Canine monoclonal antibody D1 and application thereof

Abstract

The application relates to the field of canine viral disease treatment, in particular to a full-canine-source monoclonal antibody D1 and application thereof. The heavy chain variable region amino acid sequence of the full-canine-source monoclonal antibody D1 is shown as SEQ ID No. 1, and the light chain variable region amino acid sequence is shown as SEQ ID No. 2. The antibody provided by the application is completely derived from a dog, has better reaction activity, does not cause immune rejection of the body, can be repeatedly injected, and has a neutralization titer of 3.13 mu g / mL.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of canine viral disease treatment, in particular to a canine-derived monoclonal antibody D1 and its application. BACKGROUND

[0002] Dogs are important companion animals for humans. Due to the problems of virus variation, low immune coverage, and maternal antibody interference, many viral infectious diseases, such as canine parvovirus disease, canine distemper, and canine coronavirus disease, have high clinical incidence. Injection of monoclonal antibodies is one of the most important means for treating canine viral diseases in the clinic. However, the monoclonal antibodies currently used in the clinic are derived from mice, which can neutralize viruses, but as a foreign protein, it often causes immune rejection in dogs. In order to solve the above problems, some researchers try to genetically engineer anti-parvovirus monoclonal antibodies, such as preparing single-chain antibodies or genetically engineered chimeric antibodies. These antibodies have certain advantages, but some of them are still derived from mice, and cannot completely eliminate the side effects caused by heterogeneity.

[0003] In recent years, single B cell antibody preparation technology has been widely used and has shown unique advantages and good application prospects. This technology mainly sorts single antigen-specific B cells by flow cytometry, and clones and expresses the light chain and heavy chain genes of the encoded antibody in vitro. This method ensures the natural pairing of light and heavy chain variable regions, and has the advantages of high efficiency, full natural source, and more abundant genetic diversity compared to traditional antibody preparation technology. Currently, single B cell antibody preparation technology has become a popular method for preparing human antibodies and an important tool for modern life science research. However, this technology is mainly used for the study of human and mouse antibodies, and there is no report on the preparation and application of canine monoclonal antibodies using this method.

[0004] Defects and deficiencies of the prior art:

[0005] The main treatment methods for canine parvovirus disease include specific treatment mainly by monoclonal antibody and hyperimmune serum, and symptomatic auxiliary treatment such as antidiarrheal, antibacterial anti-inflammatory and fluid replacement. Since the antibody can specifically bind to the virus, prevent the virus from invading through various ways and assist in killing the target cells infected by the virus, the antibody plays a crucial role in the clinical treatment of canine parvovirus disease. At present, the monoclonal antibody used in the clinic is derived from mice, which has the effect of neutralizing the virus. However, since the antibody is derived from heterologous animals, the antibody as an exogenous protein often causes immune rejection reaction in dogs after entering the body; at the same time, due to the heterogeneity of the Fc fragment, the antibody cannot effectively activate the activity of complement, macrophages and killer cells in the body of the dog, so that the effect of the mouse-derived antibody in the body of the dog is greatly limited. These characteristics determine the shortcomings of poor treatment effect and unsuitable for repeated use in large doses of the heterologous antibody. SUMMARY

[0006] In order to solve the above problems, the application provides a full-canine monoclonal antibody D1 and its application, and the full-canine monoclonal antibody D1 provided by the application can neutralize canine parvovirus, and the neutralization titer is 3.13 μg / mL.

[0007] In order to achieve the above purpose, the application provides the following technical scheme:

[0008] The application provides a full-canine monoclonal antibody D1, a heavy chain variable region amino acid sequence of the full-canine monoclonal antibody D1 is shown as SEQ ID No. 1, and a light chain variable region amino acid sequence is shown as SEQ ID No. 2.

[0009] Preferably, the CDR sequence of the heavy chain is shown as SEQ ID No. 3-5.

[0010] Preferably, the CDR sequence of the light chain comprises a light chain CDR1 sequence, a light chain CDR2 sequence and a light chain CDR3 sequence.

[0011] The light chain CDR1 sequence is shown as SEQ ID No. 6;

[0012] The light chain CDR2 sequence is SDG;

[0013] The light chain CDR3 sequence is shown as SEQ ID No. 7.

[0014] The application also provides application of the full-canine monoclonal antibody D1 in the above technical scheme in preparation of a drug or reagent for neutralizing canine parvovirus.

[0015] The application also provides application of the full-canine monoclonal antibody D1 in the above technical scheme in preparation of a drug for treating canine parvovirus.

[0016] The beneficial effects of the present application are as follows:

[0017] The present application uses advanced single B cell antibody preparation technology, sorts single canine parvovirus (CPV) specific B cells, and in vitro amplifies, sequences, clones and expresses the antibody sequence encoded by the B cells, and further screens the full canine source anti-parvovirus monoclonal antibody. This method is efficient and can ensure the natural pairing of the light and heavy chain variable regions of the antibody; on the one hand, since the antibody is completely derived from dogs, in addition to having better reactivity, the antibody will not cause immune rejection of the body, so it can be repeatedly injected; more importantly, the Fc fragment of the antibody can also effectively activate the complement response and the activity of phagocytes and killer cells in the dog body, helping the immune system to more effectively clear viruses and virus-infected target cells, so it will have better therapeutic effect. In addition, the antibody can be expressed and purified in large quantities by in vitro transfection of cells, so it has the conditions for large-scale production. In summary, the antibody is expected to replace the mouse-derived parvovirus antibody used in clinical practice, and has broad market application value and prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows.

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

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

[0021] Figure 3 For canine antibody heavy chain (A) and light chain (B) variable region amplification results (partially);

[0022] Figure 4 For construction of canine antibody heavy chain / light chain expression vector;

[0023] Figure 5 For expression of canine anti-CPV monoclonal antibody D1;

[0024] Figure 6 For indirect immunofluorescence method to verify the reactivity of antibody D1;

[0025] Figure 7 For ELISA to verify the reactivity of antibody D1;

[0026] Figure 8 For cell lesion map of neutralization test, A normal cells, B lesion cells. DETAILED DESCRIPTION

[0027] The application provides a full-canine-source monoclonal antibody D1, a heavy chain variable region amino acid sequence of the full-canine-source monoclonal antibody D1 is shown as SEQ ID No. 1, and a light chain variable region amino acid sequence is shown as SEQ ID No. 2.

[0028] SEQ ID No. 1:

[0029] EVQLVESGGDLVKPGGSLRLSCVVS GFTFSNYD MSWVRQAPGKGLQW VAA IRHDGSST YYTDAVKGRFTCSRDNARNTVYLQMNSLRAEDTAVYYC AKGYSSSWYLAGAFDY WGQGTLVTV. The underlined sequences are CDR1, CDR2 and CDR3 region sequences in sequence.

[0030] SEQ ID No. 3: GFTFSNYD;

[0031] SEQ ID No. 4: IRHDGSST;

[0032] SEQ ID No. 5: AKGYSSSWYLAGAFDY.

[0033] SEQ ID No. 2:

[0034] QSVLTQPASVSGSLGQRVTISCSGS TNNIGIVG ANWYQQLPGKAPKVLV Y SDG DRPSGVPDRFSGSKSGNSATLTITGLQAEDEADYYC QSSDTTLAHYV F GSGTQLTVL. The underlined sequences are CDR1, CDR2 and CDR3 region sequences in sequence.

[0035] SEQ ID No. 6: TNNIGIVG;

[0036] CDR2: SDG;

[0037] SEQ ID No. 7: QSSDTTLAHYV.

[0038] The application also provides application of the full-canine-source monoclonal antibody D1 in the above technical solution in preparation of a medicine for neutralizing canine parvovirus.

[0039] The application also provides application of the full-canine-source monoclonal antibody D1 in the above technical solution in preparation of a medicine for treating canine parvovirus.

[0040] To further illustrate the present application, the present application is described in detail below in conjunction with examples, but they should not be understood as limiting the scope of the present application.

[0041] Example 1

[0042] 1. Immunization of experimental dogs

[0043] Two 1-month-old healthy beagle dogs were purchased and repeatedly immunized 4 times with canine parvovirus live vaccine (Intervet, USA) with an interval of 21 days between each two immunizations.

[0044] 2. Sorting of virus-specific B cells and amplification of antibody heavy and light chain variable region genes

[0045] (1) Preparation of CPV-Biotin: CPV-LZ strain (CPV-2c type, VP2 gene sequence NCBI accession number OQ869252) was isolated and preserved by the laboratory, and the virus was amplified in large quantities on F81 passage cells. The specific method is as follows: F81 cells were cultured and synchronized with virus, and the cells were collected 60h after virus infection. Repeated freezing and thawing 3 times in-80℃ refrigerator, centrifuged at 12000rpm for 10min, and the supernatant was collected. After concentration with PEG6000, the virus was purified by sucrose density gradient ultracentrifugation method, and after electron microscope detection ( Figure 1 ), the virus particles were labeled with biotin labeling kit (Thermo Scientific, USA), and were recorded as CPV-Biotin.

[0046] (2) Sorting of virus-specific B cells: blood was collected from the experimental dogs after immunization by jugular vein, and peripheral blood mononuclear cells were separated by Ficoll Plus 1.077 (Solarbio) lymphocyte separation medium. After washing, the cells were resuspended with PBS solution at 10 7 cells / mL, and the cells were stained: first add anti-dog IgM-FITC (Bio-Rad, USA), anti-canine CD21-PE (Bio-Rad, USA) and CPV-Biotin, and incubate the cells at 4℃ for 30min; After washing three times, add anti-biotin-APC antibodies (Miltenyi Biotech) as secondary antibody to the cell suspension and incubate at 4℃ for 20min; In order to better distinguish antigen-specific B cells, FMO control was set at the same time (except CPV-Biotin, the cells were incubated with other antibodies at the same time). The antigen-specific CD21 + CPV + IgM single B cells were sorted by BD FACSAria II flow cytometer( Figure 2 ), first according to the FSC / SSC value to circle the live cell populationFigure 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 (Gate E, P4) were sorted into 96-well plates containing one cell per well, pre-filled with 10 μL of single-cell lysis buffer (Invitrogen). Following the instructions, cells were transferred using SuperScript. TM IVVILO TM The MasterMix (Invitrogen) kit was used to obtain single-cell cDNA via reverse transcription.

[0047] (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 3As shown, 81 heavy chain PCR products were amplified from the sorted 96 single-cell cDNA samples, of which IGHV3:IGHV4 = 63:18; 36 light chain products were amplified, of which IGLV1:IGLV3 = 35:1.

[0048] The following primers are named as SEQ ID No. 9-24.

[0049] Table 1 Primer sequences for amplifying antibody light and heavy chain variable regions

[0050] Forward primer (5'-3') Reverse primer (5'-3') LV1-outer 8: GATGACCTYCAMCATGGSMTGGT 9: GCTTGGAGGGCTTGGTGGTCTC LV1-inner 10: GCCCAGTCTRTGCTGACTC 11: CGCTGGGGTAGAAGTCGCTG LV3-outer 12: TCAGCCTCCTGGCTCTCTG 13: GCTTGGAGGGCTTGGTGGTCTC LV3-inner 14: CACAGGTTTTGTGGCCTCCTATG 15: CGCTGGGGTAGAAGTCGCTG HV3-outer 16: TTGTCRCTATTTTAMAAGGTGTCC 17: GCCGGAATTCCAGGACACAG HV3-inner 18: GTGCAGCTGGTGGARTCTGG 19: AGCCTGACACCAGGCAGG HV4-outer 20: CAGCTCCCCTGGGTGTCC 21: GCCGGAATTCCAGGACACAG HV4-inner 22: CTGAACTCACACTGCAGGAGTC 23: AGCCTGACACCAGGCAGG

[0051] 3. In vitro expression and purification of antibodies

[0052] The paired light / heavy chain variable region PCR products were ligated into pUC19 vectors (Sangon Biotech, China) and sent to Genewiz Biotechnology Co., Ltd. for sequencing. The antibodies were named according to the position of the single cell in the 96-well plate, and the antibody light / heavy chain expression vectors were constructed, the specific method was as follows Figure 4 As shown: signal peptide sequences were added at the 5' end of the antibody light / heavy chain variable region genes, and lambda light chain constant region sequences / IgG2 heavy chain constant region gene sequences and His tag sequences were added at the 3' end, and after codon optimization, they were cloned into pcDNA3.4 vectors, and the synthesis of plasmids was entrusted to the company. Recombinant heavy chain and light chain expression plasmids were transfected into ExpiCHO-S TM cells according to the mass ratio of 2:3 using ExpiFectamine TM CHO Transfection Kit (Invitrogen, USA). After 5 days of suspension culture, the cell supernatant was collected, and the AKTA protein purification instrument (GE Life Sciences) was used to purify the antibody. After eluting the antibody, it was dialyzed three times in PBS solution, and then concentrated with PEG6000 to obtain the purified antibody. Non-denaturing / denaturing SDS-PAGE electrophoresis was used to identify the purified part of the antibody Figure 5 ), which showed that the antibody was about 170KD under non-denaturing conditions, and after denaturation, the antibody was divided into heavy chain (about 55KD) and light chain (about 30KD), indicating that the antibody was correctly expressed and assembled. A total of 22 antibodies were expressed and purified, which were A1 / A4 / A5 / A6 / A8 / A9 / A10 / A11 / A12 / B2 / B6 / B10 / B11 / B12 / C12 / D1 / E1 / F1 / H8 / H10 / H11 / H12.

[0053] 4. Verification of antibody reactivity and neutralization activity.

[0054] (1) Indirect immunofluorescence test (IFA)

[0055] 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 D1 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 D1 antibody had a good reactivity with CPV-2. Figure 6 ).

[0056] The amino acid sequence of the variable region of the heavy chain of the D1 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.

[0057] The amino acid sequence of the light chain variable region of the D1 antibody is shown in SEQ ID No. 2. The CDR sequence of the light chain includes the light chain CDR1 sequence, the light chain CDR2 sequence and the light chain CDR3 sequence; the light chain CDR1 sequence is shown in SEQ ID No. 6; the light chain CDR2 sequence is SDG; and the light chain CDR3 sequence is shown in SEQ ID No. 7.

[0058] (2) Enzyme-linked immunosorbent assay (ELISA)

[0059] The 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. Each well was then blocked with 100 μL of 5% skim milk at room temperature for 2 hours. The blocking solution was discarded, and the plates were washed three times with PBST. The purified D1 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 7As shown, the cut-off value = 2.1 × negative control value. Any value greater than the cut-off value is considered a positive reaction. This indicates that D1 antibody at concentrations above 0.3 μg / mL can react with the coated viral antigen.

[0060] (3) Neutralization test

[0061] 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 8 If 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 D1 is 3.13 μg / mL.

[0062] 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 full canine monoclonal antibody D1, characterized in that, The amino acid sequence of the heavy chain variable region of the full-canine monoclonal antibody D1 is shown as SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.

2.

2. Use of the full-canine monoclonal antibody D1 of claim 1 in the preparation of a reagent for neutralizing canine parvovirus.

3. Use of the full-canine monoclonal antibody D1 of claim 1 in the preparation of a medicine for treating canine parvovirus.

Images