Human monoclonal antibody 4AC3 for resisting type 4 adenovirus and application of human monoclonal antibody 4AC3

Abstract

The invention provides a human monoclonal antibody 4AC3 for resisting type 4 adenovirus. The neutralizing antibody for resisting the 4-type adenovirus provided by the invention can be specifically combined with the 4-type adenovirus, and has very high affinity activity and very strong neutralizing activity at low concentration. The invention also provides application of the antibody in preparation of medicines for treating and / or preventing type 4 adenovirus infectious diseases, and a detection kit containing the antibody.

Description

Technical Field

[0001] The invention discloses a monoclonal antibody and belongs to the technical field of immunology and microbiology. Background Art

[0002] Human adenovirus (HAdV) is divided into seven subgenera, A to G. Within these seven distinct subgenera, subgenera B, C, and E all cause respiratory illnesses, including upper and lower respiratory tract infections and bronchitis. Some cases may also be accompanied by gastrointestinal symptoms such as diarrhea, vomiting, and abdominal pain. Most patients experience mild, self-limited infections, but severe cases can lead to pneumonia. Adenoviruses are widespread and spread rapidly. They infect a wide range of age groups, especially children, and can be fatal in severe cases. HAdV outbreaks often occur in enclosed or crowded settings. During large-scale epidemics, the mortality rate of severe pneumonia caused by HAdV is high, and even untreated severe HAdV pneumonia or disseminated disease can exceed 50%. Pneumonia caused by adenovirus is particularly severe, potentially causing not only heart and respiratory failure, but also extrapulmonary involvement, such as encephalitis, liver damage, myocarditis, or myocardial damage, and even death. The prognosis is generally poor. To this end, there is currently no effective vaccine or drug for the prevention and treatment of HAdV pneumonia, especially pneumonia caused by group E adenovirus type 4.

[0003] Adenovirus virulence factors include multiple major and minor capsid proteins. Hexon, Penton, and Fiber are the three major capsid proteins. Hexon proteins are the most abundant proteins in the adenovirus capsid and constitute the primary surface structure of the virus particle. As the primary antigen, hexon proteins can induce a strong immune response in the host, including the production of neutralizing antibodies and the initiation of cellular immune responses.

[0004] With the advancement of genetic engineering, fully human monoclonal antibodies are increasingly demonstrating advantages, significantly improving their safety, efficacy, and relevance to human diseases compared to traditional polyclonal or murine monoclonal antibodies. The present invention aims to provide a fully human monoclonal neutralizing antibody against adenovirus type 4, and further to provide its use in the preparation of a drug for the prevention or treatment of adenovirus-infected diseases. Summary of the Invention

[0005] Based on the above objectives, the present invention first provides a human monoclonal antibody against adenovirus type 4 Hexon protein, wherein the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the human monoclonal antibody against adenovirus type 4 are shown in SEQ ID NO: 1 at positions 26-33, 51-58 and 97-116, respectively; the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the light chain variable region are shown in SEQ ID NO: 3 at positions 27-33, 51-53 and 90-98, respectively.

[0006] In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the human monoclonal antibody against adenovirus type 4 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3. In the present invention, a specific embodiment having such heavy chain variable region and light chain variable region is designated as the "4AC3" antibody.

[0007] In a preferred embodiment, the amino acid sequence of the heavy chain constant region of the human monoclonal antibody against adenovirus type 4 is shown in SEQ ID NO: 5, the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 7, and the light chain is a Kappa chain.

[0008] Secondly, the present invention provides a polynucleotide encoding the heavy chain and light chain of the above-mentioned human monoclonal antibody against adenovirus type 4. The sequence of the polynucleotide encoding the heavy chain variable region of the human monoclonal antibody against adenovirus type 4 is shown in SEQ ID NO: 2, and the sequence of the polynucleotide encoding the light chain variable region is shown in SEQ ID NO: 4.

[0009] In a preferred embodiment, the sequence of the polynucleotide encoding the heavy chain constant region of the human monoclonal antibody against adenovirus type 4 is shown as SEQ ID NO: 6, and the sequence of the polynucleotide encoding the light chain constant region is shown as SEQ ID NO: 8.

[0010] Third, the present invention provides a vector containing the polynucleotide encoding the heavy and light chains of the monoclonal antibody. Any vector known in the art for expressing exogenous genes through genetic engineering, particularly eukaryotic expression vectors, can be used as the vector of the present invention. In a specific embodiment of the present invention, the vector is pcDNA3.4.

[0011] Fourth, the present invention provides a host cell containing the vector of the polynucleotide encoding the heavy and light chains of the monoclonal antibody. Host cells used for expressing foreign genes by genetic engineering in the art, especially eukaryotic host cells, can be used as the host cell of the present invention.

[0012] In a preferred embodiment, the cells are 293T cells or Expi 293F cells.

[0013] Fifth, the present invention provides a use of the aforementioned humanized monoclonal antibody against adenovirus type 4 in the preparation of a medicament for treating and / or preventing adenovirus type 4 infectious diseases. Given that antibodies against the adenovirus type 4 Hexon protein are important neutralizing antibodies, the humanized monoclonal antibody against adenovirus type 4 provided by the present invention can be prepared as a medicament for treating or preventing adenovirus infectious diseases.

[0014] Finally, the present invention provides a detection kit containing the above-mentioned human monoclonal antibody against adenovirus type 4. The kit can be used for, but not limited to, diagnosis, monitoring, and clinical efficacy evaluation of adenovirus type 4 infection.

[0015] The present invention screened a human monoclonal antibody 4AC3 against adenovirus type 4 from human serum by flow cytometry. The heavy chain variable region and light chain variable region of the antibody have unique CDR regions, which have excellent binding activity to the Hexon protein of adenovirus type 4. 50 The antibody-antigen affinity detected by SPR was KD value less than 10 -12 M. The antibody can inhibit the specific binding of adenovirus type 4 to host cell A549, and its IC 50 Reaching 0.2 μg / ml, the excellent neutralizing inhibitory activity demonstrates the promising application of the antibodies provided herein as neutralizing antibodies in the preparation of therapeutic and / or preventive drugs for adenovirus type 4 infectious diseases. Furthermore, the humanized nature of the antibodies ensures the safety of clinical use. Furthermore, the excellent antigen-binding activity and affinity give the antibodies provided herein promise as detection antibodies in immunological methods for detecting adenovirus type 4 antigens, such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, and chemiluminescent immunoassays based on antigen-antibody-specific immune reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 .Flow cytometry sorting memory B cell results; Figure 2 .SDS-PAGE detection of antibody expression results; Figure 3 Antibody and antigen binding activity detection diagram of the present invention; Figure 4 . Detection diagram of neutralization activity of antibodies and antigens of the present invention; Figure 5 .Affinity activity test diagram of the antibody of the present invention and antigen. DETAILED DESCRIPTION

[0017] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0018] In the term, antibody refers to a protein molecule that acts as a receptor for specific antigen recognition, including immunoglobulin molecules that immunoreact with specific antigens, including whole antibodies, dimers, trimers and multimers; bispecific antibodies; chimeric antibodies; recombinant and engineered antibodies, and fragments thereof, as long as the antibody includes an antigen-binding region. Immunoglobulin molecules have heavy chains and light chains, each of which contains a constant region and a variable region, wherein the variable region of each heavy chain and light chain is called a variable heavy chain (VH) and a variable light chain (VL), respectively. The variable region of the heavy chain and light chain contains four framework regions, interrupted by three hypervariable regions, also known as complementarity determining regions (CDRs), wherein the main function of CDRs is to bind to the epitope of the antigen.

[0019] Example 1. Screening and preparation of monoclonal antibodies 1. PBMC Isolation and Plasma Cell Sorting 20 ml of venous blood was collected from a volunteer infected with adenovirus type 4 into a heparinized anticoagulant tube. The 20 ml blood sample was separated into mononuclear cells (PBMCs) using Ficoll. After cell counting, the PBMCs were sorted using a Sony flow cytometer. Single memory B cells specific for the Hexon protein (Genbank No. AY594253.1) were isolated using a cell sorter (SONY, MA900). Control tubes and single-stained tubes were first loaded onto the cytometer to adjust compensation. The cells were then sorted, and morphologically intact single cells were plated into a 96-well PCR plate, ensuring that each well contained one memory B cell. The cells were then stored at -80°C until further use.

[0020] Results: As Figure 1 As shown in the figure, the gate scheme is set: lymphocytes are circled by SSC and FSC, set as A; the cells selected in A are de-adhesive by FSC-H and FSH-A; CD3 – CD19 + The cells in B are circled by PE-Cy7 and PE-CD27. + IgG + The cells in C are marked with 488 and Heoxn +The cells in D are set as D; the cell population in D (CD3 – CD19 + CD27 + IgG + HAdV-4 + ) are specific memory B cells, and a total of 376 specific memory B cells were sorted.

[0021] 2. Single-cell PCR amplification of fully human monoclonal antibodies 1) 376 hexon-specific memory B cells were sorted by reverse transcription-PCR. All of the following primers specific for each subtype of heavy chain, kappa light chain, and lambda light chain were added to each reaction system (for primer sequences, see Example 1 of CN114031685A, which is incorporated herein by reference).

[0022] The PCR reaction system contained: 6 μl of 5× buffer, 1.2 μl of dNTP, 1.2 μl of reverse transcriptase, the template was a single cell, and the volume was made up to 30 μl with water.

[0023] The PCR reaction conditions were as follows: reverse transcription at 50°C for 30 min, pre-denaturation at 95°C for 15 min, followed by 40 cycles of 95°C for 40 s, 55°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 10 min.

[0024] 2) Nested PCR uses the reverse transcription product as a template. The specific process is as follows: The PCR reaction system contained: 2.5 μl of 10× buffer, 0.5 μl of 10 mM dNTP, 0.25 μl of DNA polymerase, the primers as above, 1 μl of reverse transcription product as template, and the volume was made up to 25 μl with water.

[0025] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 4 min, followed by 40 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 45 s, and a final extension at 72°C for 10 min.

[0026] 3) Amplify the antibody variable region.

[0027] The PCR system contained: 10 ng of reverse transcription PCR product as template, 5 μl of 10× buffer, 1 μl of 10 mM dNTP, 0.5 μl of DNA polymerase (heavy chain and light chain primers were mixed separately and added to the system), and the volume was made up to 50 μl with water.

[0028] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 3 min, and a final extension at 72°C for 10 min.

[0029] 4) Amplify the antibody constant region.

[0030] The PCR system contains: 10 ng of heavy and light chain constant region template, 5 μl of 10× buffer, 1 μl of 10 mM dNTP, 0.5 μl of DNA polymerase (heavy chain and light chain primers are mixed separately and added to the system), and the volume is made up to 50 μl with water.

[0031] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 30 s, 60°C for 60 s, and 72°C for 3 min, and a final extension at 72°C for 10 min.

[0032] 3. Expression Plasmid Construction and Antibody Preparation Construct expression plasmids and prepare monoclonal antibody expression. The method is as follows: 1) Homologously recombined the amplified variable regions into pcDNA3.4 (Sangon Biotech), transformed into TOP10 competent cells, and then extracted the plasmid; 2) Cell seeding: The density is 2×10 5 1 ml of 293T cells at 5 cells / ml was seeded into a 24-well cell culture plate and cultured in a 37°C incubator for 24 h; 3) Cell co-transfection: After 24 hours, add the constructed heavy and light chain plasmids to 2 μl of Turbofect (Thermo Scientific, R0531) transfection reagent. Incubate for 15-20 minutes and then add dropwise to the culture wells of 293T cells cultured overnight. Incubate at 37°C in a cell incubator with 5% CO2 for 48 hours. Collect the cell culture supernatant and set aside.

[0033] 4. ELISA screening of antibodies with binding activity The specific steps are as follows: 1) The day before the experiment, a 96-well ELISA plate was coated with 2 µg / ml hexon protein (Genebank No. AY594253.1, self-produced), 100 µl per well, and incubated at 4°C overnight. 2) Discard the coating solution, add 100 µl of blocking solution to each well, and incubate at 37°C for 1 hour; 3) Discard the blocking solution, add 100 µl of transfected cell culture supernatant, and incubate at 37°C for 1 hour; 4) Discard the supernatant and dilute HRP-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) at 1:10,000 in diluent. Add 100 µl per well to the corresponding wells of the ELISA plate and incubate at 37°C for 1 hour. 5) Discard the supernatant and add 100 µl of TMB one-component colorimetric solution to each well. Incubate at room temperature in the dark for 6 minutes. Then, add 50 µl of stop solution to each well to terminate the reaction. 6) Detect the OD value at 450-630nm using a microplate reader and save and record the original data.

[0034] Results: A total of 4 antibodies that specifically bind to Hexon were screened, with OD values greater than 2. Based on the initial screening, the 4AC3 antibody with neutralizing activity was prepared in large quantities.

[0035] 5. Large-scale antibody preparation 293 cells were co-transfected with expression vectors for the heavy and light chains of antibodies initially screened for neutralizing activity against 4AC3. Fresh medium was replaced 6-8 hours after transfection, and the cells were incubated in a 37°C, 8% CO2 incubator for 96 hours. The transfection supernatant was collected, centrifuged at 4000 rpm for 10 minutes, and purified using Protein A affinity chromatography. Antibody expression and purification were verified using SDS-PAGE.

[0036] 6. Results The results of antibody 4AC3 are as follows Figure 2 As shown, the target antibody was obtained by SDS-PAGE, and the melted antibody light chain and heavy chain were observed, with a molecular weight of 150 kDa, which was consistent with expectations (marker 26616).

[0037] Example 2. ELISA detection of antibody binding activity 1) The day before the experiment, coat a 96-well ELISA plate with 2 µg / ml Hexon protein, 100 µl per well, at 4°C overnight.

[0038] 2) Discard the coating solution, add 100 µl of blocking solution to each well, and incubate at 37°C for 1 hour.

[0039] 3) Discard the blocking solution, add 1µg / ml antibody, dilute in a 1:3 gradient, and incubate at 37° for 1 hour.

[0040] 4) Discard the primary antibody and dilute HRP-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) at 1:10,000 in diluent. Add 100 µl per well to the corresponding wells of the ELISA plate and incubate at 37° for 1 hour.

[0041] 5) Discard the secondary antibody and add 100 µl of TMB one-component colorimetric solution to each well. Develop the color for 6 minutes at room temperature in the dark. Then, add 50 µl of stop solution to each well to terminate the reaction.

[0042] 6) Detect the OD value at 450-630nm using a microplate reader and save and record the original data.

[0043] The results are as follows Figure 3 As shown, the EC of antibody 4AC3 50 Reached 2.1ng / ml.

[0044] Example 3. Detection of antibody neutralizing activity.

[0045] Neutralization levels were determined using HAdv-4 luciferase-containing pseudovirus (purchased from Promega, catalog number E1501).

[0046] 1) Add 50 μl of 100 μg / ml antibody 4AC3 to a 96-well plate, dilute it 1:3, add an equal volume of HAdV-4 luciferase, and incubate at 37° for 1 hour. 2) After 1 hour, add 2×10 5 cells / ml of A549 cells in 100 μl and incubated at 37° for 24 h; 3) After 24 hours, add 100 μl of lysis buffer and read the value; The results are as follows Figure 4 As shown, the IC of antibody 4AC3 50 Reaching 0.2μg / ml.

[0047] Example 4. SPR detection of antibody affinity The affinity between the antibody and Hexon protein was detected using a Protein A chip using a Biacore T200 instrument. The determination method is as follows: 1) Dilute the antibody to 1 µg / mL and load it onto the Protein A chip at a flow rate of 10 µL / min for 120 s; 2) Hexon protein was diluted from 100 nM to 6.125 nM in a 2-fold series and loaded onto the antibody-captured Protein A chip at a flow rate of 30 μL / min, with binding for 120 s and dissociation for 900 s. 3) Select 5 representative curves and calculate the affinity (KD) of the antibody using the instrument configuration analysis software; 4) The results are as follows Figure 5 As shown, the KD value of antibody 4AC3 is less than 10-12 M.

[0048] Example 5 Sequence determination The present invention screened a human monoclonal antibody 4AC3 that can specifically bind to adenovirus type 4 and has high affinity and strong neutralizing activity at low concentrations. The sequence determination results are as follows: The sequence of the gene encoding the heavy chain variable region of the antibody 4AC3 is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:1. The amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region are shown in SEQ ID NO:1, positions 26-33, 51-58, and 97-116, respectively. The sequence of the gene encoding the heavy chain constant region is shown in SEQ ID NO:6, and the amino acid sequence is shown in SEQ ID NO:5. The sequence of the gene encoding the light chain variable region of the antibody 4AC3 is shown in SEQ ID NO:4, and the amino acid sequence is shown in SEQ ID NO:3. The amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown in SEQ ID NO:1, positions 27-33, 51-53, and 90-98, respectively. The sequence of the gene encoding the light chain constant region is shown in SEQ ID NO:8, and the amino acid sequence is shown in SEQ ID NO:7. The light chain is a kappa chain.

Claims

1. A human monoclonal antibody against adenovirus type 4, characterized in that: The amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the human monoclonal antibody against type 4 adenovirus are shown in SEQ ID NO: 1 at positions 26-33, 51-58 and 97-116, respectively; the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the light chain variable region are shown in SEQ ID NO: 3 at positions 27-33, 51-53 and 90-98, respectively.

2. The human monoclonal antibody against adenovirus type 4 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the human monoclonal antibody against adenovirus type 4 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

3.

3. The human monoclonal antibody against adenovirus type 4 according to claim 2, characterized in that The amino acid sequence of the heavy chain constant region of the human monoclonal antibody against adenovirus type 4 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

7.

4. A polynucleotide encoding the heavy chain and light chain of the human monoclonal antibody against adenovirus type 4 according to any one of claims 1 to 3, characterized in that: The sequence of the polynucleotide encoding the heavy chain variable region of the human monoclonal antibody against adenovirus type 4 is shown in SEQ ID NO: 2, and the sequence of the polynucleotide encoding the light chain variable region of the human monoclonal antibody against adenovirus type 4 is shown in SEQ ID NO:

4.

5. The polynucleotide according to claim 4, wherein The sequence of the polynucleotide encoding the heavy chain constant region of the human monoclonal antibody against adenovirus type 4 is shown in SEQ ID NO: 6, and the sequence of the polynucleotide encoding the light chain constant region of the human monoclonal antibody against adenovirus type 4 is shown in SEQ ID NO:

8.

6. A vector comprising the polynucleotide encoding the heavy chain and light chain of the monoclonal antibody according to claim 5.

7. A host cell containing the vector comprising the polynucleotide encoding the heavy chain and light chain of the monoclonal antibody according to claim 6.

8. The host cell according to claim 7, characterized in that The cells are 293T cells or Expi293F cells.

9. Use of the human monoclonal antibody against adenovirus type 4 according to any one of claims 1 to 3 in the preparation of a medicament for treating and / or preventing adenovirus type 4 infectious diseases.

10. A detection kit comprising the human monoclonal antibody against adenovirus type 4 according to any one of claims 1 to 3.

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