An antibody against human adenovirus hexon protein and application thereof

Abstract

This application relates to the field of biomedical technology, providing an antibody against human adenovirus hexon protein and its application. The antibody comprises a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises three heavy chain antigen complementary regions: amino acid sequences as shown in SEQ ID NO. 10-12; the light chain variable region comprises three light chain antigen complementary regions: LCDR1 with an amino acid sequence as shown in SEQ ID NO. 13, LCDR2 with an amino acid sequence of AAS, and LCDR3 with an amino acid sequence as shown in SEQ ID NO. 14. Related experiments have confirmed that this antibody can specifically recognize human adenovirus type 55 (HAdV) Hexon protein, with the antigenic epitope in the A180-A193 region, and can effectively prevent and control infection by HAdV-55 and / or HAdV-11 viruses.

Description

An antibody against human adenovirus hexagonal protein and its application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antibody against human adenovirus hexagonal protein and its application. Background Technology

[0002] Human adenovirus (HAdV) belongs to the genus Adenovirus of the family Adenoviridae. It is a non-enveloped, linear, double-stranded DNA virus with an icosahedral structure. This virus is widespread globally and can specifically infect various tissues and organs, including the respiratory tract, gastrointestinal tract, urinary tract, and eyes, making it a common infectious pathogen in clinical practice. Currently, HADV has been classified into seven subgenera (AG) based on serological and genomic characteristics, with over 100 genotypes identified. Among them, HADV-55, formed by the recombination of HADV-11 and HADV-14, has become a significant pathogen causing severe pneumonia in adults and children due to its high transmissibility and pathogenicity. Currently, treatment for HADV infection is mainly symptomatic and supportive, lacking safe and effective specific antiviral drugs. While existing broad-spectrum antiviral drugs have shown some HADV inhibitory activity in in vitro experiments, their widespread clinical use is limited by factors such as toxic side effects, drug resistance, and unstable efficacy. The capsid structure of HAdV is crucial for its infection of host cells and recognition by the immune system. It contains three main capsid proteins: hexon, penton, and fiber. Among them, hexon protein is the most abundant component of the capsid, accounting for 60% of the total capsid protein mass. Its structural characteristics directly determine the antigenicity, immunogenicity, and serotype differences of HAdV, and it is the core target for the development of HAdV prevention and control technologies.

[0003] Most existing anti-adenovirus antibodies are murine antibodies or humanized antibodies (obtained by humanizing non-human antibodies). Although they possess certain neutralizing activity, they may induce immunogenic reactions or have problems such as short half-life and adverse reactions in practical applications, thus limiting their clinical use. For example, the anti-HAdV-55 monoclonal antibody developed in patent CN116023474A is based on a murine antibody and obtained by integrating it into a human antibody backbone, enabling it to recognize human adenovirus fiber proteins. Another patent, CN118240069A, developed a neutralizing antibody against HAdV-55, using a macaque antibody as the starting sequence and humanizing it to recognize human adenovirus Hexon HVR7. Although the above antibodies improve the similarity to human antibodies to some extent, their antibody sequences inevitably retain a certain proportion of non-human framework regions or complementarity-determining regions. In subsequent clinical applications, these antibodies may induce drug-resistant antibody responses such as the HAMA response, which may lead to immunogenicity risks, resulting in reduced efficacy, shortened duration of action, or even adverse immune reactions. These problems are more pronounced when repeated administration is required or when used in individuals whose immune systems are not yet fully mature (such as children).

[0004] In contrast, fully human antibodies, whose variable and constant regions are derived from human antibody sequences, can effectively reduce immunogenicity and the possibility of developing drug-resistant antibodies at the structural level. They offer significant advantages in clinical safety, tolerability, and suitability for repeated dosing, and have gradually become one of the important directions in antibody drug development. Fully human antibodies can significantly reduce immunogenicity risks and improve drug safety, better meeting the treatment needs of severely ill adenovirus patients and high-risk groups. However, the development of human anti-adenovirus antibodies is challenging, and currently, there is a general lack of fully human anti-adenovirus antibodies that combine low immunogenicity with broad-spectrum neutralizing properties.

[0005] Compared with traditional methods, the use of single B-cell BCR sequencing technology to develop monoclonal antibodies has overwhelming advantages: maintaining natural pairing, fully human antibodies, high throughput and high efficiency, and preserving the complete antibody expression profile. It has become the preferred technical solution for developing fully human antibody drugs, especially neutralizing antibodies against infectious diseases.

[0006] Currently, AI-assisted antibody gene screening technology is becoming increasingly mature. Using tools such as AlphaFold3, IgFold, Schrödinger Maestro, ABodyBuilder, and DeepAb, antibody models can be quickly obtained by learning from a large number of known antibody sequence-structure correspondences. This allows for rapid and high-precision prediction of antibody structures, and can more accurately predict the three-dimensional structures of antibodies (Fv regions) and target antigens. Developing deep learning-based scoring functions can more accurately predict binding free energy (FV).Δ (G), thereby determining the strength of binding, and ultimately quickly and effectively screening candidate antibody sequences from massive amounts of data, accelerating the development of related antibody drugs. Summary of the Invention

[0007] Objective: The technical problem this invention aims to solve is to address the lack of fully human anti-adenovirus antibodies in existing technologies by providing an antibody against human adenovirus hexon (HAdV Hexon) and its applications. Based on a fully human antibody framework, the antibody is designed and screened targeting specific epitopes of the Hexon protein. The fully human antibody designed in this invention specifically recognizes the A180-A193 region of the human adenovirus type 55 (HAV55) Hexon protein, effectively preventing and controlling HAV infection. It can be used for the prevention and treatment of HAV infection and also exhibits neutralizing activity against human adenovirus type 11, which has similar Hexon epitope characteristics. Specifically, this invention provides the development of an antibody based on HAV Hexon and its application in the diagnosis, treatment, and prevention of HAV, particularly suitable for the prevention and control of severe HAV infection in high-risk groups such as children and immunocompromised individuals.

[0008] To address the aforementioned technical problems, this invention discloses an antibody against human adenovirus hexagonal protein and its applications. The specific technical solution is as follows:

[0009] In a first aspect, the present invention provides an antibody against human adenovirus hexagonal protein or its antigen-binding fragment (4C9), which is a fully human antibody or its binding fragment, comprising a heavy chain variable region and a light chain variable region; wherein, the heavy chain variable region comprises three heavy chain antigen complementary regions: HCDR1 with the amino acid sequence shown in SEQ ID NO. 10, HCDR2 with the amino acid sequence shown in SEQ ID NO. 11, and HCDR3 with the amino acid sequence shown in SEQ ID NO. 12; the light chain variable region comprises three light chain antigen complementary regions: LCDR1 with the amino acid sequence shown in SEQ ID NO. 13, LCDR2 with the amino acid sequence AAS, and LCDR3 with the amino acid sequence shown in SEQ ID NO. 14.

[0010] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0011] The antibody or its antigen-binding fragment comprises a heavy chain and a light chain; wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO.17, and the amino acid sequence of the light chain is shown in SEQ ID NO.18.

[0012] The antibody or its antigen-binding fragment described in this invention can recognize and / or specifically bind to specific regions A180-A193 of the adenovirus Hexon protein (amino acid sequence shown in SEQ ID NO.19).

[0013] In some embodiments of the present invention, the antibody or its antigen-binding fragment is a fully human antibody, monoclonal antibody, bispecific antibody, multispecific antibody, nanobody, single-domain antibody (sdAb), or bivalent domain antibody; the antigen-binding fragment of the antibody is selected from F(ab')2, F(ab)2, Fab', Fab, Fv, Fd, scFv, or any other antibody fragment that binds to the antigen but does not contain a complete antibody structure. In some embodiments of the present invention, the antibody is a fully human antibody.

[0014] In a second aspect, the present invention provides a nucleic acid encoding an antibody or an antigen-binding fragment thereof against the anti-human adenovirus hexaplex protein described in the first aspect.

[0015] It includes nucleotide sequences encoding three heavy chain antigen complementary regions as shown in SEQ ID NO.5~7, nucleotide sequences encoding light chain antigen complementary region LCDR1 as shown in SEQ ID NO.8, nucleotide sequences encoding light chain antigen complementary region LCDR2 with sequence GCTGCATCC, and nucleotide sequences encoding light chain antigen complementary region LCDR3 as shown in SEQ ID NO.9.

[0016] This includes a nucleotide sequence encoding the variable region of the heavy chain antigen as shown in SEQ ID NO.1, and a nucleotide sequence encoding the variable region of the light chain as shown in SEQ ID NO.2. In some embodiments of the present invention, the nucleic acid includes the nucleotide sequence encoding the heavy chain as shown in SEQ ID NO.15 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO.16.

[0017] Thirdly, the present invention provides an expression vector comprising the nucleic acid described in the second aspect.

[0018] Fourthly, the present invention provides transgenic cell lines or recombinant bacteria comprising the nucleic acid described in the second aspect or the expression vector described in the third aspect.

[0019] Fifthly, the present invention provides an immunoconjugate comprising an antibody portion and a coupling portion coupled to the antibody portion, wherein the antibody portion comprises the antibody or antigen-binding fragment thereof described in the first aspect, and the coupling portion is selected from any one or more combinations of fluorescent substances, chemiluminescent substances, colored substances, enzymes, and small molecule drugs.

[0020] In a sixth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect, or the immunoconjugate described in the fifth aspect, in the preparation of a reagent kit for detecting human adenovirus, and / or in the preparation of a drug for the prevention or treatment of diseases related to human adenovirus infection.

[0021] The human adenoviruses mentioned include human adenovirus type 55 and / or human adenovirus type 11.

[0022] Optionally, the human adenovirus infection-related diseases include upper respiratory tract infections, pharyngitis, non-purulent acute conjunctivitis, pneumonia, or pertussis.

[0023] Optionally, the drug may also include a pharmaceutically acceptable carrier and / or excipient.

[0024] Optionally, the drug is used to neutralize the virulence of adenovirus in a sample, or to prevent or treat adenovirus infection or adenovirus-related disease in a subject.

[0025] Optionally, for the above applications, the method for detecting adenovirus includes contacting the sample to be tested with a fully human antibody or an immunoconjugate, and detecting whether an antigen-antibody complex is formed or detecting the amount of antigen-antibody complex formed.

[0026] Beneficial effects

[0027] This invention discloses a fully human neutralizing antibody (4C9) against human adenovirus hexon and its applications. Specifically, it discloses the amino acid sequence and nucleic acid sequence encoding the full-molecule antibody, as well as its application in the preparation of HAdV-related drugs or diagnostic reagents. The antibody of this invention is a fully human antibody, and related experiments have confirmed that the fully human antibody of this invention can specifically recognize the hexon protein of human adenovirus type 55, and can effectively prevent and control HAdV infection. Furthermore, the fully human anti-adenovirus type 55 hexon neutralizing antibody provided by this invention can also provide good neutralizing protection against other adenovirus variants (such as human adenovirus type 11) that are highly homologous to the A180-A193 segment of the Hexon protein gene. Therefore, the fully human antibody developed in this study, while possessing the advantage of low immunogenicity, may have potential neutralizing activity against other adenovirus subtypes carrying the same or highly similar Hexon epitope sequences, exhibiting broad-spectrum neutralizing characteristics based on similar epitopes, providing new ideas and important references for the subsequent development and clinical application of broad-spectrum neutralizing antibodies against adenoviruses. Attached Figure Description

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0029] Figure 1 shows the titer detection results of HAdV positive serum samples.

[0030] Figure 2 shows HADV positive serum samples detected by ELISA.

[0031] Figure 3 shows the sorting of HAdV-55 Hexon-specific B cells. In the figure, A represents lymphocytes in PBMCs, B represents the proportion of B cells in lymphocytes, and C represents the proportion of specific B cells that can secrete IgG antibodies and specifically bind to HAdV-55 Hexon protein.

[0032] Figure 4 shows the modeling of the anti-HAdV-55 Hexon antibody molecule, where A is the modeling of the single-chain antibody molecule and B is the modeling of the single-chain antibody binding to the antigen.

[0033] Figure 5 shows the amino acid clustering analysis of the anti-HAdV-55 Hexon specific antibody.

[0034] Figure 6 shows the affinity chromatography purification of the anti-HAdV-55 Hexon specific antibody.

[0035] Figure 7 shows the SDS-PAGE electrophoresis diagram of the anti-HAdV-55 Hexon specific antibody.

[0036] Figure 8 shows the ELISA detection of anti-HAdV-55 Hexon specific antibody.

[0037] Figure 9 shows the affinity test of the anti-HAdV-55 Hexon specific antibody.

[0038] Figure 10 shows the modeling and binding energy calculation of the anti-HAdV-55 Hexon specific antibody and specific short peptide molecule, where A is the modeling of the binding of the single-chain antibody molecule to the short peptide, and B is the assessment of the binding energy of the complex.

[0039] Figure 11 shows the ELISA detection of anti-HAdV-55 Hexon specific antibody 4C9 and candidate short peptides.

[0040] Figure 12 shows the neutralization effect of antibody 4C9 on HADV-55. In the figure, A is the micro-neutralization assay for EGFP reporter virus and B is the micro-neutralization assay for SEAP reporter virus.

[0041] Figure 13 shows the broad-spectrum neutralizing effect of antibody 4C9. In this figure, A represents the neutralization detection of different HADV strains, B represents the IC50 calculation for HADV55, and C represents the IC50 calculation for HADV11. Detailed Implementation

[0042] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0043] In the following examples, the PBS is a 0.01 M pH 7.4 PBS buffer. The TBST is a pH 7.4 TBST buffer.

[0044] Example 1: Collection and Identification of HAdV Infection Positive Serum Samples

[0045] With informed consent, peripheral blood samples were collected from patients in the recovery phase (sample numbers H1-H20) who were clinically diagnosed with adenovirus infection and tested positive for HAdV infection for serological titer screening of anti-HAdV antibodies. The collected blood was allowed to stand at room temperature for 1 hour before being centrifuged at 2000 rpm for 10 minutes. The milky-yellow supernatant was collected as serum and stored at -80 °C. Serum binding capacity and titer were determined according to the following steps:

[0046] (1) HAdV-55 Hexon protein (purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number DRA298) was used for coating. The solution was dissolved in a carbonate solution at pH 9.6 and adjusted to a concentration of 2 μg / mL. 100 μL of the prepared coating solution was added to each well of a 96-well microplate. The microplate was then placed in a 4℃ refrigerator for 12–16 h.

[0047] (2) Wash the plate 5 times using an automated ELISA plate washer with TBST as the washing buffer. Add 200 μL of 2% v / v BSA to each well and block overnight;

[0048] (3) Wash the plate 5 times using an automated ELISA plate washer with TBST as the washing buffer. Serially dilute the collected serum samples with PBS, add 100 μL per well, and set up 3 replicates for each concentration. Incubate at 37 ℃ for 2 h.

[0049] (4) Wash the plate 5 times using an automated ELISA plate washer, using TBST as the washing buffer. Dilute the HRP-labeled goat anti-human secondary antibody with TBST at a ratio of 1:10000 v / v, 100 μL per well, and incubate at 37°C for 1 h;

[0050] (5) Wash the plate 5 times using an automated ELISA plate washer with TBST as the washing buffer. Add 100 μL of TMB to each well, and add 50 μL of ELISA stop solution after 7 min to terminate the reaction;

[0051] (6) Place the microplate into the microplate reader, shake the plate and read the value, record the absorbance value at 450 nm, plot and calculate.

[0052] The results showed that anti-HAdV-55 Hexon antibody expression was detected in most blood samples, with differences in antibody expression ranging from a minimum of 1:1000 to a maximum of 1:32000 (Figure 1).

[0053] The average OD value of anti-HAdV-55 Hexon IgG antibody in all samples was 2.395, and the serum titer results are shown in Figure 2. Serum samples (H1 and H5) with serum titers greater than 1:16000 and antibody OD values ​​higher than the average OD value were selected for further experimental testing.

[0054] Example 2: Isolation and sequencing of anti-HAdV-55 Hexon-specific B cells

[0055] First, PBMCs of HAdV-positive samples were isolated. Specifically, the blood sample to be separated was diluted with an equal volume of PBS. An equal volume of lymphocyte separation medium was added to a centrifuge tube, and the diluted blood sample was spread evenly on top of the lymphocyte separation medium, maintaining a clear interface between the two liquids. The tube was centrifuged at 800 rpm for 30 min at room temperature, using the slow acceleration / deceleration function. After centrifugation, the white membrane layer was carefully aspirated into a new centrifuge tube, diluted with PBS, inverted to mix, and centrifuged at 250g for 10 min at room temperature. The supernatant was discarded, and the washing was repeated twice. The cells were resuspended in PBS buffer to obtain PBMCs, which were then counted. Next, specific B cell sorting was performed. The PBMCs were added to Hexon-His protein and incubated at room temperature for 40 min, inverted every 5 min. The cells were washed with PBS, and 4 μL of anti-His tag-FITC fluorescent antibody was added. The cells were incubated at 4℃ for 30 min. After incubation, wash once with PBS, then add 3 μL each of anti-human CD3-PE, anti-human CD19-APC-Cyanine7, anti-human CD56-APC, and anti-human IgG-Brilliant violet 421, and incubate at 4 °C for 30 min. Wash once with PBS after incubation. Filter the treated cell suspension through a flow cytometer and sort single cells into 96-well plates using a flow cytometer.

[0056] The results showed that after flow cytometry separation, CD3-PE was selected from the separated PBMCs. - CD56-APC - CD19-APC-Cyanine7 + anti His tag-FITC + IgG-Brilliant violet 421 + Specific B cells (Figure 3).

[0057] GEXSCOPE single-cell sequencing library preparation reagent was pre-added to 96-well plates containing the collected and sorted individual B cells. Cell lysis buffer was added to the sorted 96-well cell culture plates. Oligo-dT was used to capture mRNA. The full-length cDNA coding strand was obtained using SMART-seq3xpress technology, and the oligo-dT index was used for subsequent cDNA library enrichment. The BCR cDNA sequence was obtained by amplification using universal PCR primers attached to the oligo. BCR product enrichment was completed through two rounds of specific semi-nested PCR. Human antibody variable region genome primers were used for PCR amplification and library preparation. Preliminary analysis of the sequencing library was performed. The BCR mapping rate of the samples and the proportion of alignment to heavy and light chains (IGH, IGκ, IGλ) were statistically analyzed. Individual cells were separated using the index and BCRV(D)J assembly was performed to obtain complete BCR sequencing results.

[0058] Example 3: Screening of variable region genes for specific anti-HAdV-55 Hexon antibodies

[0059] The paired anti-HAdV-55 Hexon antibody variable region gene nucleic acid sequences obtained in Example 2 were summarized and translated into amino acid sequences using the Expasy Translate tool. The anti-HAdV-55 Hexon antibody amino acid sequences were compared and annotated with antibody amino acid sequences in the IMGT database to determine the CDR position of the candidate antibody variable region gene. Sequences with large deletions (especially CDR regions) were removed, and repetitive sequences were merged to complete the cleaning of the antibody gene sequences to be included in the study. Homology modeling of the antibody variable region amino acids was performed using the Model antibody cascade program in Discovery Studio software to obtain a simulated spatial structure of the antibody variable region. Using the SWISS-MODEL homology modeling server, based on homologous protein templates with known structures, homology modeling of the three-dimensional structure of the HAdV-55 Hexon protein was performed to obtain a simulated spatial structure of the HAdV-55 Hexon protein. Using Schrödinger Maestro software, in the pro-pro-docking module, the antibody docking mode was selected, and the Mask non-CDR region was checked. Molecular docking was performed between the above-mentioned antibody variable region spatial structure and the HADV-55 Hexon protein spatial structure. The antigen-antibody complex binding energy was calculated using the Cluster interaction energy program. Cluster omega was used to perform cluster analysis on the amino acid sequence of the antibody variable region.

[0060] A schematic diagram of molecular modeling of anti-HAdV-55 Hexon antibody (taking clone 4C9 as an example) and molecular docking of candidate antibodies with HADV-55 Hexon are shown in Figures A and B of Figure 4. The binding energies of all candidate antibodies to the target protein were calculated. Based on the basic characteristics of antibody genes, the results of binding free energy calculation, and the results of antigen epitope binding experiments, biological evolutionary cluster analysis was performed and optimized (Figure 5). Clones with docking scores better than the average level, CDR3 lengths higher than the overall average, and polar amino acid counts higher than the average were selected as representative sequences from the clusters. Finally, representative antibodies (such as 4C9, 1A1, 4H4, 4C4, 4C12, and 4E7 as shown in Figure 5) were selected as candidate anti-HAdV-55 Hexon antibodies for full-molecule eukaryotic expression and antibody specificity verification experiments.

[0061] Example 4: Expression and purification of genetically engineered antibodies

[0062] The variable region nucleotide sequence of the candidate anti-HAdV-55 Hexon antibody obtained from the screening in Example 3 of whole-genome synthesis was used. The target DNA fragment was amplified using primers, purified using a gel extraction kit, and eluted with deionized water to obtain the heavy chain and light chain variable region gene fragments of the candidate antibody. The primer sequences used are shown in Table 1.

[0063] Table 1 Primer sequences used for amplifying the variable region of the antibody.

[0064]

[0065] The double digestion reaction system for pFUSE-CHIg-hG1 and pFUSE-CLIg-hκ template vectors (containing the constant region sequence) was (50 µL): Cut Smart buffer 10 × 5 µL, EcoRI 1.5 µL, NheRI / BsiWI 2 µL, Vector 3.3 µL (1 μg), and ddH2O 38.2 µL. The reaction conditions were: digestion overnight at 37 ℃.

[0066] The expression and purification of genetically engineered antibodies are based on anti-HAdV-55 Hexon antibody 4C9 as an example. The processing methods for other candidate antibodies are the same as for 4C9, except that the heavy chain variable region and light chain variable region sequences are replaced with the corresponding sequences of the candidate antibody. The specific methods for expression and purification of antibody 4C9 are as follows: The nucleotide gene fragment of the heavy chain variable region of antibody 4C9 (nucleotide sequence of antibody 4C9 heavy chain variable region as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.3) is recombined into the expression plasmid pFUSE-CHIg-hG1 containing the human IgG1 heavy chain constant region nucleic acid gene using in-fusion PCR to obtain the heavy chain plasmid; the nucleotide gene fragment of the light chain variable region of antibody 4C9 (nucleotide sequence of antibody 4C9 light chain variable region as shown in SEQ ID NO.2, amino acid sequence as shown in SEQ ID NO.4) is recombined into the expression plasmid pFUSE-CLIg-hκ containing the human light chain constant region nucleic acid to obtain the light chain plasmid. Transform 1 µL of the reaction solution into *E. coli* DH5α competent cells, plate them on ampicillin-resistant LB agar plates, and incubate overnight at 37°C. The next day, select positive clones and send them for sequencing. Preserve the bacterial strain of clones with correct sequencing results, expand the culture, extract plasmids to obtain heavy chain and light chain plasmids, and express them in a eukaryotic cell expression system, specifically including the following steps:

[0067] (1) Process 293 Freestyle cells, centrifuge them, resuspend them in 293F Hi-exp medium, use trypan blue to determine cell viability and count them, and aspirate an appropriate amount of cells into a culture flask to make the cell density 3×10⁻⁶. 6 The cell count was increased by 100 mL to obtain 100 mL of cells to be transfected. Cell viability was ≥97%.

[0068] (2) Take 400 µL of EZ Trans cell transfection reagent (purchased from Heyuan Liji (Shanghai) Biotechnology Co., Ltd., catalog number AC04L092) and mix it in 2100 µL of 293F Hi-exp medium. After mixing, let it stand for 5 min to obtain the transfection reagent dilution. At the same time, take 40 µg of heavy chain plasmid and 60 µg of light chain plasmid and mix them with 293F Hi-exp medium to make the total volume 2.5 mL to prepare the plasmid dilution.

[0069] (3) Slowly add the diluent of the transfection reagent to the diluent of the plasmid, mix well, and incubate at 37 °C for 30 min to fully react and form a plasmid-transfection reagent complex;

[0070] (4) Slowly add the incubated plasmid-transfection reagent complex to the cells to be transfected cultured in step (1) while shaking the shake flask. Place the flask in a shaker incubator and culture at 37℃, 8% CO2, and 125 rpm. After 22 h of transfection, add 500 µL of 5% v / v 293F Hi-exp feed and 450 µL of glucose concentrate (450 g / L) to the cell shake flask while shaking the shake flask and place it back in the shaker incubator to continue culturing.

[0071] (5) Three days after transfection, add 670 µL of glucose concentrate (450 g / L) to the cell shake flask, and continue to culture in the shaker incubator for 5-7 days. Collect the cell culture supernatant and perform nucleophilic chromatography purification of the antibody.

[0072] The affinity chromatography purification method for antibodies was as follows: The collected cell culture supernatant was filtered through a 0.22 μm filter membrane, and then PBS buffer (pH 7.4) was added at a 1:1 volume ratio. The mixture was pre-cooled at 4°C. Antibody protein purification was performed using an AKTA purifier 100 instrument with a Protein A purification column, and the eluent was collected. The ultrafiltrate was obtained by centrifugation at 4000 rpm for 20 min using an ultrafiltration tube with a 30 kDa cutoff. The antibody concentration was determined, labeled, diluted to a final volume, aliquoted, and frozen for subsequent antibody analysis experiments.

[0073] Taking antibody 4C9 as an example, Figure 6 shows the UV detection of the protein using AKTA purifier100 affinity chromatography. In the early stage, the antibody adsorbs onto the Protein A column, and a sharp elution peak is visible after antibody elution. Figure 7 shows the SDS-PAGE and Coomassie Brilliant Blue staining assays. In the 293 Freestyle cell culture supernatant lane and antibody elution lane (4C9) co-transfected with the antibody expression plasmid, distinct bands are visible at 55 kD (antibody heavy chain) and 27 kD (antibody light chain), consistent with the band size of the positive control (full-molecule, fully human IgG). Only irrelevant protein bands are visible in the 293 Freestyle cell culture supernatant flow-through lane and the negative control lane, indicating that the antibody protein was specifically purified and enriched.

[0074] Example 5 Antibody Binding Ability Detection

[0075] This embodiment utilizes the ELISA binding assay of the six candidate antibodies expressed and purified in Example 4 with recombinant HADV-55 Hexon protein (purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number DRA298), as detailed below:

[0076] (1) Dilute HADV-55 Hexon protein with carbonate solution at pH 9.6 and plate it. 100 µL per well, concentration is 0.5 µg / mL, and incubate overnight at 4℃;

[0077] (2) Wash the plate 5 times using an automated ELISA plate washer with TBST as the washing buffer. Add 200 µL of 2% v / v BSA solution to each well for blocking, and incubate at 37 ℃ for 2 h.

[0078] (3) Wash the plate 5 times using an automated ELISA plate washer, and the washing buffer was TBST. The anti-HAdV-55 Hexon antibody purified in Example 4 was serially diluted with PBS to an initial concentration of 2 µg / mL, with 100 µL per well, and 3 replicates were set up. The plate was incubated at 37°C for 2 h.

[0079] (4) Wash the plate 5 times using an automated ELISA plate washer, using TBST as the washing buffer. Dilute the HRP-labeled goat anti-human secondary antibody with TBST at a ratio of 1:10000 v / v, 100 µL per well, and incubate at 37 °C for 1 h.

[0080] (5) Wash the plate 5 times using an automated ELISA plate washer with TBST as the washing buffer. Add 100 µL of TMB to each well, and after 5 min, add 50 µL of ELISA stop solution to terminate the reaction. Immediately place the plate under a microplate reader at a wavelength of 450 nm to measure the absorbance.

[0081] The experimental results showed that among the six candidate antibodies, the anti-HAdV-55 Hexon antibody (4C9) could specifically bind to the recombinant HADV-55 Hexon protein, and the binding force of the antibody was positively correlated with the quality of the coated antigen (as shown in Figure 8), which could be used for further detection. The other five candidate antibodies did not reach the level of positive determination and had poor binding force.

[0082] Example 6 Antibody Affinity Detection

[0083] Antibody affinity was detected using a BLItz molecular interaction analyzer (purchased from Fortebio). Specifically, the Protein A sensor corresponding to BLItz was activated in PBST (pH 7.4, containing 0.2% v / v BSA) for at least 10 min. HADV-55 Hexon protein was diluted to 100 nM, 200 nM, 400 nM, 800 nM, and 1600 nM according to its molecular weight, and a 0 nM blank control was prepared. Anti-HAdV-55 Hexon antibody 4C9 was diluted to 50 µg / mL. The activated sensor was placed in the instrument, and baseline was run. After the baseline stabilized, 4 µL of anti-HAdV-55 Hexon antibody 4C9 was added for immobilization. The sensor was then placed in PBST, and baseline was run again. Different concentrations of HADV-55 Hexon protein were added to the sample wells to bind to the sensor; a new sensor was required for each binding. The sensor was placed in a glycine solution at pH 1.7 for 5 seconds, then in PBST for 5 seconds, and this process was repeated three times for regeneration. The binding and dissociation curves were analyzed to obtain the affinity constant.

[0084] The results are shown in Figure 9. Run1-6 in the figure represent the baseline values ​​used in the affinity assay of HADV-55 Hexon protein at concentrations of 100 nM, 200 nM, 400 nM, 800 nM, and 1600 nM with anti-HAdV-55 Hexon antibody 4C9, respectively. The results indicate that the affinity constant KD value of the interaction between anti-HAdV-55 Hexon antibody 4C9 and HADV-55 Hexon protein is 5.27 × 10⁻⁶. -7 M.

[0085] Example 7 Antibody Epitope Identification

[0086] First, computer simulation docking was performed on the HADV-55 Hexon protein homology model obtained in Example 3. Seven representative short peptide sequences (named A1~A7) were extracted and defined, resulting in seven simulated short peptide spatial structures. The amino acid sequences of A1~A7 are shown in Table 2. Using Schrödinger Maestro software, the antibody docking mode in the pro-pro-docking module was selected, and the Mask non-CDR region was checked. The structure of the anti-HAdV-55 Hexon protein antibody (4C9) obtained in Example 3 was docked with the short peptides A1~A7 respectively.

[0087] Table 2 HAdV-55 Hexon protein antigenic epitopes

[0088]

[0089] In addition, epitope mapping was used to detect antibody-binding epitopes, and seven overlapping short peptides (A1-A7) based on the HAdV-55 Hexon protein sequence were synthesized. The HAdV-55 Hexon-specific peptides (A1-A7) were diluted with carbonate solution at pH 9.6 and coated onto plates at 100 µL per well (100 ng / mL), incubated overnight at 4°C. Plates were washed 5 times with TBST using an automated ELISA washer. 200 µL of 2% v / v BSA solution was added to each well for blocking, and the plates were incubated at 37°C for 2 h. Plates were washed 5 times with TBST using an automated ELISA washer. The anti-HAdV-55 Hexon antibody 4C9 purified in Example 4 was diluted with PBS to a concentration of 200 µg / mL, with 100 µL per well, in triplicate, and incubated at 37°C for 2 h. Plates were washed 5 times with TBST using an automated ELISA washer. HRP-labeled goat anti-human secondary antibody was diluted with TBST at a ratio of 1:10000 v / v, 100 µL per well, and incubated at 37 ℃ for 1 h. The plate was washed 5 times using an automated ELISA plate washer with TBST as the washing buffer. 100 µL of TMB was added to each well, and the reaction was stopped by adding 50 µL of ELISA stop solution after 7 min. The absorbance was immediately measured under a 450 nm microplate reader.

[0090] Homology modeling of the HAdV-55 Hexon protein short peptides is shown in Figure 10. The results show that peptides A4 and A7 exhibit the most stable binding conformations to the anti-HAdV-55 Hexon antibody (4C9). ELISA detection using candidate short peptides showed that the anti-HAdV-55 Hexon antibody (4C9) bound to HAdV-55 Hexon-specific peptides A1-A7 (Figure 11). Among these, peptides A3-A5 showed significantly higher average OD values, and peptide A4 had a much higher OD value than A7. Based on peptide design principles, lower binding scores and higher ELISA signals indicate better epitope matching. The combined results suggest that 4C9 can specifically recognize peptide A4: KVSDEESKPIYADK (SEQ ID NO. 19).

[0091] Example 8: Detection of the neutralizing effect of anti-HAdV antibody on HAdV-55

[0092] 1. Micro-neutralization assay based on green fluorescent protein (EGFP) reporter virus

[0093] (1) Before infection, HEK293 cells were stored at a density of 3 × 10⁶ cells per well. 4 100 µL of cells were seeded into 96-well plates and cultured for 48 h. Cell status was observed and experiments were started when the cell confluence was about 90%.

[0094] (2) Antibody dilution: The antibody 4C9 was serially diluted with complete culture medium (DMEM containing 10% v / v FBS) to 200 µg / mL, 20 µg / mL, 2 µg / mL, 200 ng / mL, 20 ng / mL and 2 ng / mL. 75 µL of each concentration was added to a 96-well plate and three replicates were set for each concentration.

[0095] (3) Antibody-virus co-incubation: HADV-55-EGFP was diluted to 4×10⁻⁶ with complete medium (DMEM containing 10% v / v FBS). 7 VP / mL, use a multi-channel pipette to add 75 µL of virus solution to the above 96-well plates containing different concentrations of antibodies, mix well, and incubate at 37°C in a 5% CO2 incubator for 1 hour to obtain antibody-virus complex solution; the above steps set up control groups: use anti-RBD neutralizing antibody to prepare a control antibody-virus complex solution with no neutralizing activity (control), and use mouse anti-HAdV-55 Hexon monoclonal antibody (for research use, purchased from Thermo Scientific, catalog number MA1-7328) to prepare a positive control antibody-virus complex solution;

[0096] (4) Infecting cells: Discard the old culture supernatant in the 96-well plate from step (1), carefully add 100 µL of the above antibody-virus complex solution, and incubate at 37°C in a 5% CO2 incubator for 24 h. At this time, the final concentration of the antibody is 100 µg / mL, 10 µg / mL, 1 µg / mL, 100 ng / mL, 10 ng / mL and 1 ng / mL; among which, the final concentration of the mouse anti-HAdV-55 Hexon monoclonal antibody in the positive control is 1 µg / mL;

[0097] (5) After 24 hours, the culture supernatant was collected and photographed using a fluorescence microscope. The neutralization effect of the virus was quantified by detecting the fluorescence intensity of enhanced green fluorescent protein (EGFP).

[0098] As shown in Figure 12A, the anti-Hexon specific antibody (4C9) can specifically recognize and kill HADV-55 virus and has a good neutralizing effect.

[0099] 2. Micro-neutralization assay based on secretory phosphatase (SEAP) reporter gene

[0100] (1) Before infection, HEK293 cells were stored at a density of 3 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates (100 µL per well) and cultured for 48 h. Cell status was observed, and experiments were started when cell confluence was approximately 90%.

[0101] (2) Antibody dilution: The antibody 4C9 was serially diluted with complete culture medium (DMEM containing 10% v / v FBS) to 200 µg / mL, 20 µg / mL, 2 µg / mL, 200 ng / mL, 20 ng / mL and 2 ng / mL. 75 µL of each concentration was added to a 96-well plate and three replicates were set for each concentration.

[0102] (3) Antibody-virus co-incubation: HADV-55-SEAP was diluted to 1.6 × 10⁻⁶ with complete culture medium (DMEM containing 10% v / v FBS). 8 VP / mL, use a multi-channel pipette to take 75 µL of virus solution and add it to the 96-well plate containing different concentrations of antibodies, mix well, and incubate at 37°C in a 5% CO2 incubator for 1 hour to obtain antibody-virus complex solution;

[0103] (4) Cell Infection: Discard the old culture supernatant from the 96-well plate in step (1), carefully add 100 µL of the above antibody-virus complex solution, and incubate at 37°C in a 5% CO2 incubator for 24 h. The final antibody concentrations were 100 µg / mL, 10 µg / mL, 1 µg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL. A control group (cells without antibody, NC) was set up in the above steps. After 24 h, the culture supernatant was collected, and SEAP expression was detected using the Phospha-Light™ SEAP reporter gene detection system.

[0104] As shown in Figure 12B, the anti-Hexon specific antibody (4C9) can specifically recognize and kill HADV-55 virus and has a good neutralizing effect.

[0105] Example 9: Detection of the broad-spectrum neutralizing effect of anti-HAdV antibodies against different subtypes of HADV virus

[0106] 1. Micro-neutralization assay based on EGFP reporter virus

[0107] (1) Before infection, HEK293 cells were stored at a density of 3 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates (100 µL per well) and cultured for 48 h. Cell status was observed, and experiments were started when cell confluence was approximately 90%.

[0108] (2) Antibody dilution: Dilute antibody 4C9 with complete culture medium (DMEM containing 10% v / v FBS) to 200 µg / mL, take 75 µL and add it to a 96-well plate, and set up three replicates;

[0109] (3) Antibody-virus co-incubation: Different viruses (HAdV-3-EGFP, HADV-7-EGFP, HADV-11-EGFP, HADV-14-EGFP and HADV-55-EGFP) were diluted to 3×10⁻⁶ in complete medium (DMEM containing 10% v / v FBS). 7 VP / mL, use a multi-channel pipette to add 75 µL of virus solution to a 96-well plate containing the above antibody, mix well, and incubate at 37°C in a 5% CO2 incubator for 1 hour to obtain antibody-virus complex solution;

[0110] (4) Cell Infection: Discard the old culture supernatant from the 96-well plate in step (1), carefully add 100 µL of the above antibody-virus complex solution, and incubate at 37°C in a 5% CO2 incubator for 24 h. The final antibody concentration is 100 µg / mL. A control group (cells without antibody, NC) was set up in the above steps. After 24 h, the culture supernatant was collected, photographed using a fluorescence microscope, and the neutralizing effect of the virus was quantified by detecting the fluorescence intensity of enhanced green fluorescent protein (EGFP).

[0111] As shown in Figure 13A, the anti-Hexon specific antibody (4C9) can specifically recognize and kill HADV-11 virus.

[0112] 2. Micro-neutralization assay for SEAP-reported viruses

[0113] The experimental method here is the same as that described in "2. Micro-neutralization assay based on secretory phosphatase (SEAP) reporter gene" in Example 8. The difference is that the antibody in step (2) is diluted to a concentration of 100 µg / mL, 20 µg / mL, 2 µg / mL, 200 ng / mL, 100 ng / mL, 20 ng / mL, 2 ng / mL and 0.2 ng / mL. The virus used in step (3) is any one of HAdV-3-SEAP, HAdV-7-SEAP, HAdV-11-SEAP, HAdV-14-SEAP or HAdV-55-SEAP.

[0114] In this invention, both EGFP and SEAP reporter gene assays indicated that the anti-Hexon specific antibody (4C9) could specifically recognize and kill HAdV-55 virus with good neutralizing activity, with an IC50 of 1.685 ng / mL (Figure 13, B). Simultaneously, the anti-Hexon specific antibody (4C9) could specifically recognize and kill HAdV-11 virus, with an IC50 of 7.308 ng / mL (Figure 13, C).

[0115] This invention provides an antibody against human adenovirus hexagonal protein and its application, along with a method and approach. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. An antibody against human adenovirus hexagonal protein or its antigen-binding fragment, characterized in that, It is a fully human antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region; wherein, the heavy chain variable region comprises three heavy chain antigen complementary regions: HCDR1 with the amino acid sequence shown in SEQ ID NO.10, HCDR2 with the amino acid sequence shown in SEQ ID NO.11, and HCDR3 with the amino acid sequence shown in SEQ ID NO.12; the light chain variable region comprises three light chain antigen complementary regions: LCDR1 with the amino acid sequence shown in SEQ ID NO.13, LCDR2 with the amino acid sequence AAS, and LCDR3 with the amino acid sequence shown in SEQ ID NO.

14.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, It comprises a heavy chain and a light chain; wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO.17 and the amino acid sequence of the light chain is shown in SEQ ID NO.

18.

4. A nucleic acid encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3.

5. The nucleic acid according to claim 4, characterized in that, It contains nucleotide sequences encoding three heavy chain antigen complementary regions as shown in SEQ ID NO.5~7, nucleotide sequences encoding light chain antigen complementary region LCDR1 as shown in SEQ ID NO.8, nucleotide sequences encoding light chain antigen complementary region LCDR2 with sequence GCTGCATCC, and nucleotide sequences encoding light chain antigen complementary region LCDR3 as shown in SEQ ID NO.

9.

6. The nucleic acid according to claim 4, characterized in that, It contains the nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO.1, and the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO.

2.

7. An expression vector comprising the nucleic acid of claim 4.

8. A transgenic cell line or recombinant bacteria comprising the nucleic acid of claim 4 or the expression vector of claim 7.

9. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a kit for detecting human adenovirus and / or in the preparation of a medicament for the prevention or treatment of diseases related to human adenovirus infection; wherein the human adenovirus includes human adenovirus type 55 and / or human adenovirus type 11.

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