, the weight-bearing status of the affected hind limb was observed, and the dogs were scored according to the standing scoring criteria shown in Table 9. Each dog was simultaneously scored independently by four observers with unknown animal grouping information. Then, a contralateral hind limb lifting test was used to evaluate the maximum functional weight-bearing capacity of the affected hind limb. After the animals stood naturally, the experimenter gently lifted the unmodeled hind limb on the opposite side, allowing the affected hind limb to bear the main weight of the hindquarters, and timed the test. Each dog underwent the test four times, with approximately 2 minutes between each test. The single hind limb weight-bearing score was assessed according to the single hind limb weight-bearing scoring criteria shown in Table 10. Finally, a dynamic gait test was used to evaluate joint pain and functional changes during movement. The dogs walked along a straight path of approximately 3 meters at both natural walking and trotting speeds. Four observers with unknown animal group information independently scored the dogs according to the dynamic scoring criteria shown in Table 11. The composite score was the sum of the standing score, the single hind limb weight-bearing score, and the dynamic score.

[0112] Table 9 Standing Scoring Criteria Table 10 Single Hindlimb Weight-Bearing Scoring Standard Table 11 Dynamic Scoring Criteria The in vivo efficacy of canine-mouse chimeric monoclonal antibodies c1D4 and c3E7 was evaluated using an LPS-induced canine knee arthritis pain model. The 6-day standing score results are as follows: Figure 15 As shown, compared with the isotype control IgG group, the standing scores of the c1D4 group were significantly lower at 5h and 7h after LPS modeling, and the standing score of the c3E7 group was significantly lower at 7h after LPS modeling; the results of the single hindlimb weight-bearing score at 6d are as follows. Figure 16 As shown, compared with the isotype control IgG group, the single hind limb weight-bearing score of the c1D4 group was significantly reduced at 5h and 7h after LPS modeling, and the single hind limb weight-bearing score of the c3E7 group was significantly reduced at 7h after LPS modeling; the 6-day dynamic scoring results are as follows. Figure 17 As shown, compared with the isotype control IgG group, the dynamic scores of the c1D4 group were significantly lower at 2h, 5h and 7h after LPS modeling, and the dynamic score of the c3E7 group was significantly lower at 7h after LPS modeling; the comprehensive score results at 6d are as follows. Figure 18 As shown, compared with the isotype control IgG group, the c1D4 group showed a significant decrease in the comprehensive score at 2h, 5h, and 7h after LPS modeling, while the c3E7 group showed a significant decrease in the comprehensive score at 7h after LPS modeling. The 28-day standing score results are shown below. Figure 19 As shown, compared with the isotype control IgG group, the standing scores of the c1D4 group were significantly lower at 2h, 5h and 7h after LPS modeling, and the standing score of the c3E7 group was significantly lower at 7h after LPS modeling; the results of the single hindlimb weight-bearing score at 28 days are as follows. Figure 20 As shown, compared with the isotype control IgG group, the single hind limb weight-bearing score of the c1D4 group was significantly reduced at 2h, 5h and 7h after LPS modeling, and the single hind limb weight-bearing score of the c3E7 group was significantly reduced at 5h after LPS modeling; the 28-day dynamic scoring results are as follows. Figure 21 As shown, compared with the isotype control IgG group, the dynamic scores of the c1D4 group were significantly lower at 2h, 5h and 7h after LPS modeling, and the dynamic score of the c3E7 group was significantly lower at 7h after LPS modeling; the comprehensive score results at 28 days are as follows. Figure 22 As shown, compared with the isotype control IgG group, the overall score of the c1D4 group was significantly reduced at 2h, 5h, and 7h after LPS modeling, while the overall score of the c3E7 group was significantly reduced at 7h after LPS modeling. In summary, this invention demonstrates that both c1D4 and c3E7 have therapeutic activity in the canine LPS osteoarthritis model, and that c1D4 has a superior therapeutic effect compared to Bedinvetmab.

[0113] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A chimeric monoclonal antibody c1D4 that specifically binds to canine nerve growth factor, characterized in that, The canine-mouse chimeric monoclonal antibody c1D4 includes a mouse heavy chain variable region VH, a mouse light chain variable region VL, a canine heavy chain constant region CH, and a canine light chain constant region CL. The mouse-derived heavy chain variable region VH includes heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO.7, heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO.8, and heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO.9; The mouse-derived light chain variable region VL includes light chain CDR1 with the amino acid sequence shown in SEQ ID NO.14, light chain CDR2 with the amino acid sequence shown in SEQ ID NO.15, and light chain CDR3 with the amino acid sequence shown in SEQ ID NO.16; The amino acid sequence of the constant region CH of the canine heavy chain is shown in SEQ ID NO.11; The amino acid sequence of the constant region CL of the canine light chain is shown in SEQ ID NO.

18.

2. The canine-mouse chimeric monoclonal antibody c1D4 as described in claim 1, characterized in that, The amino acid sequence of the variable region VH of the mouse heavy chain is shown in SEQ ID NO. 6; The amino acid sequence of the variable region VL of the mouse light chain is shown in SEQ ID NO.

13.

3. The canine-mouse chimeric monoclonal antibody c1D4 as described in claim 1, characterized in that, The nucleotide sequence of the gene encoding the variable region VH of the mouse heavy chain is shown in SEQ ID NO.5; The nucleotide sequence of the gene encoding the variable region VL of the mouse light chain is shown in SEQ ID NO.

12.

4. The use of the canine-mouse chimeric monoclonal antibody c1D4 as described in any one of claims 1-3 in the preparation of a medicament for treating osteoarthritis and / or osteoarthritis-related pain.

5. A medicament for treating osteoarthritis and / or osteoarthritis-related pain, characterized in that, The drug uses the canine-mouse chimeric monoclonal antibody c1D4 as the active ingredient as described in any one of claims 1-3.

6. A chimeric monoclonal antibody c3E7 that specifically binds to canine nerve growth factor, characterized in that, The canine-mouse chimeric monoclonal antibody c3E7 includes a mouse heavy chain variable region VH, a mouse light chain variable region VL, a canine heavy chain constant region CH, and a canine light chain constant region CL. The mouse-derived heavy chain variable region VH includes heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO.21, heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO.22, and heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO.23; The mouse-derived light chain variable region VL includes light chain CDR1 with the amino acid sequence shown in SEQ ID NO.26, light chain CDR2 with the amino acid sequence shown in SEQ ID NO.27, and light chain CDR3 with the amino acid sequence shown in SEQ ID NO.28; The amino acid sequence of the constant region CH of the canine heavy chain is shown in SEQ ID NO.11; The amino acid sequence of the constant region CL of the canine light chain is shown in SEQ ID NO.

18.

7. The canine-mouse chimeric monoclonal antibody c3E7 as described in claim 6, characterized in that, The amino acid sequence of the variable region VH of the mouse heavy chain is shown in SEQ ID NO.20; The amino acid sequence of the variable region VL of the mouse light chain is shown in SEQ ID NO.

25.

8. The canine-mouse chimeric monoclonal antibody c3E7 as described in claim 6, characterized in that, The nucleotide sequence of the gene encoding the variable region VH of the mouse heavy chain is shown in SEQ ID NO.19; The nucleotide sequence of the gene encoding the variable region VL of the mouse light chain is shown in SEQ ID NO.

24.

9. The use of the canine-mouse chimeric monoclonal antibody c3E7 as described in any one of claims 6-8 in the preparation of a medicament for treating osteoarthritis and / or osteoarthritis-related pain.

10. A medicament for treating osteoarthritis and / or osteoarthritis-related pain, characterized in that, The drug uses the canine-mouse chimeric monoclonal antibody c3E7 as the active ingredient as described in any one of claims 6-8.

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