A canine monoclonal antibody A10 and its applications and pharmaceuticals

Abstract

This invention relates to the field of whole viral disease treatment technology, and in particular to a canine monoclonal antibody A10, its applications, and pharmaceutical uses. The amino acid sequence of the heavy chain variable region of the canine monoclonal antibody A10 is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2. The antibody provided by this invention is entirely derived from dogs. Besides exhibiting better reactivity, this antibody does not induce an immune rejection response in the body, therefore it can be repeatedly injected, and its neutralizing titer is 3.13 μg / mL.

Description

Technical Field

[0001] This invention relates to the field of whole viral disease treatment technology, and in particular to a canine monoclonal antibody A10, its application, and a drug thereof. Background Technology

[0002] Dogs are important companion animals to humans. Due to viral mutations, low immunization coverage, and interference from maternal antibodies, the clinical incidence of many viral infectious diseases, such as canine parvovirus, canine distemper, and canine coronavirus disease, remains high. Monoclonal antibody injection is currently one of the most important clinical treatments for canine viral diseases. However, the monoclonal antibodies currently used clinically are derived from mice. Although they have a neutralizing effect on viruses, as foreign proteins, they often induce immune rejection in dogs. To address these issues, some researchers have attempted to genetically engineer anti-parvovirus monoclonal antibodies, such as preparing single-chain antibodies or genetically engineered chimeric antibodies. While these antibodies possess certain advantages, a portion of them still originates from mice, failing to completely eliminate the side effects caused by heterologous origin.

[0003] In recent years, single-B-cell antibody preparation technology has been widely applied, demonstrating unique advantages and promising application prospects. This technology primarily involves sorting individual antigen-specific B cells using flow cytometry and then cloning and expressing the light and heavy chain genes encoding antibodies in vitro. Monoclonal antibodies produced using this method ensure the natural pairing of the light and heavy chain variable regions, offering advantages over traditional antibody preparation techniques such as high efficiency, all-natural origin, and richer gene diversity. Currently, single-B-cell antibody preparation technology has become a popular method for preparing human antibodies and an important tool in modern life science research. However, this technology is currently mainly used in the research of human and murine antibodies; there are no reports of its application in the preparation and use of canine monoclonal antibodies.

[0004] The main treatments for canine parvovirus disease include specific therapy primarily using monoclonal antibodies and hyperimmune serum, along with symptomatic adjunctive therapy such as antidiarrheal medication, antibacterial and anti-inflammatory drugs, and fluid replacement. Antibodies play a crucial role in the clinical treatment of canine parvovirus disease because they can directly bind to the virus, preventing viral invasion and assisting in the killing of virus-infected target cells through multiple pathways. Currently, clinically used monoclonal antibodies are derived from mice. While they have a neutralizing effect on the virus, their foreign origin often leads to immune rejection in dogs. Furthermore, due to the heterologous nature of their Fc fragments, these antibodies cannot effectively activate the activity of complement, macrophages, and cytotoxic cells in dogs, significantly limiting the effectiveness of murine antibodies in the canine body. These characteristics determine the disadvantages of heterologous antibody therapy, such as poor efficacy and unsuitability for repeated high-dose use. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a canine monoclonal antibody A10, its applications, and a pharmaceutical formulation. The canine monoclonal antibody A10 provided by this invention can neutralize canine parvovirus with a neutralizing titer of 3.13 μg / mL.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The present invention provides a canine monoclonal antibody A10, wherein the amino acid sequence of the heavy chain variable region of the canine monoclonal antibody A10 is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2.

[0008] Preferably, the CDR sequence of the heavy chain is shown in SEQ ID No. 3 to 5.

[0009] Preferably, the CDR sequence of the light chain includes a light chain CDR1 sequence, a light chain CDR2 sequence, and a light chain CDR3 sequence;

[0010] The light chain CDR1 sequence is shown in SEQ ID No. 6;

[0011] The light chain CDR2 sequence is GSS;

[0012] The light chain CDR3 sequence is shown in SEQ ID No. 7.

[0013] This invention also provides the application of the canine monoclonal antibody A10 described in the above technical solution in the preparation of drugs for neutralizing canine parvovirus.

[0014] The present invention also provides the application of the canine monoclonal antibody A10 described in the above technical solution in the preparation of drugs for treating canine parvovirus.

[0015] The present invention also provides a drug for treating canine parvovirus, comprising the canine monoclonal antibody A10 described in the above technical solution and a pharmaceutically acceptable vector.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention utilizes advanced single-cell antibody preparation technology. By sorting individual canine parvovirus (CPV)-specific B cells and amplifying, sequencing, cloning, and expressing their encoded antibody sequences in vitro, a fully canine anti-parvovirus monoclonal antibody is screened. This method is highly efficient and ensures the natural pairing of the antibody's light and heavy chain variable regions. Furthermore, since the antibody is entirely derived from dogs, it not only exhibits better reactivity but also does not induce immune rejection, allowing for repeated injections. More importantly, the Fc fragment of this antibody effectively activates the complement response and the activity of phagocytes and cytotoxic cells in dogs, helping the immune system more effectively clear the virus and virus-infected target cells, thus resulting in better therapeutic effects. In addition, the antibody can be expressed and purified in large quantities through in vitro cell transfection, thus enabling large-scale production. In summary, this antibody has the potential to replace clinically used murine parvovirus antibodies, possessing broad market application value and prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 For electron microscopy observation of purified virus particles;

[0020] Figure 2 For the sorting of virus-specific B cells in PBMCs;

[0021] Figure 3 Partial amplification results of the variable regions of the canine antibody heavy chain (A) and light chain (B);

[0022] Figure 4 Construction of a canine antibody heavy / light chain expression vector;

[0023] Figure 5 Expression of canine anti-CPV monoclonal antibody;

[0024] Figure 6 To verify the reactivity of antibody A10 using an indirect immunofluorescence method;

[0025] Figure 7 To verify the reactivity of antibody A10 using ELISA;

[0026] Figure 8 The image shows cytopathic effects in a neutralization test. A represents normal cells, and B represents diseased cells. Detailed Implementation

[0027] IgG antibodies are Y-shaped molecules composed of two identical heavy chains (H chains) and two identical light chains (L chains). Based on the degree of amino acid variation, both the heavy and light chains can be divided into variable regions (V regions) and constant regions (C regions). Amino acid variations in the variable regions are mainly concentrated in the hypervariable regions. Each variable region of both the heavy and light chains contains three hypervariable regions. These hypervariable regions together form a three-dimensional spatial structure that binds complementary to the antigenic epitope; therefore, it is also called the "complementarity-determining region" (CDR). Because the CDR is the region where the antibody directly binds to the antigenic determinant, it generally has high specificity and is of greater significance.

[0028] The present invention provides a canine monoclonal antibody A10, wherein the amino acid sequence of the heavy chain variable region of the canine monoclonal antibody A10 is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2.

[0029] SEQ ID No. 1:

[0030] EVQLVESGGDLVKPGGSLRLSCVAS GFTFSASD MSWIRQAPGKGLQWV AY ITNDGSTT NYADAVKGRFTISRDNAKNTLYLQMNSLRTEDTAVYYC ADY VQDIYGIWIDL WGQGTLVTV. The underscores represent the CDR1, CDR2, and CDR3 region sequences, respectively.

[0031] SEQ ID No. 3: GFTFSASD;

[0032] SEQ ID No. 4: ITNDGSTT;

[0033] SEQ ID No. 5: ADYVQDIYGIWIDL.

[0034] SEQ ID No. 2:

[0035] QSVLTQPASVSGSLGQRVTISCTGS NSNVGYGNY VGWYQQLPGTGPRTL IY GSS YRPSGVPDRFSGSSSGSSATLTISGLQAEDEADYYC SSYDSSLSGIV FG GGSHLTVL. The underscores represent the CDR1, CDR2, and CDR3 region sequences, respectively.

[0036] SEQ ID No. 6: NSNVGYGNY;

[0037] CDR2: GSS;

[0038] SEQ ID No. 7: SSYDSSLSGIV.

[0039] This invention also provides the application of the canine monoclonal antibody A10 described in the above technical solution in the preparation of drugs for neutralizing canine parvovirus.

[0040] The present invention also provides the application of the canine monoclonal antibody A10 described in the above technical solution in the preparation of drugs for treating canine parvovirus.

[0041] This invention provides a drug for treating canine parvovirus, comprising the fully canine monoclonal antibody A10 described in the above-described technical solution and a pharmaceutically acceptable carrier. This invention does not specifically limit the type of drug or its preparation method; those skilled in the art can use conventional methods.

[0042] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] 1. Immunization of experimental dogs

[0045] Purchase two healthy 1-month-old Beagle puppies and vaccinate them four times with canine parvovirus live vaccine (Intervet, USA), with an interval of 21 days between each vaccination.

[0046] 2. Sorting of virus-specific B cells and amplification of antibody heavy and light chain variable region genes.

[0047] (1) Preparation of CPV-Biotin: The CPV-LZ isolate (CPV-2c type, NCBI accession number for its VP2 gene sequence is OQ869252) was amplified in large quantities in F81 passaged cells. The specific method is as follows: F81 cells were cultured and simultaneously inoculated with the virus. Cells were harvested 60 h after inoculation, subjected to three freeze-thaw cycles at -80℃, and centrifuged at 12000 rpm for 10 min. The supernatant was collected. After concentration with PEG6000, the virus was purified by sucrose density gradient ultracentrifugation and analyzed by electron microscopy. Figure 1 Afterwards, the virus particles were labeled with a biotin labeling kit (Thermo Scientific, USA), and designated as CPV-Biotin.

[0048] (2) Sorting of virus-specific B cells: Blood was collected from the jugular vein of immunized experimental dogs, and peripheral blood mononuclear cells were separated using Ficoll Plus 1.077 lymphocyte separation medium (Solarbio). After washing, the cells were sorted according to a 10... 7 Cells were resuspended in PBS and stained: anti-dog IgM-FITC (Bio-Rad, USA), anti-canine CD21-PE (Bio-Rad, USA), and CPV-Biotin were added, and the cells were incubated at 4°C for 30 min. After washing three times, anti-biotin-APC antibodies (Miltenyi Biotech) were added to the cell suspension as secondary antibodies and incubated at 4°C for 20 min. To better distinguish antigen-specific B cells, an FMO control was also set up (cells were incubated with other antibodies in the same way except for CPV-Biotin). Subsequently, antigen-specific CD21 cells were sorted using a BDFACSAria II flow cytometer. + CPV + IgM-inducing single B cells ( Figure 2 First, the viable cell population is delineated based on the FSC / SSC value. Figure 2 (A, P1 phylum), then exclude adherent cells according to the FSC-A / FSC-H diagram, and circle the single live cells ( Figure 2 (B, P2 phylum), then based on CD21 / IgM staining, exclude naïve B cells and circle CD21 cells. + IgM-cells (mainly including plasma cells and memory B cells, such as...) Figure 2 (C, P3 gates), and finally according to FMO comparison ( Figure 2 The marker status of D and CPV was used to circle antigen-specific B cells. Figure 2 Cells from the P4 gate (E, P4 phylum) were sorted into 96-well plates containing 10 μL of single-cell lysis buffer (Invitrogen), one cell per well; cells were then processed using SuperScript according to the manufacturer's instructions. TM IVVILO TM The MasterMix (Invitrogen) kit was used to obtain cellular cDNA via reverse transcription.

[0049] (3) Amplification of antibody heavy chain and light chain variable region genes: Based on the usage preference of the V gene of the canine antibody heavy chain variable region (IGHV3 and IGHV4 were used most frequently, at 79.1% and 20.6%, respectively), upstream primers (HV3 and HV4) and downstream primers (common) for IGHV3 and IGHV4 were designed respectively; based on the usage preference of the V gene of the light chain variable region (IGLVs:IGKVs were approximately 10:1; among IGLVs, IGLV1 was the most commonly used [77.2%], followed by IGLV8 [15.3%] and IGLV3 [5.9%]), κ chain primers (not listed), upstream primers (LV1 and LV3) and downstream primers (common) for IGLV1 and IGLV3 were designed respectively, and the primers are shown in Table 1. Nested PCR was used with single-cell cDNA per well as a template. The antibody heavy chain variable region sequence was amplified using upstream and downstream primers for IGHV3 and IGHV4, respectively, and the antibody light chain variable region sequence was amplified using κ chain primers (not listed), IGLV1, and IGLV3 upstream and downstream primers, respectively. The first-round PCR amplification system was as follows: 25 μl of 2×PhantaMaxMasterMix, 1 μl each of outer upstream and downstream primers (OuterF / R), 5 μl of single-cell cDNA sample (template), and 18 μl of ddH2O. The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 72℃ extension for 50 s, 30 cycles; 72℃ extension for 10 min. Using the first-round PCR product as a template, the second round of amplification was performed using inner upstream and downstream primers (InnerF / R), with the same reaction program as the first round. Electrophoresis images of the amplification products in each well (partial) are shown below. Figure 3 As shown, a total of 81 heavy chain PCR products were amplified from 96 single-cell cDNA samples, of which IGHV3:IGHV4 = 63:18; at the same time, 36 light chain products were amplified, of which IGLV1:IGGLV3 = 35:1.

[0050] The following primers are named SEQ ID No. 9-24.

[0051] Table 1 Primer sequences for amplifying the variable regions of the antibody light and heavy chains.

[0052]

[0053]

[0054] 3. In vitro expression and purification of antibodies

[0055] The paired light / heavy chain variable region PCR products were ligated into the pUC19 vector (Sangon Biotech, China) and sent to Genewiz Biotechnology Co., Ltd. for sequencing. Antibodies were named according to their location on single cells in 96-well plates, and antibody light / heavy chain expression vectors were constructed using the following method: Figure 4 As shown: A signal peptide sequence was added to the 5' end of the antibody light / heavy chain variable region gene, and a λ light chain constant region sequence / IgG2 heavy chain constant region gene sequence and His tag sequence were added to the 3' end. After codon optimization, the gene was cloned into the pcDNA3.4 vector, and plasmid synthesis was commissioned to a company. The recombinant heavy chain and light chain expression plasmids were mixed at a 2:3 mass ratio using ExpiFectamine. TM ExpiCHO-STM cells were transfected using an Invitrogen (USA) transfection kit. After 5 days of suspension culture, the cell supernatant was collected, and the antibody was purified using an AKTA protein purification system (GELifeSciences). After eluting the antibody, the cells were dialyzed three times in PBS solution, followed by concentration with PEG6000 to obtain the purified antibody. The purified antibody fraction was identified by non-denaturing / denaturing SDS-PAGE electrophoresis. Figure 5 As can be seen, the antibody under non-denaturing conditions is approximately 170 kDa, while after denaturation, the antibody separates into heavy chains (approximately 55 kDa) and light chains (approximately 30 kDa), indicating that the antibodies were correctly expressed and assembled. A total of 22 antibody strains were expressed and purified, namely A1 / A4 / A5 / A6 / A8 / A9 / A10 / A11 / A12 / B2 / B6 / B10 / B11 / B12 / C12 / D1 / E1 / F1 / H8 / H10 / H11 / H12.

[0056] 4. Verification of antibody reactivity and neutralizing activity.

[0057] (1) Indirect immunofluorescence assay (IFA)

[0058] After passage of F81 cells, at a rate of 2 × 10⁶ 5 Cells were seeded in 24-well plates and simultaneously inoculated with CPV-LZ at 1 MOI. After 48 h, cells were fixed with paraformaldehyde at room temperature for 15 min. The fixative was discarded, and 0.1% Tritonx-100 was added, followed by permeabilization at room temperature for 5 min. The permeabilization solution was discarded, and cells were washed three times with PBS, then blocked with 5% BSA solution at room temperature for 2 h. The blocking solution was discarded, and cells were washed three times with PBS, then purified A10 antibody (approximately 2 ng / μL) was added and incubated at room temperature for 2 h. The primary antibody was discarded, and cells were washed three times with PBS, then HRP-labeled anti-canine IgG antibody (Bio-Rad Laboratories) was added and incubated at room temperature for 1 h. After washing cells four times with PBS, the results were observed under a fluorescence microscope. Green fluorescence was observed in the cells, indicating that the A10 antibody had a good reactivity with CPV-2. Figure 6 ).

[0059] The amino acid sequence of the variable region of the heavy chain of the A10 antibody is shown in SEQ ID No. 1, and the CDR sequence of the heavy chain is shown in SEQ ID No. 3 to 5.

[0060] The amino acid sequence of the light chain variable region of the A10 antibody is shown in SEQ ID No. 2. The CDR sequences of the light chain are light chain CDR1, light chain CDR2 and light chain CDR3. The light chain CDR1 sequence is shown in SEQ ID No. 6; the light chain CDR2 sequence is GSS; and the light chain CDR3 sequence is shown in SEQ ID No. 7.

[0061] (2) Enzyme-linked immunosorbent assay (ELISA)

[0062] Purified CPV-2 virus was diluted to 1 ng / μL with PBS and coated onto 96-well microplates (100 μL / well) overnight at 4°C. The coating solution was discarded, and the plates were washed three times with PBST. 100 μL of 5% skim milk was added to each well for blocking at room temperature for 2 hours. The blocking solution was discarded, and the plates were washed three times with PBST. The purified A10 antibody was first diluted to 10 μg / mL, then serially diluted 2-fold. 1 -2 10 Then, use 100 μL of HRP-labeled anti-canine IgG antibody (Bio-Rad Laboratories) as the primary antibody and incubate at room temperature for 2 hours. Discard the primary antibody, wash the plate three times with PBST, and incubate at room temperature for 1 hour with HRP-labeled anti-canine IgG antibody (Bio-Rad Laboratories). Discard the secondary antibody, wash the plate four times with PBST, add 100 μL of TMB substrate solution per well, and incubate for 5 minutes. Add 100 μL of stop solution per well and measure the OD using a microplate reader. 450 Absorbance values ​​for each well at the specified wavelength. Two replicates were set up for each antibody dilution, along with a negative control (primary antibody plus PBS). Results are as follows: Figure 7 As shown, the cut-off value = 2.1 × negative control value. Values ​​greater than the cut-off value were considered positive reactions, indicating that A10 antibodies at concentrations above 0.3 μg / mL can react with the coated viral antigens.

[0063] (3) Neutralization test

[0064] The neutralizing capacity of antibodies was determined at the cellular level using a micro-neutralization assay. The specific procedure was as follows: 100 μg / mL of purified antibody was added from 2... 1 -2 9 Perform a 2-fold serial dilution; take 50 μL of the diluted antibody and mix with an equal volume of 100 TCID50. 50 The virus-antibody mixture was mixed with the virus solution and incubated at 37°C for 1 hour; the virus-antibody mixture was then added to an equal volume (100 μL) of freshly digested F81 cells (approximately 2 × 10⁻⁶ cells). 4 Cells (100 cells) were seeded in 96-well plates and incubated at 37°C in a 5% CO2 incubator; a blank cell control and 0.1 / 100 TCID45 cells were also included. 50 For the virus-infected cell control, each antibody dilution and control was prepared in 10 replicates. Cytopathic effects were observed daily during culture. Figure 8If the antibody has a neutralizing effect, the amount that can prevent 50% of 100 TCID32 antibodies can be calculated using the Reed-Muench method. 50 The antibody dilution for the cytopathic effect caused by the virus is calculated by dividing the initial and final antibody concentrations by this dilution, which gives the antibody neutralizing titer. The final calculated neutralizing titer of A10 is 3.13 μg / mL.

[0065] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A canine monoclonal antibody A10, characterized in that, The amino acid sequence of the heavy chain variable region of the canine monoclonal antibody A10 is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

2. The use of the canine monoclonal antibody A10 as described in claim 1 in the preparation of a drug for neutralizing canine parvovirus.

3. The use of the canine monoclonal antibody A10 as described in claim 1 in the preparation of a drug for treating canine parvovirus.

4. A drug for treating canine parvovirus, characterized in that, It includes the canine monoclonal antibody A10 as described in claim 1 and a pharmaceutically acceptable vector.

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