Monoclonal antibody based on chimpanzee adenovirus type 68 and methods of making and uses thereof

Abstract

The application discloses a hybridoma cell secreting a monoclonal antibody against Hexon protein of AdC68 obtained by immunizing mice with chimpanzee 68 type adenovirus hexon protein as an antigen, and corresponding sequences are obtained by sequencing variable regions of the antibody, and the sequences are constructed into an expression vector plasmid, so that a recombinant monoclonal antibody with stable and efficient expression is obtained. The recombinant antibody is used for virus titration of chimpanzee 68 type adenovirus and verification, and the specificity, linear range, repeatability, intermediate precision, and durability of the recombinant monoclonal antibody obtained by the application for detecting chimpanzee 68 type adenovirus all meet the requirements.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody based on chimpanzee adenovirus type 68, its preparation method and application, belonging to the field of recombinant protein biotechnology. Background Technology

[0002] Replication-defective adenoviruses have been widely used as gene therapy drug vectors and vaccine vectors due to their low toxicity, multi-tissue tropism, high heterologous gene expression levels, strong induced immune responses, and ease of preparation and production. Among them, traditional adenovirus vectors, represented by human adenovirus type 5, have not achieved ideal clinical results due to defects such as a high pre-existing antibody ratio in the population. Therefore, non-human adenovirus serotype vectors have gradually become a focus of attention. Chimpanzee adenovirus type 68 is the first chimpanzee adenovirus vector reported in the literature for vaccine development. It possesses immunogenicity similar to human adenovirus type 5, a low pre-existing antibody ratio in the population, and advantages such as not being cross-neutralized by human adenovirus antibodies. Its application in vaccine and gene therapy drug development is increasing. Commonly used quantitative methods for adenovirus include ultraviolet spectrophotometry for detecting viral particle count, quantitative real-time PCR for detecting viral genome copy number, and plaque assay or TCID50 assay for detecting viral titer. Ultraviolet spectrophotometry is convenient and rapid, but it is easily affected by the matrix in the sample and is only suitable for the quantification of highly purified adenovirus. Quantitative real-time PCR has a wide quantitative range and high sensitivity. The nucleic acid extraction step of the sample can reduce or eliminate the interference of the sample matrix. However, like ultraviolet spectrophotometry, it cannot detect the number of infectious adenoviruses

[12] . Plaque assay or TCID50 assay calculates the number of infectious adenoviruses by inoculating adenoviruses into cell cultures and observing the cytopathic effect. However, the experimental cycle is long (usually more than 10 days) and the reproducibility of the experiment is poor.

[0003] Hexon protein is one of the most important structural proteins of adenovirus. It has a highly conserved core structure and seven hypervariable regions. It has species-specific and serum-specific determinant sites and is also the main target of serotype-specific neutralizing antibodies.

[0004] In gene therapy and vaccine development, the titer of adenovirus vectors is an important quality control indicator. Developing accurate and specific titer detection methods is of great significance for gene therapy and vaccine research and development process development and production process monitoring. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to prepare a recombinant monoclonal antibody that specifically recognizes chimpanzee adenovirus type 68 (Hexon protein) using chimpanzee adenovirus type 68 as an antigen. This antibody is used for the titer determination of chimpanzee adenovirus type 68 vector by cell immunostaining, providing guidance for the research and development of gene therapy drugs and vaccines using chimpanzee adenovirus type 68 as a viral vector.

[0006] For the reasons mentioned above, this invention provides a monoclonal antibody based on chimpanzee adenovirus type 68, which can effectively detect the viral titer of chimpanzee adenovirus type 68, thereby solving the above problems.

[0007] To achieve one of the above-mentioned objectives, this invention provides a monoclonal antibody based on chimpanzee adenovirus type 68. The antibody comprises a heavy chain and a light chain, the heavy chain and light chain each containing 3 CDR regions and 4 FR regions, respectively. The amino acid sequence of the CDR1 region of the heavy chain is GLPFNTKA (SEQ ID NO:1), the amino acid sequence of the CDR2 region of the heavy chain is IRTKSNNYAT (SEQ ID NO:2), the amino acid sequence of the CDR3 region of the heavy chain is VRDEAS (SEQ ID NO:3), the amino acid sequence of the FR1 region of the heavy chain is EVQLVETGGGLVQPKGSLKLSCAVS (SEQ ID NO:4), the amino acid sequence of the FR2 region of the heavy chain is MDWVRQAPGEGLEWVAR (SEQ ID NO:5), the amino acid sequence of the FR3 region of the heavy chain is YYADSVKDRFTISRDDAQSMLYLQMTNLKTEDTAMYYC (SEQ ID NO:6), and the amino acid sequence of the FR4 region of the heavy chain is WGHGTLVTVSA (SEQ ID NO:6). The amino acid sequence of the CDR1 region of the light chain is QSLVHSDGNTY (SEQ ID NO: 8), the amino acid sequence of the CDR2 region of the light chain is KVS (SEQ ID NO: 9), and the amino acid sequence of the CDR3 region of the light chain is SQSTHFPWT (SEQ ID NO: 10); the amino acid sequence of the FR1 region of the light chain is DIVMTQIPLSLPVSLGDQASISCRSS (SEQ ID NO: 11), the amino acid sequence of the FR2 region of the light chain is LHWYLQKPGQSPKLLIY (SEQ ID NO: 12), the amino acid sequence of the FR3 region of the light chain is NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO: 13), and the amino acid sequence of the FR4 region of the light chain is FGGGTKLEIK (SEQ ID NO: 14).

[0008] Preferably, the antibody is an anti-AdC68 antibody, and the sequence of the antibody heavy chain is: EVQLVETGGGLVQPKGSLKLSCAVSGLPFNTKAMDWVRQAPGEGLEWVARIRTKSNNYATYYADSVKDRFTISRDDAQSMLYLQMTNLKTEDTAMYYCVRDEASWGHGTLVTVSA (SEQ ID NO:15);

[0009] The sequence of the light chain is:

[0010] DIVMTQIPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHFPWTFGGGTKLEIK (SEQ ID NO: 16).

[0011] Preferably, the Hexon protein sequence of the above-mentioned chimpanzee adenovirus type 68 is as follows:

[0012] DGETATEKTYTYGNAPVQGINITKDGIQLGTDTDDQPIYADKTYQPEPQVGDAEWH

[0013] DITGTD

[0014] EKYGGRALKPDTKMKPCYGSFAKPTNKEGGQANVKTGTGTTKEYDIDMAFFDNR

[0015] SAAAAGL APEIVLYTENVDLETPDTHIVYKAGTDDSSSSINLG (SEQ ID NO: 17).

[0016] Preferably, the antibody is an IgG antibody.

[0017] To achieve one of the above-mentioned objectives, the technical solution for preparing monoclonal antibodies based on chimpanzee adenovirus type 68 adopted in this invention is as follows:

[0018] The method provided by this invention uses chimpanzee adenovirus type 68 hexagonal protein as an antigen to immunize hybridoma cells, constructs them into an expression vector plasmid, and obtains recombinant monoclonal antibodies.

[0019] The method further includes the following steps:

[0020] a) Using the Hexon protein sequence of chimpanzee adenovirus type 68, and according to the codon preference of E. coli, the target gene insertion expression vector pET-32a was synthesized, transformed into E. coli, and positive clones were selected.

[0021] b) Antigen expression and purification;

[0022] c) Animal immunization;

[0023] d) Cell fusion and subcloning screening to obtain monoclonal hybridoma cell lines;

[0024] e) Expand the culture of monoclonal hybridoma cell lines and purify them.

[0025] Preferably, the preparation method further includes an identification step, using a monoclonal antibody subtype kit to detect the antibody subtype in the hybridoma cell supernatant; specifically: the cell supernatant is added to 6 wells of an ELISA plate coated with 50 ng AdC68-Hexon protein, 100 μL / well. The plate is incubated at 37°C for 1 h, washed three times with PBST, and then diluted enzyme-labeled secondary antibodies against mouse IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 are added to the above 6 wells, incubated at 37°C for 1 h, washed three times with PBST, and then developed with TMB.

[0026] More preferably, step b, antigen expression, specifically involves: transforming the constructed plasmid into BL21 DE3 competent cells, inoculating them onto resistant LB agar plates (ampicillin), and culturing overnight at 37°C; picking single colonies, inoculating them into 3 ml of resistant liquid LB agar (ampicillin), and culturing at 37°C and 220 rpm until the OD reaches 1.0; transferring 60 μl to a 200 ml Erlenmeyer flask containing resistant liquid LB agar, and culturing overnight at 37°C and 200 rpm; the next day, adding 800 mL of fresh resistant liquid LB agar to the Erlenmeyer flask, and culturing for 1–2 h until the OD at 600 nm is 0.5–0.6; adding 200 μl of 1 M IPTG to the Erlenmeyer flask, and inducing at 37°C for 3.5 h.

[0027] More preferably, step b, antigen purification, specifically involves: centrifuging at 4000 rpm for 15 min at 4°C to collect bacterial cells, discarding the supernatant, resuspending the bacterial cells in 30 ml of PBST, adding PMSF to a final concentration of 1 mM, and sonicating at 200 W for 6 min on ice. Then, centrifuging at 8000 rpm for 15 min at 4°C, collecting the supernatant, purifying the target protein using nickel column affinity chromatography, and dialyzing the purified target protein with PBS buffer.

[0028] More preferably, step c specifically involves immunizing BALB / c mice four times with an equal volume mixture of purified AdC68-Hexon protein and complete and incomplete Freund's adjuvant, each time at two-week intervals. The first injection uses Freund's complete adjuvant at a dose of 100 μg protein per mouse, and the subsequent three booster injections use Freund's incomplete adjuvant at a dose of 50 μg protein per mouse. Seven days after the fourth immunization, blood is collected from the tail vein of the mice to separate antiserum.

[0029] More preferably, step d specifically involves: selecting BALB / c mice with high antiserum titers and boosting immunization with Freund's incomplete adjuvant at a dose of 50 μg protein per mouse; sacrificing the mice the next day, removing the spleen, collecting and counting the spleen cells, and mixing myeloma cells and spleen cells at a ratio of 1:3 to 1:10 for cell fusion; using AdC68-Hexon protein as the coating antigen, detecting whether antibodies against AdC68-Hexon protein are secreted in the cell supernatant using indirect ELISA, selecting positive clones, and then performing two rounds of subcloning on the positive clones using limiting dilution; using wild-type chimpanzee adenovirus type 68 and human adenovirus type 5 as screening strains, selecting stable positive monoclonal hybridoma cell lines that recognize wild-type chimpanzee adenovirus type 68 by cell immunostaining, expanding the culture, and cryopreserving the strains.

[0030] More preferably, step e specifically involves: expanding the cell culture obtained in step d and injecting it into the peritoneal cavity of Balb / C mice pre-sensitized with Freund's incomplete adjuvant. After 10 days, observe significant abdominal distension in the mice and aspirate ascites. Purify the ascites obtained from cell-immunized mice using a Protein A column. Load the Protein A packing material into a gravity purification column and wash three times with 10 column volumes of PBS solution. Add the ascites to the column and incubate at 4°C for 4 hours. Release the flow-through fluid and wash three times with 10 column volumes of PBS solution. Elute the antibody with pre-cooled HCl-Glycine (pH 3.0) elution buffer, and immediately neutralize the collected antibody with 10*PBS neutralization buffer. Detect the antibody concentration in the collection tube using the OD280 method, combine the high-concentration collection tubes, and dialyze overnight at 4°C with PBS solution.

[0031] To achieve one of the above-mentioned objectives, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the aforementioned antibody.

[0032] To achieve one of the above-mentioned objectives, the present invention provides a recombinant vector containing any of the aforementioned nucleotides.

[0033] To achieve one of the above-mentioned objectives, the present invention provides a cell line containing the above-mentioned recombinant vector, or having the above-mentioned nucleotides integrated into its genome.

[0034] To achieve one of the above-mentioned objectives, this invention provides the application of monoclonal antibodies in the detection of chimpanzee adenovirus type 68.

[0035] The antibody of this invention can specifically bind to the Hexon protein of chimpanzee virus type 68, effectively detecting the content of Hexon protein. It has high sensitivity and a linear range of 104.56 to 109.57 IFU / mL, which can meet the needs of quantitative detection of vaccines.

[0036] Compared with existing technologies, this invention uses the hexagonal protein of chimpanzee adenovirus type 68 as an antigen to immunize mice, obtaining hybridoma cells that secrete monoclonal antibodies against AdC68 hexon protein. The variable region of the antibody is sequenced to obtain the corresponding sequence, which is then constructed into an expression vector plasmid, resulting in a stable and highly efficient recombinant monoclonal antibody. The recombinant antibody is used for viral titration and verification of chimpanzee adenovirus type 68. The specificity, linear range, repeatability, intermediate precision, and robustness of the recombinant monoclonal antibody obtained by this invention for detecting chimpanzee adenovirus type 68 all meet the requirements. In the verification experiment of this embodiment, the cell immunostaining method involves inoculating cells with serially diluted chimpanzee adenovirus type 68 for 48 hours, then staining and counting the infected cells with a specific monoclonal antibody to calculate the infectious adenovirus titer. The entire experimental cycle takes only 3 days, and the staining spot count is accurate. Combined with a cell imaging counter, high-throughput fully automated spot counting determination can be achieved. Using recombinant anti-chimpanzee adenovirus type 68 monoclonal antibody for staining and counting can distinguish chimpanzee adenovirus type 68 from other adenovirus vectors, especially human adenovirus type 5 vectors. This enables specific, accurate, and efficient determination of the infectivity titer of chimpanzee adenovirus type 68, supporting the process development and production monitoring of chimpanzee adenovirus type 68 gene therapy drugs and vaccines.

[0037] The recombinant monoclonal antibody obtained in this invention has better specificity than currently commercialized adenovirus antibodies, and has the advantages of clear and well-defined antibody sequences, large-scale production using cells, and good batch-to-batch consistency. It is of great significance for the qualitative identification and quantitative analysis of gene drugs and vaccines using chimpanzee adenovirus type 68 as a vector. Attached Figure Description

[0038] Figure 1 Image showing the SDS-PAGE identification results of AdC68 Hexon protein;

[0039] Figure 2The image shows the cell immunostaining results of subclonal screening of the hybridoma cell line obtained in this invention. In the image, A to E are the viral immunostaining results of wild-type human adenovirus type 5 with antibodies 20#, 22#, 36#, 42# and commercial adenovirus, respectively. a to e are the viral immunostaining results of wild-type chimpanzee adenovirus type 68 with antibodies 20#, 22#, 36#, 42# and commercial adenovirus, respectively.

[0040] Figure 3 The image shows the Western Blot results for the specificity assessment of the monoclonal antibody obtained in this invention. Lane 1: Wild-type chimpanzee adenovirus type 68; Lane 2: Wild-type human adenovirus type 5.

[0041] Figure 4 The image shows the results of the reduced SDS-PAGE method for determining the purity of recombinant monoclonal antibodies. Detailed Implementation

[0042] The preparation method of the monoclonal antibody provided by the present invention and its application will be further described in detail and completely below with reference to the embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0043] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all commercially available.

[0044] The main biological materials involved in the embodiments of this invention are as follows: wild-type chimpanzee adenovirus type 68, wild-type human adenovirus type 5, HEK293A cells and myeloma cells SP2 / 0 are all derived from ATCC, BL21 DE3 competent cells are derived from Thermo Scientific, and pET-32a plasmid is preserved in the laboratory of Wuhan Bowo Biotechnology Co., Ltd.

[0045] The reagents and buffers involved in this invention are as follows: ampicillin, IPTG, PMSF, and complete and incomplete Freund's adjuvants are products of Sigma-Aldrich; Protein A column and nickel column are products of Cytiva; and commercial adenovirus antibody is a product of CellBiolabs. HRP-labeled goat anti-mouse IgG antibody, BCA protein quantification reagent, and Trizol were purchased from ThermoFisher Scientific; TMB substrate solution was purchased from SeraCare; and monoclonal antibody subtype identification reagent was purchased from Southern Biotech. Other common biochemical reagents were domestically produced analytical grade reagents.

[0046] Example

[0047] I. Preparation of Monoclonal Antibodies

[0048] 1. Constructing a carrier

[0049] Based on the Hexon protein sequence of chimpanzee adenovirus type 68 and in accordance with the codon preference of E. coli, the target gene insertion expression vector pET-32a was synthesized, transformed into E. coli, positive clones were selected, and sequencing was used to verify the correctness of the synthesized sequence.

[0050] 2. Antigen expression

[0051] The constructed plasmid was transformed into BL21 DE3 competent cells and inoculated onto resistant LB agar plates (ampicillin) and cultured overnight at 37°C. Single colonies were picked and inoculated into 3 ml of resistant liquid LB broth (ampicillin), and cultured at 37°C and 220 rpm until the OD reached 1.0. 60 μl of the inoculum was then transferred to a 200 ml Erlenmeyer flask containing resistant liquid LB broth and cultured overnight at 37°C and 200 rpm. The next day, 800 mL of fresh resistant liquid LB broth was added to the Erlenmeyer flask, and the cells were cultured for 1–2 h until the OD at 600 nm reached 0.5–0.6. 200 μl of 1 M IPTG was added to the Erlenmeyer flask, and the cells were induced at 37°C for 3.5 h.

[0052] 3. Antigen purification

[0053] The bacterial cells were collected by centrifugation at 4000 rpm for 15 min at 4℃, the supernatant was discarded, and the cells were resuspended in 30 ml of PBST. PMSF was then added to a final concentration of 1 mM, and the cells were sonicated at 200 W for 6 min on ice. The cells were then centrifuged at 8000 rpm for 15 min at 4℃, the supernatant was collected, and the target protein was purified by nickel column affinity chromatography. The purified protein was then dialyzed with PBS buffer. SDS-PAGE was used to identify the molecular weight and purity of the AdC68-Hexon protein. The results are shown below. Figure 1 As shown; the protein concentration was determined using the BCA method, and the total amount of target antigen was 3 mg.

[0054] 4. Animal immunization

[0055] BALB / c mice were immunized four times with equal volumes of purified AdC68-Hexon protein mixed with complete and incomplete Freund's adjuvant, each two weeks apart. The first injection used Freund's complete adjuvant at a dose of 100 μg protein per mouse, and the subsequent three booster injections used Freund's incomplete adjuvant at a dose of 50 μg protein per mouse. Seven days after the fourth immunization, blood was collected from the tail vein of the mice to separate antiserum (blood was incubated overnight at 4°C and centrifuged at 4000 rpm for 10 min to obtain the supernatant). The antiserum titer was detected by indirect ELISA using AdC68-Hexon protein as the coating antigen. The results are shown in Table 1, with the highest titer reaching 1:32000.

[0056] Table 1. Serum anti-Hexon protein IgG antibody titers in mice

[0057]

[0058] 5. Determination of anti-AdC68-Hexon antibody titer by indirect ELISA.

[0059] Dilute AdC68-Hexon protein to 2 μg / ml with coating buffer, add 100 μL / well to the microplate, and incubate overnight at 4°C. The next day, discard the liquid in the wells, wash the plate three times with PBST, add blocking buffer (150 μL / well), and block at room temperature for 0.5 h. Wash three times with PBST. Serially dilute the antiserum to be tested at a starting ratio of 1:2000, add 100 μL to each well, and incubate at 37°C for 1 h. Use blank serum as a negative control. Wash three times with PBST, add HRP-labeled anti-mouse IgG secondary antibody (100 μL / well), and incubate at 37°C for 40 min.

[0060] Wash the plate 5 times with PBST washing buffer and 2 times with distilled water. Add 100 μl of TMB substrate solution per well, incubate at room temperature in the dark for 15 min, then add 50 μl of stop solution per well. Measure the absorbance of each well at 450 nm using a microplate reader. Use 2.1 times the OD value of the negative control as the cutoff value, and the maximum dilution factor by which the OD value of the antiserum exceeds the cutoff value as the antiserum titer.

[0061] 6. Cell fusion and subcloning

[0062] BALB / c mice with high antiserum titers were selected and boosted with Freund's incomplete adjuvant at a dose of 50 μg protein per mouse. The mice were sacrificed the following day, and spleens were harvested. Spleen cells were collected and counted, and myeloma cells and spleen cells were mixed at a ratio of 1:3 to 1:10 for cell fusion. Using AdC68-Hexon protein as the coating antigen, an indirect ELISA method was used to detect whether antibodies against AdC68-Hexon protein were secreted in the cell supernatant. Positive clones were selected, and then two rounds of subcloning were performed using a limiting dilution method.

[0063] Stable positive monoclonal hybridoma cell lines were selected using a cell immunostaining method, with wild-type chimpanzee adenovirus type 68 as a positive control and wild-type human adenovirus type 5 as a negative control. The 293A cells that formed a monolayer were digested with trypsin to prepare a suspension with a cell concentration of 2.5 × 10⁵–3.0 × 10⁵ cells / ml, and seeded into 24-well culture plates. After one day, a monolayer formed, and the culture medium in the 24-well plates was removed. Perform 10-fold serial dilutions of the test sample. Inoculate 293A cells with an appropriate dilution at 0.1 ml / well. Set up separate cell control and antibody control wells. Incubate the culture plate at 37°C and 5% CO2 for 2 hours (shaking the plate once every 15 minutes). Remove the virus solution from the 24-well plate, then add 1 ml of culture medium to each well. Continue incubation at 37°C and 5% CO2 for 2 days. Fix with methanol for 10 minutes, wash the plate, add recombinant monoclonal antibody and incubate for 1 hour, wash the plate, add enzyme-labeled secondary antibody and incubate for 1 hour, wash the plate, develop color for 15-20 minutes, count the staining spots under a microscope, and calculate the IFU based on the average number of staining spots at each dilution.

[0064] See results Figure 2 In the figure, A to E represent the immunostaining results of wild-type human adenovirus type 5 (HPV) against cells 20#, 22#, 36#, 42#, and commercial adenovirus antibodies, respectively. a to e represent the immunostaining results of wild-type chimpanzee adenovirus type 68 (HPV) against cells 20#, 22#, 36#, 42#, and commercial adenovirus antibodies, respectively. In the cell supernatants secreted by the four monoclonal hybridoma cell lines, except for cell 42# which could simultaneously recognize both wild-type chimpanzee adenovirus type 68 and wild-type human adenovirus type 5 (HPV), cells 20#, 22#, and 36# could specifically recognize wild-type chimpanzee adenovirus type 68 (HPV). Three stable positive monoclonal hybridoma cell lines that single-recognize wild-type chimpanzee adenovirus type 68 (HPV) were screened out, expanded in culture, and cryopreserved for preservation.

[0065] 7. Monoclonal antibody subtype identification

[0066] The antibody subtypes in hybridoma cell supernatant were detected using a Southern Biotech monoclonal antibody subtype identification kit. Cell supernatant was added to six wells of an ELISA plate coated with 50 ng Hexon protein (100 μL / well). The plates were incubated at 37°C for 1 h, washed three times with PBST, and then diluted secondary antibodies against mouse IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 were added to the same six wells. The plates were incubated at 37°C for 1 h, washed three times with PBST, and developed with TMB. The subtype of the secondary antibody corresponding to a positive well was the antibody subtype. The antibody subtypes in the positive hybridoma cell supernatant were detected using the monoclonal antibody subtype identification kit. The results are shown in Table 2, all of which were the IgG1 subtype.

[0067] Table 2. Determination of Antibody Subtypes in Positive Hybridoma Cells

[0068]

[0069] 8. Ascites preparation and antibody purification

[0070] Select a positive cell line for expanded culture and inject it into the peritoneal cavity of Balb / C mice that have been pre-sensitized with Freund's incomplete adjuvant. Ten days later, observe that the mouse's abdomen is significantly distended and aspirate the ascites.

[0071] Ascites fluid obtained from mice immunized with positive cells was purified using a Protein A column for affinity purification. Protein A packing material was loaded into a gravity purification column and washed three times with 10 column volumes of PBS. Ascites fluid was added to the column and incubated at 4°C for 4 hours. The flow-through was drained and washed three times with 10 column volumes of PBS. Antibodies were eluted with pre-cooled pH 3.0 HCl-Glycine elution buffer, and the collected antibodies were immediately neutralized with 10x PBS neutralization buffer. The antibody concentration in the collection tubes was determined using the OD280 method. High-concentration collection tubes were combined and dialyzed overnight at 4°C with PBS.

[0072] The concentration of monoclonal antibodies after dialysis was detected by the BCA method, and the titer of monoclonal antibodies was determined by the indirect ELISA method. Using wild-type chimpanzee adenovirus and human adenovirus type 5 as antigens, Western blotting was used to investigate the specificity of monoclonal antibodies in recognizing chimpanzee adenovirus.

[0073] Monoclonal antibody specificity evaluation

[0074] Prepare a 10% separating gel and a 5% stacking gel. Mix 30 μl of wild-type chimpanzee adenovirus type 68 and wild-type human adenovirus type 5 with 10 μl of 4× reducing loading buffer, respectively. Incubate in boiling water for 5 min, then load 3 μl of each mixture, followed by 15 μl of pre-stained marker. Electrophoresis is performed at 90 V for 30 min, then at 150 V for 60 min. Wet transfer is performed at 90 V for 1 h, followed by washing three times with PBST for 5 min each time. Blocking is then performed with 10% skim milk at 37°C for 1 h. Washing is repeated three times with PBST for 5 min each time. The AdC68-Hexon monoclonal antibody is diluted 10,000 times with 10% skim milk, and the membrane is immersed in the primary antibody dilution solution and incubated overnight at 4°C. The membrane was removed and washed four times with PBST for 3 minutes each time. HRP-labeled goat anti-mouse IgG antibody was diluted 10,000 times with 10% skim milk. The membrane was then immersed in the secondary antibody dilution buffer and incubated at 37°C for 1 hour. The chemiluminescent substrate solution was then uniformly added to the membrane. After standing for 1 minute, the membrane was developed on a chemiluminescence imaging device with an exposure time of 3 seconds. The results are as follows: Figure 3 As shown, the monoclonal antibody can specifically recognize wild-type chimpanzee adenovirus type 68, but does not recognize the Hexon protein of wild-type human adenovirus type 5.

[0075] Monoclonal antibody variable region sequencing

[0076] RNA was extracted from anti-AdC68-Hexon monoclonal hybridoma cell lines using the Trizol method, and cDNA was obtained by reverse transcription using the 5' RACE method. Primers were designed based on the 3' constant region of the antibody, along with TSO primer sequences, to obtain the heavy and light chain variable regions. The cDNA was cloned into the pCE2 vector, and the variable region sequences VH and VL of the anti-AdC68-Hexon monoclonal antibody were obtained using first-generation sequencing.

[0077] Specifically, RT-PCR was used to amplify and sequence the heavy and light chain variable region genes from total RNA of hybridoma cell lines secreting anti-AdC68-Hexon antibody. Based on BLAST alignment, the heavy chain variable region gene VH was identified as 345 bp long, encoding 115 amino acids, and the light chain variable region gene VL was identified as 336 bp long, encoding 112 amino acids.

[0078] The antibody is an anti-AdC68 antibody, and the sequence of the antibody heavy chain is as follows:

[0079] EVQLVETGGGLVQPKGSLKLSCAVSGLPFNTKAMDWVRQAPGEGLEWVARIRTKS NNYATYYADSVKDRFTISRDDAQSMLYLQMTNLKTEDTAMYYCVRDEASWGHGTLVTV SA(SEQ ID NO:15);

[0080] The sequence of the light chain is:

[0081] DIVMTQIPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIYKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHFPWTFGGGTKLEIK (SEQ ID NO: 16).

[0082] 9. Construction, expression, and purification of recombinant monoclonal antibody plasmids

[0083] The constant region sequences of the mouse IgG1 heavy chain (IgG1CH, GenBank No.: NC_000078.7) and the constant region sequences of the mouse IgGκ light chain (IgGκCL, GenBank No.: NC_000072.7) were synthesized. Recombinant PCR was used to splice the anti-AdC68-Hexon mouse monoclonal antibody VH (VL) and mouse IgG1CH (IgGκCL), respectively, to obtain the full-length heavy and light chain sequences of the anti-AdC68-Hexon mouse monoclonal antibody. The obtained PCR products were introduced into the commercially available eukaryotic expression vectors pcDNA3.1(+) and pcDNA3.1 / zeo(+), respectively. Enzyme digestion identified positive anti-AdC68-Hexon heavy and light chain eukaryotic expression vectors MH pcDNA3.1(+) and ML pcDNA3.1 / zeo(+) (where MH represents the mouse heavy chain and ML represents the mouse light chain), which were then sent to a sequencing company for sequencing.

[0084] Recombinant PCR was used to splice anti-AdC68-Hexon mouse monoclonal antibody VH with mouse IgG1CH, and anti-AdC68-Hexon mouse monoclonal antibody VL with mouse IgGκCL, to obtain the full-length heavy chain sequence (4328 bp) and the full-length light chain sequence (656 bp) of the anti-AdC68-Hexon mouse monoclonal antibody. The full-length heavy and light chain sequences were then introduced into the eukaryotic expression vectors pcDNA3.1(+) and pcDNA3.1 / zeo(+), respectively, and sequencing results showed successful ligation.

[0085] After co-transfection of HEK293 eukaryotic cells with the anti-AdC68-Hexon heavy-light chain eukaryotic expression vector, the expressed mouse monoclonal antibody heavy-light chain molecules assembled into IgG1k and were secreted extracellularly. Cell supernatant was collected and purified using a Protein A column to obtain recombinant anti-AdC68-Hexon mouse monoclonal antibody. The concentration of the recombinant anti-AdC68-Hexon mouse monoclonal antibody was determined using the BCA method, and its purity was >90% as determined by reducing SDS-PAGE. The results are as follows: Figure 4 As shown, the binding specificity of the parental mouse antibody to AdC6-Hexon protein was determined by indirect ELISA, and its titer against AdC6-Hexon protein was 6561000.

[0086] II. Validation of Monoclonal Antibody Applications

[0087] The monoclonal antibody obtained in this embodiment can effectively and specifically recognize chimpanzee adenovirus type 68, but does not recognize human adenovirus type 5 or wild-type respiratory syncytial virus. The following experiments are used to verify the application of this monoclonal antibody.

[0088] (1) Experimental steps

[0089] 293A cells that had formed a monolayer were digested with trypsin to prepare a suspension with a cell concentration of 2.5 × 10⁵ to 3.0 × 10⁵ cells / ml, and seeded into 24-well culture plates. After one day, a monolayer formed, and the culture medium in the 24-well plates was removed. The test sample was serially diluted 10-fold, and 0.1 ml of the appropriate dilution was seeded into 293A cells per well. Cell control and antibody control wells were also included. The culture plates were incubated at 37°C and 5% CO₂ for 2 hours (shaking once every 15 minutes). The virus solution in the 24-well plates was removed, and 1 ml of culture medium was added to each well. The plates were incubated at 37°C and 5% CO₂ for another 2 days. After fixation with methanol for 10 minutes, the plates were washed, and recombinant monoclonal antibody was added and incubated for 1 hour. After washing, enzyme-labeled secondary antibody was added and incubated for 1 hour. After washing, the plates were developed for 15–20 minutes, and the staining spots were counted under a microscope. IFU was calculated based on the average number of staining spots at each dilution.

[0090] (2) Methodological validation

[0091] 2.1 Specificity

[0092] Wild-type respiratory syncytial virus (RSV) and human adenovirus type 5 (ACV) were used as negative controls. Wild-type chimpanzee adenovirus type 68 (test sample 1) and recombinant chimpanzee adenovirus type 68 modified with wild-type ACV as a vector (test sample 2) were used as test samples. The recombinant chimpanzee adenovirus type 68 was prepared by Wuhan Bowo Biotechnology Co., Ltd., by inserting a gene fragment GFP for expressing green fluorescent protein (GFP) into wild-type chimpanzee adenovirus type 68. The presence of staining spots was observed to verify the antibody's specific recognition ability against chimpanzee adenovirus type 68. The test samples were diluted 10-fold using both viral dilution buffer and various matrices from the test samples as dilution buffers, and their titers were measured. The coefficients of variation of the titer values ​​measured using the two dilution methods were compared to verify that the matrix components in the test samples did not affect the titer of chimpanzee adenovirus measured by this antibody.

[0093] The experimental results are shown in Table 4. The test samples were diluted 10-fold using both the virus diluent and the matrix of each test sample as diluents. The titers were determined using recombinant monoclonal antibodies. The coefficients of variation of the titer values ​​of each test sample determined by the two dilution methods were all less than 1%, indicating that this antibody is not affected by the matrix components in the test sample in the titer determination of chimpanzee adenovirus type 68, and has good specificity.

[0094] Table 4. Specificity of Chimpanzee Adenovirus Type 68 Titration (Cellular Immunostaining Method)

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[0096] 2.2 Linearity and Range

[0097] The recombinant chimpanzee adenovirus type 68 reference sample was serially diluted 10-fold using viral dilution buffer to determine six different dilutions (dilution factor: 10). 0 10 1 10 2 10 3 10 4 and 10 5 The theoretical titers were 9.50, 8.50, 7.50, 6.50, 5.50, and 4.50 lg IFU / mL. Each dilution reference was measured in triplicate. A regression curve was plotted for the titers of the same dilution reference and their corresponding dilution factors. The regression coefficient (r²), the measured titers of each dilution reference, and the coefficient of variation of the titer measurements were calculated.

[0098] The test sample titer showed good linearity in the range of 4.56–9.57 lg IFU / mL, with the linear equation being y = -0.436 * ln(x) + 9.5849, R0. 2 The titer was 1.0000; the CV values ​​of the titers of the six reference concentrations were all ≤1%; the titers of the six reference concentrations were all within ±0.10lg IFU / ml of the theoretical titer, indicating that the antibody has good linearity, accuracy and precision in determining the titer of chimpanzee adenovirus type 68 in the range of 4.56 to 9.57lg IFU / ml.

[0099] 2.3 Precision

[0100] A batch of recombinant chimpanzee adenovirus type 68 was used as the test sample. The same group of researchers performed six titer measurements on the same day, and the coefficient of variation of the six measurements was calculated to verify repeatability. Two groups of researchers performed 24 titer measurements on this batch of test sample on different days, and the coefficient of variation of the six measurements was calculated to verify intermediate precision.

[0101] The results are shown in Table 5. The coefficient of variation of the results of six parallel determinations of the same batch of test samples by the same group of researchers on the same day ranged from 0.18% to 0.27%. The coefficient of variation of the results of six parallel determinations of the same batch of test samples by different groups of researchers on different days was 0.39%, indicating that the antibody has good repeatability and intermediate precision in determining the titer of chimpanzee adenovirus type 68.

[0102] Table 5 Precision of Chimpanzee Adenovirus Type 68 Titration (Cellular Immunostaining Method)

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[0104] 2.4 Durability

[0105] The titers of the same batch of test samples were determined using different primary antibody incubation times (30 min, 60 min, and 90 min), with each incubation time measured in triplicate. The coefficient of variation (COP) of the titer values ​​for different incubation times was calculated to demonstrate the robustness of the primary antibody incubation time. The titers of the same batch of test samples were also determined using different antibody concentrations (1:1500, 1:2000, and 1:2500), with each staining concentration measured in triplicate. The COP of the titer values ​​for different antibody concentrations was calculated to demonstrate the robustness of the staining time.

[0106] The results are shown in Table 6. The same batch of test samples was measured three times in parallel with primary antibody incubation times of 30 min, 60 min and 90 min respectively, and the coefficient of variation was 0.28%. The same batch of test samples was measured three times in parallel with primary antibody concentrations of 1:1500, 1:2000 and 1:2500 respectively, and the coefficient of variation was 0.14%. This indicates that the antibody has good robustness in determining the titer of chimpanzee adenovirus type 68.

[0107] Table 6. Durability of Chimpanzee Adenovirus Type 68 Titration (Cellular Immunostaining Method)

[0108]

[0109] In summary, this invention uses the hexagonal protein of chimpanzee adenovirus type 68 as an antigen to immunize mice, obtaining hybridoma cells that secrete monoclonal antibodies against AdC68 hexon protein. The variable region of the antibody was sequenced to obtain the corresponding sequence, which was then constructed into an expression vector plasmid, resulting in a stable and highly efficient recombinant monoclonal antibody. The recombinant antibody was used for viral titration and validation of chimpanzee adenovirus type 68. The specificity, linear range, repeatability, intermediate precision, and robustness of the recombinant monoclonal antibody obtained by this invention for detecting chimpanzee adenovirus type 68 all met the requirements. The cell immunostaining method validated in the above experiments calculates the infectious adenovirus titer by staining and counting the virus-infected cells with a specific monoclonal antibody after inoculating them with serially diluted chimpanzee adenovirus type 68 for 48 hours. The entire experimental cycle takes only 3 days, and the staining spot count is accurate. Combined with a cell imaging counter, high-throughput fully automated spot counting determination can be achieved. Using recombinant anti-chimpanzee adenovirus type 68 monoclonal antibody for staining and counting can distinguish chimpanzee adenovirus type 68 from other adenovirus vectors, especially human adenovirus type 5 vectors. This enables specific, accurate, and efficient determination of the infectivity titer of chimpanzee adenovirus type 68, supporting the process development and production monitoring of chimpanzee adenovirus type 68 gene therapy drugs and vaccines.

[0110] The recombinant monoclonal antibody obtained in this invention has better specificity than currently commercialized adenovirus antibodies, and has the advantages of clear and well-defined antibody sequences, large-scale production using cells, and good batch-to-batch consistency. It is of great significance for the qualitative identification and quantitative analysis of gene drugs and vaccines using chimpanzee adenovirus type 68 as a vector.

[0111] Finally, it is necessary to state that the above embodiments are only used to further illustrate the technical solution of the present invention in detail, and should not be construed as limiting the scope of protection of the present invention. Any changes, modifications, substitutions, combinations, or simplifications made by those skilled in the art without departing from the scope of the technical solution of this application using the above-disclosed technical content should be considered as equivalent substitutions and are included within the scope of protection of the present invention.

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Claims

1. A monoclonal antibody based on chimpanzee adenovirus type 68, characterized in that: The antibody comprises a heavy chain and a light chain, the heavy chain and the light chain each containing 3 CDR regions and 4 FR regions, wherein the amino acid sequence of the CDR1 region of the heavy chain is shown in SEQ ID NO:1, the amino acid sequence of the CDR2 region of the heavy chain is shown in SEQ ID NO:2, the amino acid sequence of the CDR3 region of the heavy chain is shown in SEQ ID NO:3, the amino acid sequence of the FR1 region of the heavy chain is shown in SEQ ID NO:4, the amino acid sequence of the FR2 region of the heavy chain is shown in SEQ ID NO:5, the amino acid sequence of the FR3 region of the heavy chain is shown in SEQ ID NO:6, the amino acid sequence of the FR4 region of the heavy chain is shown in SEQ ID NO:7, the amino acid sequence of the CDR1 region of the light chain is shown in SEQ ID NO:8, the amino acid sequence of the CDR2 region of the light chain is shown in SEQ ID NO:9, the amino acid sequence of the CDR3 region of the light chain is shown in SEQ ID NO:10; the amino acid sequence of the FR1 region of the light chain is shown in SEQ ID NO:11, and the amino acid sequence of the FR2 region of the light chain is shown in SEQ ID NO:

11. As shown in NO:12, the amino acid sequence of the FR3 region of the light chain is shown in SEQ ID NO:13, and the amino acid sequence of the FR4 region of the light chain is shown in SEQ ID NO:

14.

2. The antibody according to claim 1, characterized in that: The antibody is an anti-AdC68 antibody, and the sequence of the antibody heavy chain is shown in SEQ ID NO:15, and the sequence of the light chain is shown in SEQ ID NO:

16.

3. The antibody according to claim 1 or 2, characterized in that, The antibody specifically recognizes the Hexon protein of chimpanzee adenovirus type 68, but does not recognize the Hexon protein of wild-type human adenovirus type 5.

4. A nucleic acid, characterized in that: It contains a nucleotide sequence encoding an antibody according to any one of claims 1-3.

5. A recombinant vector, characterized in that: The plasmid comprises the nucleic acid as described in claim 4.

6. A cell line, characterized in that: It contains the recombinant vector of claim 5, or the nucleic acid of claim 4 is integrated into its genome.

7. A detection reagent, characterized in that, Using the antibody as described in claim 1 or 2 as the detection active ingredient, the detection reagent is used for the determination of chimpanzee adenovirus type 68 by cell immunostaining.

8. The detection reagent according to claim 7, characterized in that, The detection reagent is used to determine the viral titer of chimpanzee adenovirus type 68 by cellular immunostaining.

9. The detection reagent according to claim 7, characterized in that, The detection reagent is used for virus titer quality control during the production process of adenovirus vector vaccines or gene therapy drugs.

10. A method for preparing the antibody according to claim 1 or 2, characterized in that, Includes the following steps: (a) The nucleotide sequence encoding the variable region of the antibody heavy chain was spliced ​​with the mouse IgG1 heavy chain constant region sequence, and the nucleotide sequence encoding the variable region of the antibody light chain was spliced ​​with the mouse IgGκ light chain constant region sequence. The sequences were then introduced into eukaryotic expression vectors to construct recombinant expression vectors. (b) The recombinant expression vector was co-transfected into HEK293 cells; (c) Culture the HEK293 cells to express and assemble the antibody heavy and light chains; (d) Collect cell cultures and purify the antibody using Protein A affinity chromatography.

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