Monoclonal antibody fab5 recognizing gb protein of EB viruses and use thereof

Abstract

Disclosed are a monoclonal antibody Fab5 recognizing the gB protein of EB viruses and the use thereof, belonging to the technical field of antibodies. The monoclonal antibody or an antigen-binding fragment thereof has high affinity for the gB protein and can significantly inhibit the EB virus infection of epithelial cells and B cells, and the epitope of the Fab5 binding to the gB is clarified by structural analysis. The antibody can be used for detecting the presence or level of the gB protein in specimens, detecting EB viruses, diagnosing diseases caused by EB virus infection, preventing EB virus infection, and / or treating and / or preventing diseases caused by EB virus infection.

Description

A monoclonal antibody Fab5 that recognizes the EB virus gB protein and its applications Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to a monoclonal antibody Fab5 that recognizes the gB protein of EB virus and its applications. Background Technology

[0002] Epstein-Barr virus (EBV), also known as human herpesvirus 4, was first established in 1964 by Epstein and Barr through in vitro suspension culture of Burkitt lymphoma cells. It is a DNA oncogenic virus belonging to the genus Lymphovirus of the subfamily Gamma subfamily. EBV particles consist of four structural components: a core protein, a capsid, an outer shell, and an envelope. The core is a core protein wrapped with DNA.

[0003] Epstein-Barr virus (EBV) primarily exists as a latent infection, with over 90% of EBV-infected individuals experiencing lifelong latent infection. The virus can be activated and lead to cancer under certain conditions. Current research has found a close relationship between human EBV and the development of malignant tumors such as nasopharyngeal carcinoma, infectious mononucleosis, Hodgkin's and non-Hodgkin's lymphomas, Burkitt's lymphoma, and epithelial cell carcinomas including gastric cancer, as well as autoimmune diseases such as multiple sclerosis. EBV initially replicates in the oropharynx, subsequently growing and multiplying within B lymphocytes and oral epithelial cells, then infecting more B lymphocytes. These infected cells enter the bloodstream in large numbers, causing systemic infection. When the body's immune function declines, latent EBV is activated, leading to recurrent infection.

[0004] Currently, there is no effective vaccine against EBV, and there are no specific treatments for diseases caused by EBV infection. Treatment for infectious mononucleosis mostly involves antiviral drugs such as acyclovir. While these drugs can alleviate symptoms to some extent, they cannot eliminate EBV from B lymphocytes and the pharyngeal epithelium. Treatment for EBV-related tumors primarily involves chemotherapy and radiotherapy, but these are less effective for patients with metastases or relapses.

[0005] Monoclonal antibodies can be mass-produced, and their high affinity and specificity for binding to antigens significantly reduce adverse reactions in clinical applications. These antibody molecules can also be modified to increase their antiviral efficacy. Antibodies, with their specificity and flexibility of use, are a very promising approach to the treatment of infectious diseases; however, to date, no monoclonal antibody targeting EBV envelope glycoprotein gB has been commercialized. Therefore, developing anti-EBV monoclonal antibodies will provide a more effective means of prevention and treatment for EBV-related diseases. Summary of the Invention

[0006] The first aspect of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof.

[0007] A second aspect of the present invention is to provide a recombinant protein.

[0008] A third aspect of the present invention aims to provide biological materials related to the monoclonal antibody or its antigen-binding fragment of the first aspect of the present invention or the recombinant protein of the second aspect of the present invention.

[0009] The fourth aspect of this invention aims to provide a method for preparing the monoclonal antibody or its antigen-binding fragment of the first aspect of this invention or the recombinant protein of the second aspect.

[0010] The fifth aspect of the present invention is to provide a coupling material.

[0011] The sixth aspect of this invention aims to provide the use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect, the recombinant protein of the second aspect, the biomaterial of the third aspect, and / or the conjugate of the fifth aspect in the preparation of products.

[0012] The seventh aspect of this invention is to provide a medicine.

[0013] The object of the eighth aspect of the present invention is to provide a vaccine.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0015] In a first aspect, the present invention provides a monoclonal antibody against EB virus gB or an antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region;

[0016] The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3;

[0017] The CDR-H1, CDR-H2, and CDR-H3 are amino acid sequences as shown in SEQ ID NO: 4, namely CDR1, CDR2, and CDR3.

[0018] The light chain variable region includes CDR-L1, CDR-L2 and CDR-L3;

[0019] The CDR-L1, CDR-L2, and CDR-L3 are amino acid sequences as shown in SEQ ID NO: 17, namely CDR1, CDR2, and CDR3.

[0020] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 are as shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and the amino acid sequence of CDR-L2 is DTS. The CDR is defined using the IMGT definition scheme.

[0021] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are as shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 19, respectively, and the CDR is defined using the Kabat definition scheme.

[0022] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are as shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 19, respectively, and the CDR is defined according to the Chothia definition scheme.

[0023] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are as shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, and the CDR is defined using the Contact definition scheme.

[0024] Preferably, the amino acid sequence of the heavy chain variable region comprises:

[0025] a1)SEQ ID NO: 4; or

[0026] a2) An amino acid sequence obtained from SEQ ID NO: 4 by substitution and / or deletion and / or addition of one or more amino acids, and having the same function as the protein shown in SEQ ID NO: 4; or

[0027] a3) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with SEQ ID NO: 4, and has the same functional amino acid sequence as the protein shown in SEQ ID NO: 4.

[0028] Preferably, the amino acid sequence of the light chain variable region comprises:

[0029] b1)SEQ ID NO: 17; or

[0030] b2) An amino acid sequence obtained from SEQ ID NO: 17 by substitution and / or deletion and / or addition of one or more amino acids, and having the same function as the protein shown in SEQ ID NO: 17; or

[0031] b3) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with SEQ ID NO: 17, and has the same functional amino acid sequence as the protein shown in SEQ ID NO: 17.

[0032] Preferably, the monoclonal antibody or its antigen-binding fragment comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, and multispecific antibody.

[0033] Preferably, the heavy chain further includes a heavy chain constant region; and / or, the light chain further includes a light chain constant region.

[0034] Preferably, the amino acid sequence of the heavy chain constant region comprises:

[0035] c1) An amino acid sequence consisting of amino acids 138–467 of SEQ ID NO: 3; or

[0036] c2) An amino acid sequence obtained from the amino acid sequence described in c1) by substitution and / or deletion and / or addition of one or more amino acids, and having the same function as the protein with the amino acid sequence described in c1); or

[0037] c3) has 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology with the amino acid sequence described in c1), and the protein with the amino acid sequence described in c1) has the same function.

[0038] Preferably, the amino acid sequence of the light chain constant region comprises:

[0039] d1) An amino acid sequence consisting of amino acids 127 to 232 of SEQ ID NO: 16; or

[0040] d2) An amino acid sequence obtained from the amino acid sequence described in d1) by substitution and / or deletion and / or addition of one or more amino acids, and which has the same function as the amino acid sequence described in d1); or

[0041] d3) has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in d1), and the protein with the same function as the amino acid sequence described in d1) has an amino acid sequence.

[0042] Preferably, the heavy chain further comprises a heavy chain signal peptide; and / or the light chain further comprises a light chain signal peptide.

[0043] Preferably, the amino acid sequence of the heavy chain signal peptide comprises:

[0044] e1) An amino acid sequence consisting of amino acids 1 to 19 of SEQ ID NO: 3; or

[0045] e2) An amino acid sequence obtained from the amino acid sequence described in e1) by substitution and / or deletion and / or addition of one or more amino acids, and having the same function as the protein with the amino acid sequence described in e1); or

[0046] e3) has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in e1), and the protein with the same function as the amino acid sequence described in e1) has an amino acid sequence.

[0047] Preferably, the amino acid sequence of the light chain signal peptide comprises:

[0048] f1) An amino acid sequence consisting of amino acids 1 to 19 of SEQ ID NO: 16; or

[0049] f2) An amino acid sequence obtained from the amino acid sequence described in f1) by substitution and / or deletion and / or addition of one or more amino acids, and having the same function as the protein with the amino acid sequence described in f1); or

[0050] f3) has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in f1), and the protein with the same function as the amino acid sequence described in f1) has an amino acid sequence.

[0051] Preferably, the amino acid sequence of the EB protein gB comprises:

[0052] g1) An amino acid sequence consisting of amino acids 25 to 685 of SEQ ID NO: 1; or

[0053] g2) An amino acid sequence obtained from the amino acid sequence described in g1) by substitution and / or deletion and / or addition of one or more amino acids, and having the same function as the protein with the amino acid sequence described in g1); or

[0054] g3) has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in g1), and the protein with the amino acid sequence described in g1) has the same function.

[0055] A second aspect of the present invention provides a recombinant protein comprising: the monoclonal antibody or its antigen-binding fragment as described in the first aspect of the present invention; and optionally a tag sequence for assisting expression and / or purification.

[0056] Preferably, the tag sequence is selected from at least one of the following: histidine (His) tag, GGGS sequence (SEQ ID NO: 25), FLAG tag; more preferably His tag; and even more preferably 6×His tag.

[0057] A third aspect of the invention provides biological materials relating to the monoclonal antibody or its antigen-binding fragment of the first aspect of the invention, or the recombinant protein of the second aspect of the invention, said biological material comprising at least one of h1) to h16):

[0058] h1) A nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention, or a recombinant protein of the second aspect of the present invention;

[0059] h2) contains an expression cassette containing the nucleic acid molecule described in h1);

[0060] h3) A carrier containing the nucleic acid molecule described in h1);

[0061] h4) A carrier containing the expression box described in h2);

[0062] h5) A transgenic cell line containing the nucleic acid molecules described in h1);

[0063] h6) Transgenic cell lines containing the expression cassette described in h2);

[0064] h7) A transgenic cell line containing the vector described in h3);

[0065] h8) A transgenic cell line containing the vector described in h4);

[0066] h9) Microorganisms containing the nucleic acid molecules described in h1);

[0067] h10) contains microorganisms containing the expression cassette described in h2);

[0068] h11) contains microorganisms containing the carrier described in h3);

[0069] h12) contains microorganisms containing the carrier described in h4);

[0070] h13) is a virus containing the nucleic acid molecule described in h1);

[0071] h14) contains the expression cassette described in h2);

[0072] h15) contains a virus with the vector described in h3);

[0073] h16) contains a virus with the vector described in h4).

[0074] Preferably, the transgenic cell line does not contain propagation material.

[0075] Preferably, the nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment as described in the first aspect of the invention comprises:

[0076] Nucleic acid molecules encoding the heavy chain of a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0077] A nucleic acid molecule encoding the light chain of a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0078] A fourth aspect of the present invention provides a method for preparing a monoclonal antibody or its antigen-binding fragment of the first aspect of the present invention or a recombinant protein of the second aspect of the present invention, the method comprising the following steps: expressing and purifying the biological material described in the third aspect of the present invention.

[0079] A fifth aspect of the present invention provides a conjugate comprising: at least one of a monoclonal antibody or an antigen-binding fragment thereof from the first aspect of the present invention and a recombinant protein from the second aspect of the present invention, and a conjugation portion thereof; said conjugation portion comprising at least one of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

[0080] Preferably, the detectable marker is selected from radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, or any combination thereof.

[0081] Preferably, the conjugate is selected from: fluorescent substances, chemiluminescent markers, colored substances, radioactive isotopes, magnetic resonance imaging (MRI) or computed tomography (CT) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes, chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0082] In a sixth aspect of the present invention, the use of the monoclonal antibody of the first aspect or its antigen-binding fragment, the recombinant protein of the second aspect, the biomaterial of the third aspect, and / or the conjugate of the fifth aspect in the preparation of a product is provided.

[0083] The product includes at least one of the following: drug, reagent, test plate, reagent kit, and test chip.

[0084] Preferably, the drug has at least one of the functions i1) to i2):

[0085] i1) Prevention of EB virus infection;

[0086] i2) Treatment and / or prevention of diseases caused by EB virus infection.

[0087] Preferably, the reagent, detection plate, detection chip, or kit has at least one function among j1) to j3):

[0088] j1) Detect the presence or level of gB protein in the sample;

[0089] j2) Detection of EB virus;

[0090] j3) Diagnose diseases caused by EB virus infection.

[0091] Preferably, the disease includes at least one of the following: nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disorders, and infectious mononucleosis.

[0092] Preferably, the drug comprises pharmaceutically acceptable excipients.

[0093] Preferably, the drug comprises a vaccine.

[0094] Preferably, the vaccine includes a pharmaceutically acceptable adjuvant.

[0095] A seventh aspect of the present invention provides a product comprising at least one of k1) to k3):

[0096] k1) The monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention;

[0097] k2) The recombinant protein of the second aspect of the present invention;

[0098] k3) The coupling of the fifth aspect of the present invention;

[0099] The product includes at least one of reagents, test plates, reagent kits, and test chips.

[0100] Preferably, the product has at least one function among j1) to j3):

[0101] j1) Detect the presence or level of gB protein in the sample;

[0102] j2) Detection of EB virus;

[0103] j3) Diagnose diseases caused by EB virus infection.

[0104] Preferably, the disease includes at least one of the following: nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disorders, and infectious mononucleosis.

[0105] An eighth aspect of the present invention provides a medicament comprising at least one of 11) to 14):

[0106] l1) The monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention;

[0107] l2) The recombinant protein of the second aspect of the present invention;

[0108] l3) Benzene inventions in the third aspect of biomaterials;

[0109] l4) The coupling of the fifth aspect of the present invention.

[0110] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0111] Preferably, the drug has at least one of the functions i1) to i2):

[0112] i1) Prevention of EB virus infection;

[0113] i2) Treatment and / or prevention of diseases caused by EB virus infection.

[0114] Preferably, the disease includes at least one of the following: nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disorders, and infectious mononucleosis.

[0115] Preferably, the drug comprises a vaccine.

[0116] Preferably, the vaccine comprises at least one of l1) to l4) and an adjuvant:

[0117] l1) The monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention;

[0118] l2) The recombinant protein of the second aspect of the present invention;

[0119] l3) Benzene inventions in the third aspect of biomaterials;

[0120] l4) The coupling of the fifth aspect of the present invention.

[0121] The beneficial effects of this invention are:

[0122] This invention provides a monoclonal antibody against EBV gB or its antigen-binding fragment thereof. The monoclonal antibody or its antigen-binding fragment has a high affinity for EBV gB protein and can significantly inhibit EBV infection of epithelial cells and B cells. It can be used to detect the presence or level of gB protein in a sample, detect EBV, diagnose diseases caused by EBV infection, prevent EBV infection and / or treat and / or prevent diseases caused by EBV infection. Attached Figure Description

[0123] Figure 1 shows the affinity test results between the monoclonal antibody Fab5 and the gB protein.

[0124] Figure 2 shows the results of the monoclonal antibody Fab5 blocking EBV infection in epithelial cells.

[0125] Figure 3 shows the results of the monoclonal antibody Fab5 blocking EBV infection of B cells.

[0126] Figure 4 shows the cryo-electron microscopy structure of the monoclonal antibody Fab5 and gB protein complex. Detailed Implementation

[0127] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0128] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, all materials and reagents used in these examples are commercially available.

[0129] Example 1: Preparation, animal immunization, and antibody screening of anti-EBV gB protein (EBV gB)

[0130] 1.1 Expression and purification of EBV gB antigen protein

[0131] 1.1.1 Experimental Materials

[0132] (1) Expression vector: eukaryotic expression vector pcDNA3.1(+)(Thermo Fisher).

[0133] (2) Expression system: eukaryotic expression system cells HEK293F(ATCC).

[0134] (3) Reagents and consumables: Plasmid extraction kit (MN), cell transfection reagent PEI (Polyscience), 293F medium (Union), histidine tag protein purification agarose beads (Roche), and other conventional reagents and consumables were all purchased commercially.

[0135] (4) Gene: The gB gene of EB virus (M81 strain) was optimized and synthesized by Nanjing Genscript Biotech Co., Ltd., and inserted into the pcDNA3.1 vector to construct a recombinant plasmid, hereinafter referred to as pcDNA3.1-gB.

[0136] The gB extracellular recombinant protein consists of 661 amino acid residues (excluding *), with the signal peptide marked in bold. The specific sequence is as follows:

[0137] Its DNA expression sequence consists of 2071 base pairs, with the bolded part indicating the signal peptide. The specific sequence is as follows:

[0138] 1.1.2 Protein Expression

[0139] Step 1: Transformation and Selection of Single Clones

[0140] (1) The synthesized plasmid was added to the frozen-thawed Escherichia coli DH5α and placed on ice for 5 minutes.

[0141] (2) Place it in a vibrating metal bath and heat shock it at 42℃ and 120rpm for 60-90 seconds.

[0142] (3) Let it stand on ice to cool down for 5 minutes.

[0143] (4) Use a sterile spreader to spread the bacterial solution onto a Terrific Broth (TB) medium plate containing ampicillin resistance and incubate overnight.

[0144] (5) The next day, use a sterile pipette tip to pick up a single colony on the surface of the plate and put it into 500 mL of culture medium. Add 0.1 mg / mL ampicillin and incubate at 37°C and 220 rpm for 12-16 hours.

[0145] Step 2: Plasmid extraction

[0146] (1) Centrifuge the cultured bacterial solution at 4000 rpm and 4℃ and discard the supernatant. Collect the bacterial precipitate and extract plasmids according to the instructions of the MN extraction kit. The following is a simplified plasmid extraction process.

[0147] (2) Resuspend the precipitate using the RES resuspension buffer (containing RNAase A) from the MN extraction kit.

[0148] (3) Add LYS lysis buffer and lyse for 5 minutes.

[0149] (4) Add NEU neutralization buffer to neutralize. At this time, a precipitate should be formed and the solution should be clear and yellow.

[0150] (5) Equilibrate the hollow fiber column with EQU equilibration buffer, making sure the solution flows in from the top of the fiber column inlet to completely wet the entire fiber sleeve.

[0151] (6) Transfer the neutralized solution along with the precipitate into the hollow fiber column after equilibrium.

[0152] (7) Add EQU equilibration buffer to re-equilibrate the hollow fiber column so that the solution below is clear, colorless and transparent.

[0153] (8) Discard the middle fiber sleeve and leave the outer adsorption column.

[0154] (9) Add Wash washing buffer to wash the adsorption membrane below the adsorption column.

[0155] (10) Place centrifuge tubes under the adsorption column to collect the effluent, then add an appropriate amount of Elu elution buffer to elute the plasmid, and finally add isopropanol to precipitate the plasmid.

[0156] (11) The precipitated plasmid solution was centrifuged at 4000 rpm for 30 minutes, the supernatant was discarded, and the precipitate was resuspended in 70% ethanol and centrifuged again.

[0157] (12) After centrifugation, discard the supernatant in a clean bench, resuspend the plasmid in an appropriate amount of sterile double-distilled water, and store the resuspended plasmid at -20℃.

[0158] Step 3: Plasmid transfection of 293F cells

[0159] (1) 293F cells were cultured in medium at 37℃, 120 rpm, and 5% CO2 with shaking, and their density was maintained at 0.5-5*10. 6 Cells / mL (0.5-5X). If the density is exceeded, the cells should be cultured in separate bottles or transfected in a timely manner.

[0160] (2) Take out the plasmid and dilute it with 293 medium. Generally, the transfection amount is calculated as 1 mg of plasmid per 1 L of 1X cells.

[0161] (3) Take out the dissolved PEI and dilute it with 293 medium. Generally, add 5mg PEI to each 1mg plasmid to prepare the transfection system.

[0162] (4) Mix the plasmid diluted with 293 medium with PEI and let it stand for 10-15 minutes.

[0163] (5) Dilute the density of 293F cells to 1X, then add them to the transfection mixture and continue to culture at 37℃, 120rpm and 5%CO2 for 5-7 days.

[0164] Step 4: Purification of secretory proteins from eukaryotic culture supernatant

[0165] (1) After culturing, 293F cells were centrifuged at 8000 rpm and 4℃ for 1 hour, and the supernatant was collected. Since the synthetic plasmid contains a signal peptide sequence, the target protein is secreted into the supernatant, so the supernatant was used for protein purification.

[0166] (2) The cell supernatant was filtered twice using a 0.22 μm filter membrane to remove impurities.

[0167] (3) Take a gravity purification column, add 1 mL of His-tagged protein purification agarose beads to it, and add double-distilled water filtered through a 0.22 μm filter membrane (all liquids added to the purification column during the purification stage need to be filtered through a 0.22 μm filter membrane) to wash the magnetic beads. Then, use phosphate buffered saline (PBS) to equilibrate the purification column.

[0168] (4) Use the siphon method to transfer the supernatant to the purification column so that the supernatant passes completely through the purification column. If multiple recovery is required, the supernatant can be recovered in this step and purified again.

[0169] (5) Equilibrate the purification column with PBS at three column volumes.

[0170] (6) Elute the purification column with PBS containing 300mM imidazole for two column volumes and collect the eluent.

[0171] (7) Collect samples of supernatant, equilibration solution and elution solution respectively, and verify their protein expression by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0172] (8) Use an ultrafiltration tube with a suitable molecular weight cutoff to concentrate the eluent so that the volume after concentration is less than 1 mL. Centrifuge the concentrated eluent at 12000 g and 4 °C for 5 minutes to remove the precipitate.

[0173] (9) The concentrated eluent was purified by molecular sieve using an AKTA purifier equipped with a Superdex 200Increase 10 / 300GL molecular sieve column. Protein samples at the molecular sieve elution peaks were collected and verified by SDS-PAGE with the aforementioned samples.

[0174] 1.2 EBV gB mouse immunization experiment

[0175] 1.2.1 Vaccine preparation

[0176] (1) Remove gB protein and freeze-thaw it. The amount of gB is 5 μg / mouse. Use sterile PBS as solvent to prepare antigen solution.

[0177] (2) Mix 100 μg of antigen solution with 100 μL of Complete Freund's Adjuvant (CFA), emulsify to form a dose of 200 μL / mouse, place it on a rotating shaker, mix at 4°C overnight.

[0178] 1.2.2 Mouse Immunization and Spleen Collection

[0179] (1) Mice were fed with conventional feed and water, and were immunized at 6 weeks of age. The strain was Balb / C.

[0180] (2) Fix the mouse, use a 1mL syringe to draw up the vaccine after manually shaking it again, and administer it via subcutaneous injection in the abdomen.

[0181] (3) Observe the mice for 3-5 minutes, and release them after they resume normal activity.

[0182] (4) The vaccine immunization process is from week 0 to week 3 to week 6 to week 8, and the spleen is harvested in week 10.

[0183] (5) When collecting the spleen of a mouse, the mouse was euthanized by vertebral dislocation in accordance with animal welfare and standard animal experimental procedures, and its blood was collected and its spleen was dissected.

[0184] 1.3 Isolation and Cloning of EBV gB Mouse B Cells (gB-Specific Cells)

[0185] 1.3.1 Steps for separating spleen lymphocytes:

[0186] (1) Mice were euthanized by cervical dislocation in the 10th week after immunization, and their peripheral lymph nodes and spleen were separated and placed in EP tubes.

[0187] (2) Add appropriate amounts of DNase and type IV collagenase to 1640 culture medium to prepare tissue digestion solution, and add appropriate amounts of digestion solution according to the size of lymph nodes and spleen.

[0188] (3) Place the tissue on a 70μm cell sieve and crush it with the pressure plug of a sterile syringe until the tissue is almost broken and eroded. Then rinse the cell sieve with an appropriate amount of fresh culture medium so that all the remaining cells are collected in the 50mL centrifuge tube below.

[0189] (4) Add red blood cell lysis buffer and incubate at room temperature for 10 minutes.

[0190] (5) Centrifuge at 2000g and 18℃ for 5min and discard the supernatant.

[0191] (6) Wash twice with fresh culture medium.

[0192] 1.3.2 B lymphocyte antigen staining and sorting

[0193] (1) Wash the cells with PBS and adjust to 1*102 8 Centrifuge each cell / flow cytometer at 3000g and 18°C ​​for 5 minutes, and discard the supernatant.

[0194] (2) Add 5 μL of Fc receptor blocking antibody solution to 100 μL PBS, resuspend the cells and incubate on ice for 30 minutes.

[0195] (3) Prepare flow cytometry antibody dilution buffer using PBS according to cell grouping. When using the antibody for the first time, different dilution gradients can be set up, with antibody:PBS ratios of 1:20, 1:50, 1:100, 1:200, and 1:500, and the optimal gradient can be selected. Take 100 μL of the diluted flow cytometry antibody and mix it with cell viability dye, resuspend the cells for staining, and incubate at 4°C in the dark for 30 minutes. Mouse IgG and B220 antibodies are used as B cell separation reagents, and gB is used as a gB-specific B cell separation reagent.

[0196] (4) Wash the cells with PBS, centrifuge at 3000g and 4℃ for 5 minutes, repeat the washing 1 to 2 times, and discard the supernatant.

[0197] (5) Resuspend the cells in PBS into flow cytometry tubes and sort the cells by flow cytometry using a BD Rhaspody single-cell sorter.

[0198] (6) Add IgG + B220 + The 0.2% of cells with the strongest gB positive signal were sorted into 96-well plates.

[0199] 1.3.3 B cell sequence cloning and amplification

[0200] (1) By using universal cloning primers for mouse IgG heavy and light chains, PCR was performed on B cells in a 96-well plate to obtain the heavy and light chain sequences of a single B cell.

[0201] (2) A recombinant fragment capable of expressing the complete heavy chain was constructed by linking the upstream of the variable region of the antibody heavy chain to a cytomegalovirus (CMV) fragment and the downstream of a mouse IgG1 constant region; conversely, a recombinant fragment capable of expressing the complete light chain was constructed by linking the upstream of the variable region of the antibody light chain to a CMV fragment and the downstream of a light chain κ / λ constant region. Plasmids containing the full-length sequences of the antibody heavy and light chains were co-transfected into 293F cells to achieve antibody expression. Antibodies were purified using protein A beads, yielding a total of eight antibodies, Fab1-Fab8, through this method.

[0202] (3) The obtained antibodies were subjected to gB affinity assay using ELISA, and the Fab5 antibody with the highest affinity was selected for further validation.

[0203] Example 2: Preparation of monoclonal antibody (mAb) against EBV gB protein (EBV gB)

[0204] A recombinant fragment expressing the complete heavy chain was constructed by linking the upstream of the variable region of the antibody heavy chain to a CMV fragment and the downstream of a constant region of human IgG1. Similarly, a recombinant fragment expressing the complete light chain was constructed by linking the upstream of the variable region of the antibody light chain to a CMV fragment and the downstream of a constant region of the light chain κ / λ. Co-transfection of plasmids containing the full-length sequences of both antibody heavy and light chains into 293F cells resulted in antibody expression. Protein A beads were used for antibody purification.

[0205] The full-length Fab5 heavy chain consists of 467 amino acid residues (excluding *), and its specific sequence is as follows:

[0206] The underlined portion of the sequence represents the amino acid sequence of the heavy chain variable region (SEQ ID NO: 4). The italicized portions represent the amino acid sequences (IMGT definition scheme) of the three complementary regions CDR-H1 (SEQ ID NO: 5), CDR-H2 (SEQ ID NO: 6), and CDR-H3 (SEQ ID NO: 7) within the heavy chain variable region. The bolded portion represents the signal peptide sequence. Amino acids 138–467 constitute the heavy chain constant region. * indicates a stop codon.

[0207] The sequences of CDR-H1, CDR-H2, and CDR-H3 in the variable region of the heavy chain using other defined CDR schemes are shown in Table 11.

[0208] Table 11 shows the sequences of CDR-H1, CDR-H2, and CDR-H3 in the variable region of the heavy chain using other defined CDR schemes.

[0209] The full-length light chain of Fab5 consists of 232 amino acid residues (excluding *), and its specific sequence is as follows:

[0210] The underlined portion of the sequence represents the amino acid sequence of the light chain variable region (SEQ ID NO: 17). The underlined and bolded portions represent the amino acid sequences (IMGT definition scheme) of the three complementarity-determining regions CDR-L1 (SEQ ID NO: 18), CDR-L2 (DTS), and CDR-L3 (SEQ ID NO: 19) within the light chain variable region. The bolded portion represents the signal peptide sequence. Amino acids 127–232 constitute the light chain constant region. * indicates a stop codon.

[0211] The sequences of CDR-H1, CDR-H2, and CDR-H3 in the variable region of this light chain using other defined CDR schemes are shown in Table 12.

[0212] Table 12 Sequences of CDR-H1, CDR-H2, and CDR-H3 using other defined CDR schemes in the variable region of light chains.

[0213] Example 3: Affinity determination of Fab5

[0214] The affinity of antibody Fab5 was determined using bio-layer interferometry (BLI).

[0215] BLI can be performed according to conventional methods in the art. In this embodiment, the specific operation is as follows: the biosensor (Sartorius, Germany) is used... The SA probe was equilibrated in a buffer solution (a mixture of KB buffer, 0.1 wt% BSA, and 0.02 v / v% Tween 20). It was then removed and immersed in a solution containing 5 μg / mL gB-Biotin (biotin-labeled gB protein). The gB antigen in the solution bound to the streptavidin (SA) bioprobe surface, increasing the surface membrane thickness. A biosensor with a known concentration of cured gB antigen was then immersed in the buffer solution, and baseline signals were recorded. Immersing the biosensor with a known concentration of cured antigen in a sample solution containing 31.3–500 nM Fab5 antibody for approximately 120 seconds resulted in an increase in membrane thickness due to the specific binding between antigen and antibody. Immersing the biosensor with the bound Fab5 antibody in the buffer for approximately 180 seconds allowed dissociation, causing the analyte antibody (Fab5 antibody) to detach from the biosensor surface, reducing the membrane thickness. Real-time monitoring of the biosensor biomembrane thickness during the experiment allowed the determination of the kinetic constants of the analyte sample (Fab5 antibody). The results are shown in Figure 1: KD(M) of the Fab5 antibody < 10. -12 M indicates that the Fab5 antibody has a very high affinity for the gB antigen.

[0216] Example 4: Neutralization activity assay of Fab5 antibody

[0217] (1) Preparation of EBV virus:

[0218] 1) CNE2 cells infected with EBV-GFP were cultured in RPMI 1640 medium at 37°C (5 v / v % CO2) using 5 v / v % FBS medium. (The virus has been disclosed in the literature: An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus). When the cell density reached 90% (10 cm dish), phorbol-12-myristate-13-acetate (TPA or PMA) at a final concentration of 20 ng / mL and sodium butyrate (NaB) at a final concentration of 2.5 mM were added for induction. The culture medium was replaced after 12 hours.

[0219] 2) Collect the culture supernatant 48–72 h after changing the medium and isolate and purify the virus. Directly aspirate the supernatant, centrifuge it, and filter it through a 0.45 μm filter. After concentrating the filtrate, resuspend it in serum-free RPMI 1640. Immediately use the resuspended solution for infection or store it at -80°C.

[0220] (2) Detection of neutralizing activity of Fab5 monoclonal antibody in epithelial cells

[0221] 1) Lay 1x10mm PVC in each well of a 96-well plate. 6 Add 100 μL of DMEM medium containing 10% FBS to each well of 293T epithelial cells.

[0222] 2) On the second day, take the Fab5 monoclonal antibody from the above examples and adjust its concentration to 2 mg / mL. Add 60 μL of DMEM medium to each well of a new 96-well plate, and add 120 μL of Fab5 antibody diluted with DMEM at 12.5 μg / mL to the first well (the first well does not contain RPMI 1640 medium).

[0223] 3) After a 2-fold serial dilution (the serial dilution steps are: aspirate 60 μL from the first well and add it to the second well, and so on, discarding 60 μL from the last well, resulting in a final volume of 60 μL per well); add 60 μL of virus diluent to each well (the virus is diluted with DMEM medium to a titer of approximately 4*10). 6 The incubation solution ( / mL) was added to 293T cells plated the previous day after incubation at 37°C for 2 hours. The cells were then incubated at 37°C for 48 hours before analysis.

[0224] 4) After digesting 293T cells with trypsin to prepare a cell suspension, the infection rate was detected by flow cytometry. The inhibition rate (neutralization efficiency, %) of the antibody in the 293T epithelial cell infection model was calculated by detecting the reduction in the number of GFP-positive cells in the antibody-treated group compared to the infection control group (which received an equal volume of DMEM). The IC50 of the Fab5 monoclonal antibody was obtained using Prism software for plotting and calculation. 50 .

[0225] The results are shown in Figure 2: IC5 of monoclonal antibody Fab5 in the epithelial cell infection model. 50 The concentration was 0.30 μg / mL, indicating that the monoclonal antibody Fab5 can significantly inhibit EBV infection of epithelial cells.

[0226] Example 5: Detection of neutralizing activity of Fab5 monoclonal antibody in B cells

[0227] 1) Take the Fab5 monoclonal antibody from the above examples and adjust its concentration to 2 mg / mL. Add 60 μL of RPMI 1640 medium to each well of a new 96-well plate. Add 90 μL of 100 μg / mL Fab5 antibody diluted with RPMI 1640 to the first well (the first well does not contain RPMI 1640 medium).

[0228] 2) After a 3-fold serial dilution (the serial dilution steps are: aspirate 30 μL from the first well and add it to the second well, and so on, discarding 30 μL from the last well, resulting in a final volume of 60 μL per well); add 60 μL of virus diluent to each well (the virus is diluted with DMEM medium to a titer of approximately 4*10). 6 After incubating at 37°C for 2 hours, add 1*10 mL to each well. 6 Raji cells were incubated at 37°C for 48 hours before analysis.

[0229] 3) Raji cells were aspirated and prepared into a cell suspension. The infection rate was detected by flow cytometry. The inhibition rate (neutralization efficiency, %) of the antibody in the Raji B cell infection model was calculated by detecting the reduction in the number of GFP-positive cells in the antibody-treated group compared with the infection control group (which had an equal volume of RPMI 1640 added). The IC50 of the Fab5 monoclonal antibody was obtained by plotting and calculating using Prism software. 50 .

[0230] The results are shown in Figure 3: IC5 of monoclonal antibody Fab5 in the B cell infection model. 50 The concentration was 0.9 μg / mL, indicating that the monoclonal antibody Fab5 can significantly inhibit EBV infection of B cells.

[0231] Example 6: Cryo-electron microscopy structures of monoclonal antibody Fab5 and gB protein

[0232] Brief steps for cryo-electron microscopy imaging:

[0233] (1) 1 mg / mL of the Fab5 / gB complex sample was dropped into a glow-treated Quantifoil Cu R1.2 / 1.3 300 mesh copper mesh at a molar ratio of 1:1 (M / M).

[0234] (2) The Vitrobot Mark IV automated freezing robot was used for freezing samples. The freezing time was 7 seconds and the humidity was 100%.

[0235] (3) The sample was observed using a 300kV Titan Krios transmission electron microscope. If the sample preparation was not up to standard, the sample was frozen again.

[0236] (4) For qualified samples, K2Summit direct detection is used to take pictures and record the original images.

[0237] Brief steps for single-particle analysis using cryo-electron microscopy:

[0238] (1) The photo data is aligned using motionCor2.

[0239] (2) Use PatchCTF to calculate the contrast transfer function (CTF) value of cryo-electron microscopy data.

[0240] (3) Use Blob Picker for parameterless automatic particle picking to generate 2D classification control phase.

[0241] (4) Based on the best set in the control phase, further select the reference density to adjust the particles with parameters and perform 2D classification.

[0242] (5) Homologous reconstruction of particles from the best 2D category set is performed, and the resolution is determined according to the gsFSC standard.

[0243] The results, shown in Figure 4, reveal that Fab5 binds to the DI domain of the gB protein. The Fab5 heavy chain is a medium-depth gray, while the light chain is a lighter gray. Further analysis of the binding interface provided precise information on the key interaction regions between the heavy and light chains and gB. This binding to the DI region significantly distinguishes Fab5 from previously reported antibodies 3A3 and 3A5 (Zhang et al., PNAS, 2023), highlighting Fab5 as a novel EBV neutralizing antibody. The structure of the Fab5-gB protein complex provides crucial molecular biological evidence for the significant gB affinity and high EBV neutralizing activity of antibody Fab5.

Claims

1. A monoclonal antibody against EB virus gB protein or an antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3; The CDR-H1, CDR-H2, and CDR-H3 are amino acid sequences as shown in SEQ ID NO: 4, namely CDR1, CDR2, and CDR3. The light chain variable region includes CDR-L1, CDR-L2 and CDR-L3; The CDR-L1, CDR-L2, and CDR-L3 are amino acid sequences as shown in SEQ ID NO: 17, namely CDR1, CDR2, and CDR3.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that: The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 are shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 18, and SEQ ID NO: 19, respectively. The sequence of CDR-L2 is DTS, and the CDR is defined according to the IMGT definition scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 19, respectively. The CDRs are defined using the Kabat scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 19, respectively. The CDRs are defined according to the Chothia scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively. The CDR is defined using the Contact definition scheme.

3. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that: The monoclonal antibody or its antigen-binding fragment comprises at least one of the following: full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, and multispecific antibody.

4. A recombinant protein comprising: a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 3; and optionally a tag sequence for assisting expression and / or purification.

5. A biological material relating to the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3, or the recombinant protein as described in claim 4, wherein the biological material comprises at least one of h1) to h16): h1) A nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3, or the recombinant protein as described in claim 4; h2) contains an expression cassette containing the nucleic acid molecule described in h1); h3) A carrier containing the nucleic acid molecule described in h1); h4) A carrier containing the expression box described in h2); h5) A transgenic cell line containing the nucleic acid molecules described in h1); h6) Transgenic cell lines containing the expression cassette described in h2); h7) A transgenic cell line containing the vector described in h3); h8) A transgenic cell line containing the vector described in h4); h9) Microorganisms containing the nucleic acid molecules described in h1); h10) contains microorganisms containing the expression cassette described in h2); h11) contains microorganisms containing the carrier described in h3); h12) contains microorganisms containing the carrier described in h4); h13) is a virus containing the nucleic acid molecule described in h1); h14) contains the expression cassette described in h2); h15) contains a virus with the vector described in h3); h16) contains a virus with the vector described in h4).

6. A method for preparing the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3, or the recombinant protein as described in claim 4, comprising the following steps: It is obtained by expression and purification using the biological material described in claim 5.

7. A conjugate comprising: at least one of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3 and the recombinant protein as described in claim 4; And a coupling portion, the coupling portion comprising at least one of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

8. The application of at least one of (1) to (4) in the preparation of the product; (1) The monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3; (2) The recombinant protein according to claim 4; (3) The biomaterial as described in claim 5; (4) The coupling compound according to claim 7; The product includes at least one of the following: drugs, vaccines, reagents, test strips, test kits, and test chips.

9. A drug comprising at least one of (l1) to (l4): (l1) The monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3; (l2) The recombinant protein according to claim 4; (l3) The biomaterial according to claim 5; (l4) The coupling compound according to claim 7; Preferably, the drug further comprises a pharmaceutically acceptable carrier.

10. A kit for detecting EB virus, the kit comprising at least one of (l1) to (l4) and an adjuvant: (l1) The monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3; (l2) the recombinant protein according to claim 4; (l3) the biomaterial according to claim 5; (l4) the conjugate according to claim 7.

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