Monoclonal antibody Fab5 for identifying EB virus gB protein and application thereof

Abstract

The invention belongs to the technical field of antibodies, and discloses a monoclonal antibody Fab5 for recognizing EB virus gB protein and application of the monoclonal antibody Fab5. The monoclonal antibody or the antigen binding fragment thereof has high affinity to gB protein and can significantly inhibit EBV infection to epithelial cells and B cells, and epitopes of Fab5 and gB binding are defined through structural analysis. The antibody can be used for detecting the existence or level of gB protein in a sample, detecting EB virus, diagnosing diseases caused by EB virus infection, preventing EB virus infection and / or treating and / or preventing diseases caused by EB virus infection.

Description

Technical Field

[0001] The invention belongs to the technical field of antibodies, and in particular relates to a monoclonal antibody Fab5 for recognizing Epstein-Barr virus gB protein and an application thereof. Background Art

[0002] Epstein-Barr virus (EBV), also known as human herpesvirus type 4, was first established in 1964 by Epstein and Barr from Burkitt lymphoma cells through in vitro suspension culture. It is a DNA oncogenic virus of the genus Lymphotropic virus of the γ subfamily. EB virus particles are composed of four structural components: core protein, capsid, capsid, and envelope. The core is the core protein wrapped with DNA.

[0003] EBV is most commonly latently infected, with more than 90% of EBV-infected people being latently infected for life. The virus is activated under certain conditions and causes cancer. Current studies have found that human EBV is closely related to malignant tumors such as nasopharyngeal carcinoma, infectious mononucleosis, Hodgkin's and non-Hodgkin's lymphomas, Burkitt's lymphomas, and epithelial cell cancers including gastric cancer, as well as autoimmune diseases such as multiple sclerosis. The initial site of EBV replication is the oropharynx, where it grows and multiplies in B lymphocytes and oral epithelial cells, and then infects B lymphocytes. These cells enter the blood circulation in large numbers and cause systemic infection. When the body's immune function is low, latent EBV is activated to form a recurrent infection.

[0004] There is currently no effective vaccine for EBV, and there is also a lack of specific treatment for diseases caused by EBV infection. Infectious mononucleosis is mostly treated with antiviral drugs such as acyclovir. Although these drugs can relieve symptoms to a certain extent, they cannot eliminate the EBV in B lymphocytes and throat epithelium. The treatment of EBV-related tumors is mainly chemotherapy and radiotherapy, but the efficacy is poor for patients with metastasis or recurrence.

[0005] Monoclonal antibodies can be mass-produced, and their high affinity and high specificity for antigen binding greatly reduce adverse reactions during clinical application. These antibody molecules can also be modified to increase their antiviral efficacy. Antibodies have become a very promising means for the treatment of infectious diseases with their specificity and flexibility of use, but there is still no monoclonal antibody against EBV envelope glycoprotein gB on the market. Therefore, the development of anti-EBV monoclonal antibodies will provide more effective prevention and treatment means for EBV infection-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] The second aspect of the present invention aims to provide a recombinant protein.

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

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

[0010] The fifth aspect of the present invention aims to provide a conjugate.

[0011] The purpose of the sixth aspect of the present invention is 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 a product.

[0012] The seventh aspect of the present invention aims to provide a medicine.

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

[0014] In order to achieve the above object, the technical solution adopted by the present invention is:

[0015] The first aspect of the present invention provides a monoclonal antibody or an antigen-binding fragment thereof against Epstein-Barr virus gB, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region;

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

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

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

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

[0020] Preferably, the amino acid sequences of the 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 amino acid sequence of the CDR-L2 is: DTS, and the CDR is defined according to 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 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 CDRs are defined according to 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 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 CDRs are 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 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 CDRs are defined according to 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 in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 4 and which has the same function as the protein shown in SEQ ID NO: 4; or

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

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

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

[0030] b2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 17 and which has the same function as the protein shown in SEQ ID NO: 17; or

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

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

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

[0034] The light chain also comprises a light chain constant region.

[0035] Preferably, the amino acid sequence of the heavy chain constant region comprises:

[0036] c1) an amino acid sequence consisting of amino acids 138 to 467 of SEQ ID NO::3; or

[0037] c2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in c1) and which has the same function as the protein of the amino acid sequence described in c1); or

[0038] c3) An amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in c1) and has the same function as the protein of the amino acid sequence described in c1).

[0039] Preferably, the amino acid sequence of the light chain constant region comprises:

[0040] d1) an amino acid sequence consisting of amino acids 127 to 232 of SEQ ID NO: 16; or

[0041] d2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in d1) and which has the same function as the protein of the amino acid sequence described in d1); or

[0042] d3) An amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in d1) and has the same function as the protein of the amino acid sequence described in d1).

[0043] Preferably, the heavy chain further comprises a heavy chain signal peptide; and / or

[0044] The light chain also comprises a light chain signal peptide.

[0045] Preferably, the amino acid sequence of the heavy chain signal peptide comprises:

[0046] e1) an amino acid sequence consisting of amino acids 1 to 19 in SEQ ID NO: 3; or

[0047] e2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in e1) and which has the same function as the protein of the amino acid sequence described in e1); or

[0048] e3) An amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in e1) and has the same function as the protein of the amino acid sequence described in e1).

[0049] Preferably, the amino acid sequence of the light chain signal peptide comprises:

[0050] f1) an amino acid sequence consisting of amino acids 1 to 19 of SEQ ID NO: 16; or

[0051] f2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in f1) and which has the same function as the protein of the amino acid sequence described in f1); or

[0052] f3) is an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in f1) and has the same function as the protein of the amino acid sequence described in f1).

[0053] Preferably, the amino acid sequence of the EB protein gB comprises:

[0054] g1) an amino acid sequence consisting of amino acids 25 to 685 of SEQ ID NO: 1; or

[0055] g2) an amino acid sequence obtained by replacing and / or deleting and / or adding one or more amino acids to the amino acid sequence described in g1) and having the same function as the protein of the amino acid sequence described in g1); or

[0056] g3) An amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in g1) and has the same function as the protein of the amino acid sequence described in g1).

[0057] A second aspect of the present invention provides a recombinant protein comprising: the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and

[0058] Optional tag sequence to facilitate expression and / or purification.

[0059] Preferably, the tag sequence is selected from at least one of the following groups: a His tag, a GGGS sequence, a FLAG tag; further a His tag; and further a 6×His tag.

[0060] The third aspect of the present invention provides a biological material related to the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention, or the recombinant protein of the second aspect of the present invention, wherein the biological material comprises at least one of h1) to h16):

[0061] h1) a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of the first aspect, or the recombinant protein of the second aspect of the present invention;

[0062] h2) an expression cassette comprising the nucleic acid molecule described in h1);

[0063] h3) a vector comprising the nucleic acid molecule described in h1);

[0064] h4) a vector comprising the expression cassette described in h2);

[0065] h5) a transgenic cell line comprising the nucleic acid molecule described in h1);

[0066] h6) a transgenic cell line comprising the expression cassette described in h2);

[0067] h7) a transgenic cell line comprising the vector described in h3);

[0068] h8) a transgenic cell line comprising the vector described in h4);

[0069] h9) a microorganism comprising the nucleic acid molecule described in h1);

[0070] h10) A microorganism comprising the expression cassette described in h2);

[0071] h11) a microorganism comprising the vector described in h3);

[0072] h12) a microorganism comprising the vector described in h4);

[0073] h13) A virus comprising the nucleic acid molecule described in h1);

[0074] h14) A virus comprising the expression cassette described in h2);

[0075] h15) A virus comprising the vector described in h3);

[0076] h16) A virus comprising the vector described in h4).

[0077] Preferably, the transgenic cell line comprises no reproductive material.

[0078] Preferably, the nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain of the monoclonal antibody or antigen-binding fragment thereof described in the first aspect of the present invention and a nucleic acid molecule encoding the light chain of the monoclonal antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0079] The fourth aspect of the present invention provides a method for preparing the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention or the recombinant protein of the second aspect of the present invention, which is obtained by expression and purification using the biological material described in the third aspect of the present invention.

[0080] A fifth aspect of the present invention provides a conjugate comprising: at least one of the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention and the recombinant protein according to the second aspect of the present invention;

[0081] and a coupling portion, wherein the coupling portion comprises at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

[0082] Preferably, the detectable marker is selected from a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or any combination thereof.

[0083] Preferably, the conjugate is selected from: fluorescent substances, chemiluminescent markers, colored substances, radioactive isotopes, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activated enzymes, chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0084] The sixth aspect of the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the invention, 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;

[0085] The product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip.

[0086] Preferably, the drug has at least one of the functions i1) to i2):

[0087] i1) Prevent EB virus infection;

[0088] i2) Treating and / or preventing diseases caused by EB virus infection.

[0089] Preferably, the reagent, detection plate, detection chip or kit has at least one of the functions j1) to j3):

[0090] j1) detecting the presence or level of gB protein in the sample;

[0091] j2) Detection of Epstein-Barr virus;

[0092] j3) Diagnosis of diseases caused by EB virus infection.

[0093] Preferably, the disease comprises at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T cell lymphoma, lymphoproliferative disease, and infectious mononucleosis.

[0094] Preferably, the medicament comprises pharmaceutically acceptable excipients.

[0095] Preferably, the medicament comprises a vaccine.

[0096] Preferably, the vaccine comprises a pharmaceutically acceptable adjuvant.

[0097] A seventh aspect of the present invention provides a product, wherein the product comprises at least one of k1) to k3):

[0098] k1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0099] k2) the recombinant protein according to the second aspect of the present invention;

[0100] k3) The conjugate according to the fifth aspect of the present invention;

[0101] The product comprises at least one of a reagent, a detection plate, a test kit, and a detection chip.

[0102] Preferably, the product has at least one of the functions j1) to j3):

[0103] j1) detecting the presence or level of gB protein in the sample;

[0104] j2) Detection of Epstein-Barr virus;

[0105] j3) Diagnosis of diseases caused by EB virus infection.

[0106] Preferably, the disease comprises at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T cell lymphoma, lymphoproliferative disease, and infectious mononucleosis.

[0107] The eighth aspect of the present invention provides a drug, which comprises at least one of 11) to 14):

[0108] l1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0109] 12) The recombinant protein according to the second aspect of the present invention;

[0110] l3) benzene The biomaterial of the third aspect of the invention;

[0111] 14) The conjugate according to the fifth aspect of the present invention.

[0112] Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

[0113] Preferably, the drug has at least one of the functions i1) to i2):

[0114] i1) Prevent EB virus infection;

[0115] i2) Treating and / or preventing diseases caused by EB virus infection.

[0116] Preferably, the disease comprises at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T cell lymphoma, lymphoproliferative disease, and infectious mononucleosis.

[0117] Preferably, the medicament comprises a vaccine.

[0118] Preferably, a vaccine comprises at least one of 11) to 14) and an adjuvant:

[0119] l1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0120] 12) The recombinant protein according to the second aspect of the present invention;

[0121] l3) benzene The biomaterial of the third aspect of the invention;

[0122] 14) The conjugate according to the fifth aspect of the present invention.

[0123] The beneficial effects of the present invention are:

[0124] The present invention provides an anti-EBV gB monoclonal antibody or an antigen-binding fragment thereof. The monoclonal antibody or the antigen-binding fragment thereof has a high affinity for the EBV gB protein, can significantly inhibit the infection of EBV to epithelial cells and B cells, and can be used to detect the presence or level of the gB protein in a sample, detect EB virus, diagnose diseases caused by EB virus infection, prevent EB virus infection and / or treat and / or prevent diseases caused by EB virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 This is the affinity test result of monoclonal antibody Fab5 and gB protein.

[0126] Figure 2 This is a diagram showing the results of monoclonal antibody Fab5 blocking EBV infection of epithelial cells.

[0127] Figure 3 This is a diagram showing the results of monoclonal antibody Fab5 blocking EBV infection of B cells.

[0128] Figure 4 This is the cryo-electron microscopy structure result of the monoclonal antibody Fab5 and gB protein. DETAILED DESCRIPTION

[0129] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0130] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0131] Example 1 Preparation of anti-EB virus gB protein (EBV gB), animal immunization and antibody screening

[0132] 1.1 Expression and purification of EBV gB antigen protein

[0133] 1.1.1 Experimental Materials

[0134] (1) Expression vector: Eukaryotic expression vector: pcDNA3.1(+) (ThermoFisher).

[0135] (2) Expression system: eukaryotic expression system cells HEK293F (ATCC).

[0136] (3) Reagents and consumables: Plasmid midi extraction kit (MN), cell transfection reagent PEI (Polyscience), 293F culture medium (Union), histidine tag protein purification agarose beads (Roche), and other common reagents and consumables were purchased from commercial sources.

[0137] (4) Gene: The gB gene of Epstein-Barr virus (M81 strain) was optimized and synthesized by Nanjing GenScript Biotech Co., Ltd. and inserted into the pcDNA3.1 vector, hereinafter referred to as pcDNA3.1-gB.

[0138] The total length of the gB extracellular segment recombinant protein is 661 amino acid residues (excluding *), of which the bold marks are signal peptides, specifically:

[0139] MPMGSLQPLATLYLLGMLVASCLGQTPEQPAPPATTVQPTATRQQTSFPFRVCELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKIVTNILIYNGHRADSVTNRHEEKFSVESYETDQMDTIYQCYNAVKMTKDGLTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGRVEATYRTRTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNTETFHERADSFHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQHWQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKTMHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELTTPTSSPPSSPSPPAPPAARGSTSAAVLRRRRRNAGNATTPVPPAAPGKSLGTLNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKINPTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSETMCYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNEIHVYNDYHHFKTIELDGIATLQTFISLNTSLIENIDFASLELYSRDEQRASNVFDLEGIFREYNFQAQNIAGLRKDLDNAVSHHHHHH*(SEQ ID NO:1).

[0140] Its DNA expression sequence is a total of 2,071 base pairs, with the signal peptide marked in bold, specifically:

[0141]

[0142] 1.1.2 Protein expression

[0143] Step 1: Transformation and selection of clones

[0144] (1) Add the synthesized plasmid to the frozen-thawed DH5a bacteria and let it stand on ice for 5 minutes.

[0145] (2) Place the bacteria with the plasmid in a shaking metal bath and heat shock at 42°C and 120 rpm for 60-90 seconds.

[0146] (3) Let the bacteria cool on ice for 5 minutes.

[0147] (4) Use a spreading stick to spread the bacteria onto a TB medium plate containing ampicillin resistance and culture overnight.

[0148] (5) The next day, use a sterile pipette to pick a single colony from the surface and transfer it to 500 mL of culture medium. Add 0.1 mg / mL ampicillin and culture at 37°C and 220 rpm for 12-16 hours.

[0149] Step 2: Plasmid extraction

[0150] (1) The cultured bacteria were centrifuged at 4000 rpm and 4°C to discard the culture medium and retain the bacterial precipitate. The plasmid was extracted according to the instructions of the NM midi extraction kit. The following is a brief plasmid extraction process.

[0151] (2) Resuspend the precipitate using the RES resuspension buffer (containing RNAase A) in the NM extraction kit.

[0152] (3) Add LYS lysis buffer and lyse for 5 minutes.

[0153] (4) Add NEU neutralization buffer to neutralize. A precipitate should form and the solution should be clear yellow.

[0154] (5) Use EQU balancing buffer to equilibrate the hollow fiber column. Note that the solution should flow into the fiber column from the top inlet to completely wet the entire fiber sleeve.

[0155] (6) Add the neutralized solution and precipitate together into the equilibrated hollow fiber column.

[0156] (7) Add EQU equilibration buffer to equilibrate the hollow fiber column again so that the solution below is clear, colorless and transparent.

[0157] (8) Discard the middle fiber sleeve and leave the outer adsorption column.

[0158] (9) Add wash buffer to wash the adsorption film under the adsorption column.

[0159] (10) Place a centrifuge tube under the adsorption column for collection, then add an appropriate amount of Elu elution buffer to elute the plasmid, and finally add isopropanol to precipitate the plasmid.

[0160] (11) The precipitated plasmid solution was centrifuged at 4000 rpm for 30 minutes, the supernatant was discarded, and 70% ethanol was added for resuspending and centrifugation was performed again.

[0161] (12) Place the centrifuged solution in a clean bench, discard the supernatant, and resuspend the plasmid with an appropriate amount of sterile double-distilled water. After resuspending the plasmid, it should be stored at -20°C.

[0162] Step 3: Plasmid transfection into 293F cells

[0163] (1) 293F cells should be cultured in a shaking culture medium at 37°C, 120 rpm, and 5% CO2. The density should be maintained at 0.5-5*10 ^6 The density should be between 0.5-5X / mL. If the density exceeds this value, the cells should be cultured in separate bottles or allocated for transfection in a timely manner.

[0164] (2) Take out the plasmid and dilute it with 293 culture medium. The transfection dosage is generally calculated based on 1 mg of plasmid per 1L of 1X cells.

[0165] (3) Take out the dissolved PEI and dilute it with 293 culture medium. Generally, 5 mg PEI is added for every 1 mg plasmid to prepare the transfection system.

[0166] (4) Mix the plasmid diluted with 293 medium and PEI and let it stand for 10-15 minutes.

[0167] (5) Dilute 293F cells to 1X density, then add the transfection mixture, and then return to 37°C, 120 rpm, 5% CO2 for shaking culture for 5-7 days.

[0168] Step 4: Purification of secretory proteins from eukaryotic culture supernatant

[0169] (1) The cultured 293F cells were centrifuged at 8000 rpm and 4°C for 1 hour to collect the supernatant. Since a signal peptide sequence was added to the plasmid, the protein was expressed in the supernatant. The supernatant was used for protein purification.

[0170] (2) Collect the cell supernatant and filter it twice using a 0.22 μm filter to remove impurities.

[0171] (3) Take out the gravity purification column, add 1 mL of His-tag purification agarose beads, and add 0.22 μm filtered double distilled water (the liquid added to the purification column during the purification stage needs to be filtered through a 0.22 μm filter membrane), wash the beads, and then balance the purification column with PBS.

[0172] (4) Use the siphon method to transfer the supernatant to the purification column, so that the supernatant completely passes through the purification column. If repeated recovery is required, the supernatant can be recovered in this step and re-purified.

[0173] (5) Equilibrate the purification column with PBS for three column volumes.

[0174] (6) The purification column was eluted with PBS containing 300 mM imidazole for two column volumes to obtain the eluate.

[0175] (7) Samples of the supernatant, equilibrium solution, and eluate were collected for SDS-PAGE to verify their protein expression.

[0176] (8) The eluate was concentrated using an ultrafiltration tube of corresponding size to a volume less than 1 mL. The concentrated eluate was centrifuged at 12,000 g and 4° C. for 5 min to remove the precipitate.

[0177] (9) The concentrated eluate was purified by molecular sieve purification using an Akta purifier equipped with a Superdex200 Increase 10 / 300GL molecular sieve column, and the protein sample at the molecular sieve peak was collected and compared with the previous sample for SDS-PAGE verification.

[0178] 1.2 EBV gB mouse immunization experiment

[0179] 1.2.1 Vaccine configuration

[0180] (1) The gB protein was taken out and frozen and thawed, with gB at 5 μg / mouse. Sterile PBS was used as the solvent for the antigen.

[0181] (2) 100 μg of antigen solution was mixed with 100 μL of complete Freund's adjuvant to form a dose of 200 μL per mouse, and the mixture was placed on a rocking table at 4°C for mixing overnight.

[0182] 1.2.2 Mouse immunization and spleen collection

[0183] (1) The mice were fed with conventional feed and drinking water and were 6 weeks old at the time of immunization. They were of the Balb / C strain.

[0184] (2) Fix the mouse and use a 1 mL syringe to manually shake the vaccine, then draw it up and inject it into the mouse subcutaneously in the abdomen.

[0185] (3) Observe the mouse for 3-5 minutes and release it after it resumes normal activities.

[0186] (4) The vaccination process is 0 weeks-3 weeks-6 weeks-8 weeks, and the spleen collection time is 10 weeks.

[0187] (5) When collecting the spleen of mice, the mice were killed by spinal dislocation according to animal welfare and standard animal experimental procedures, and their blood was collected and their spleens were obtained by dissection.

[0188] 1.3 EBV gB mouse B cell gB-specific cell isolation and cloning

[0189] 1.3.1 Splenic lymphocyte isolation steps:

[0190] (1) Ten weeks after immunization, mice were sacrificed by cervical dislocation, and their peripheral lymph nodes and spleen were isolated and placed in EP tubes.

[0191] (2) Add appropriate amounts of DNase and type IV collagenase to 1640 medium to prepare tissue digestion solution. Add appropriate amounts of digestion solution according to the size of the lymph nodes and spleen.

[0192] (3) Using the pressure plug in a sterile syringe, place the tissue on a 70 μm cell sieve and crush it until the tissue is almost broken and minced. Then, rinse the cell sieve with an appropriate amount of fresh culture medium so that all remaining cells are rinsed into the 50 mL centrifuge tube below.

[0193] (4) Add red blood cell lysis buffer and incubate at room temperature for 10 minutes.

[0194] (5) Centrifuge at 2000 g and 18°C ​​for 5 min.

[0195] (6) Add fresh culture medium and wash twice.

[0196] 1.3.2 B lymphocyte antigen staining and sorting

[0197] (1) Wash the cells with PBS until the number of cells is 1*10^8, transfer to a flow cytometer, centrifuge at 3000g and 18°C ​​for 5 minutes, and discard the supernatant.

[0198] (2) Add 5 μL of Fc receptor blocking antibody solution to 100 μL of PBS, resuspend the cells and incubate on ice for 30 min.

[0199] (3) According to the cell grouping situation, use PBS to prepare the flow cytometry antibody dilution solution. Different dilution gradients can be set for the first use of antibodies. The optimal gradient is selected according to the ratio of antibody to PBS of 1:20, 1:50, 1:100, 1:200, and 1:500. Add 100 μL of diluted flow cytometry antibody cells and cell death and viability dye to 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.

[0200] (4) Wash the cells with PBS and centrifuge at 3000 g and 4°C for 5 minutes. Repeat 1 to 2 times and discard the supernatant.

[0201] (5) Resuspend the cells in PBS into a flow cytometry tube and perform flow cytometry sorting on the cells using a BD Rhaspody single cell sorter.

[0202] (6) Add IgG + B220 + The 0.2% cells with the strongest gB positive signal were sorted into a 96-well plate.

[0203] 1.3.3B cell sequence cloning and expansion

[0204] (1) Using universal cloning primers for mouse IgG heavy and light chains, PCR was performed on B cells in 96-well plates to obtain the heavy and light chain sequences corresponding to a single B cell.

[0205] (2) After connecting the variable region of the heavy chain of the antibody to the CMV fragment upstream and the constant region of mouse IgG1 downstream, the fragment of the complete heavy chain can be expressed; and after connecting the variable region of the light chain of the antibody to the CMV fragment upstream and the constant region of the light chain κ / λ downstream, the fragment of the complete light chain can be expressed. The expression of the antibody can be achieved by co-transfecting the plasmids carrying the full-length sequences of the heavy and light chains of the above antibodies into 293F cells, and the antibody can be purified by using protein A beads. The expression of a total of 8 antibodies, Fab1-Fab8, was obtained by this method.

[0206] (3) The obtained antibodies were subjected to gB affinity determination using ELISA, and the Fab5 antibody with the highest affinity was screened for further verification.

[0207] Example 2 Preparation of monoclonal antibodies (mAbs) against Epstein-Barr virus gB protein (EBV gB)

[0208] After connecting the upstream of the antibody heavy chain variable region with the CMV fragment and the downstream with the constant region of human IgG1, the complete heavy chain fragment can be expressed; and after connecting the upstream of the antibody light chain variable region with the CMV fragment and the downstream with the constant region of the light chain κ / λ, the complete light chain fragment can be expressed. The plasmids carrying the full-length sequences of the heavy and light chains of the above antibodies can be co-transfected into 293F cells to achieve antibody expression, and the antibody can be purified using protein A beads.

[0209] The Fab5 full-length heavy chain has a total of 467 amino acid residues (excluding *), specifically:

[0210] QVQLQQPGAELVRPGASVKLSCKAS INWVKQRPGQGLEWIGN NYNQKFKDKATLTVDKSSSTAYMLLSSPPTSEDSAVYYC WGQGTTLTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO: 3);

[0211] Among them, the underlined part in the sequence is the amino acid sequence of the heavy chain variable region (SEQ ID NO: 4). The italic parts are the amino acid sequences of the three complementary regions CDR-H1 (SEQ ID NO: 5), CDR-H2 (SEQ ID NO: 6) and CDR-H3 (SEQ ID NO: 7) in the heavy chain variable region (IMGT definition scheme). The bold part is the signal peptide. The amino acids 138 to 467 are the heavy chain constant region. * indicates the stop codon.

[0212] CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region using other CDR definition schemes are shown in Table 11.

[0213] Table 11 CDR-H1, CDR-H2, CDR-H3 in the heavy chain variable region with other CDR definition schemes

[0214]

[0215] The Fab5 full-length light chain has a total of 232 amino acid residues (excluding *), specifically:

[0216] QIVLTQSPAIMSASPGEKVTMTCSAS MYWYQQKPGSSPRLLIY NLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYC FGGGT LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(SEQ ID NO: 16);

[0217] Among them, the underlined part in the sequence is the amino acid sequence of the light chain variable region (SEQ ID NO: 17). The underlined and bold parts are the amino acid sequences of the three complementary determining regions CDR-L1 (SEQ ID NO: 18), CDR-L2 (DTS) and CDR-L3 (SEQ ID NO: 19) in the light chain variable region (IMGT definition scheme). The bold part in bold is the signal peptide. The amino acids 127 to 232 are the light chain constant region. * indicates a stop codon.

[0218] CDR-H1, CDR-H2, and CDR-H3 in the light chain variable region using other CDR definition schemes are shown in Table 12.

[0219] Table 12 CDR-H1, CDR-H2, CDR-H3 in the light chain variable region with other CDR definition schemes

[0220]

[0221] Example 3 Affinity determination of Fab5

[0222] The affinity of antibody Fab5 was determined by biofilm interferometry (BLI).

[0223] BLI can be performed according to conventional methods in the art. In this embodiment, the specific operation is as follows: a biosensor (Sartorius, Germany) SA probe) is immersed in a buffer solution (a mixture of KB buffer, 0.1wt% BSA and 0.02v / v% Tween20) for equilibrium. It is then taken out and immersed in a solution containing 5μg / mL gB-Biotin (biotin-labeled gB protein). The gB antigen in the solution will bind to the surface of the SA (streptavidin) biological probe, increasing the thickness of the surface film. The biosensor with a solidified antigen of known concentration is then immersed in a buffer solution as a baseline. The solidified biosensor with a known antigen concentration is immersed in a sample solution containing 31.3-500nM Fab5 antibodies for about 120 seconds. Since the specific binding between antigen and antibody will lead to an increase in the thickness of the film layer, the biosensor bound to the Fab5 antibody is immersed in a buffer solution for dissociation for about 180 seconds. The antibody to be tested (Fab5 antibody) will fall off the surface of the biosensor, resulting in a decrease in the thickness of the film layer. The kinetic constant of the sample to be tested (Fab5 antibody) can be obtained by real-time monitoring of the biosensor biofilm thickness during the experiment. The results are shown in Figure 1 Shown: KD(M) of Fab5 antibody <10 -12 M; This indicates that the Fab5 antibody has a very high affinity with the gB antigen.

[0224] Example 4 Neutralizing Activity Determination of Fab5 Antibody

[0225] (1) Preparation of EBV virus:

[0226] 1) CNE2 cells infected with EBV-GFP (the virus has been disclosed in the literature: An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tr opic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus) were cultured in RPMI1640 + 5v / v% FBS at 37 degrees incubator (5v / v% CO2). When the cells grew to 90% density (10 cm dish), TPA with a final concentration of 20ng / mL and NaB (sodium butyrate) with a final concentration of 2.5mM were added for induction, and the medium was changed after 12 hours.

[0227] 2) 48-72 hours after the medium is replaced, the culture supernatant is collected to separate and purify the virus. The supernatant is directly aspirated, centrifuged and filtered with a 0.45 μm filter. After concentration, the supernatant is resuspended in serum-free RPMI1640 and immediately used for infection or stored at -80 degrees.

[0228] (2) Neutralization activity assay of Fab5 monoclonal antibody in epithelial cells

[0229] 1) 1x10 cells / well were plated in each well of a 96-well plate. 6 293T epithelial cells were cultured in each well and 100 μL of DMEM medium containing 10% FBS was added to each well.

[0230] 2) On the second day, take the Fab5 monoclonal antibody in the above example and adjust the concentration to 2 mg / mL. Add 60uL DMEM medium to each well of a new 96-well plate, and add 120uL 12.5μg / mL Fab5 antibody diluted with DMEM to the first well (the first well does not contain RPMI1640 medium).

[0231] 3) After 2-fold gradient dilution (gradient dilution will aspirate 60 μL from the first well and add it to the second well, and so on, aspirate 60 μL from the last well and discard it, and the final volume of each well is 60uL); add 60 μL of virus diluent to each well (the virus is diluted with DMEM medium, the titer is about 4*10 6 / mL), incubated at 37 degrees for 2 hours, and then added to the 293T cells plated the day before. Placed in a 37-degree incubator for 48 hours before detection.

[0232] 4) After digesting 293T cells with trypsin, prepare cell suspension, detect infection rate by flow cytometry, and calculate the inhibition rate (neutralization efficiency, %) of antibody in 293T epithelial cell infection model by detecting the reduction ratio of GFP-positive cells in antibody-treated group compared with infection control group (adding equal volume of DMEM). IC value of Fab5 monoclonal antibody was calculated and plotted using Prism 50 .

[0233] The results are as follows Figure 2 Shown: IC of monoclonal antibody Fab5 in epithelial cell infection model 50 is 0.30μg / mL; monoclonal antibody Fab5 can significantly inhibit EBV infection of epithelial cells.

[0234] Example 5 Detection of B cell neutralizing activity of Fab5 monoclonal antibody

[0235] 1) Take the Fab5 monoclonal antibody in the above example and adjust the concentration to 2 mg / mL. Add 60uL RPMI1640 medium to each well of a new 96-well plate, and add 90uL 100μg / mL Fab5 antibody diluted with RPMI1640 to the first well (the first well does not contain RPMI1640 medium).

[0236] 2) After 3-fold gradient dilution (gradient dilution will aspirate 30 μL from the first well and add it to the second well, and so on, aspirate 30 μL from the last well and discard it, and the final volume of each well is 60 μL); add 60 μL of virus dilution solution to each well (the virus is diluted with DMEM medium, the titer is about 4*10 6 / mL), and then 1*10 6 Raji cells were placed in a 37°C incubator for 48 hours before testing.

[0237] 3) Raji cells were aspirated to prepare cell suspension, and 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 ratio of the number of GFP-positive cells in the antibody-treated group compared with the infection control group (adding an equal volume of RPMI1640). The IC value of the Fab5 monoclonal antibody was calculated and drawn using Prism. 50 .

[0238] The results are as follows Figure 3 Shown: IC of monoclonal antibody Fab5 in B cell infection model 50 The concentration of monoclonal antibody Fab5 was 0.9 μg / mL, which could significantly inhibit the infection of EBV to B cells.

[0239] Example 6 Cryo-EM structure of monoclonal antibody Fab5 and gB protein

[0240] Brief steps for cryo-electron microscopy photography:

[0241] (1) 1 mg / mL of Fab5 / gB composite particle sample at a molar ratio of 1:1 (M / M) was dropped onto a glow-irradiated Quantifoil Cu R1.2 / 1.3 300-mesh copper mesh.

[0242] (2) Use an automatic freezing sample preparation robot Vitrobot Mark IV for freezing sample preparation, with a freezing time of 7 seconds and a humidity of 100%.

[0243] (3) The samples were observed using a 300 kV Titan Krios transmission electron microscope. If the sample preparation failed, the samples were frozen again.

[0244] (4) Qualified samples are photographed using K2 Summit direct detection and their original photos are recorded.

[0245] Brief steps for single particle analysis by cryo-EM:

[0246] (1) Use motionCor2 to align the camera data.

[0247] (2) Use PatchCTF to calculate the CTF value of frozen data.

[0248] (3) Use Blob Picker to automatically pick particles without parameters to generate 2D classification images.

[0249] (4) Based on the best set in the comparison photograph, reference density is further selected to adjust the particles and perform 2D classification.

[0250] (5) Perform homologous reconstruction on the best 2D Class set particles and determine the resolution using the gsFSC standard.

[0251] The results are as follows Figure 4 The structure shown shows that Fab5 binds to the DI domain of the gB protein, where the Fab5 heavy chain is pink and the light chain is gray. Through further binding interface analysis, accurate information on the key interaction regions between the light and heavy chains and gB was obtained. At the same time, the feature of Fab5 binding to the DI region is significantly different from the previously reported antibodies 3A3 and 3A5 (Zhang et al. PNAS, 2023), highlighting that Fab5 is a brand-new EB virus neutralizing antibody. The structure of the Fab5 and gB protein complex provides important molecular biological evidence for the antibody Fab5's significant gB affinity and high EB virus neutralization.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof against Epstein-Barr virus gB protein, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3; The CDR-H1, CDR-H2 and CDR-H3 are CDR1, CDR2 and CDR3 with amino acid sequences as shown in SEQ ID NO: 4; The light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3; The CDR-L1, CDR-L2 and CDR-L3 are CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO:

17.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequences of the 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 the CDR-L2 is DTS, and the CDRs are 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, and the CDRs are defined according to the Kabat 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: 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 CDRs are defined according to the Chothia 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: 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 CDRs are defined according to the Contact definition scheme.

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

4. A recombinant protein comprising: the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; and an optional tag sequence for facilitating expression and / or purification.

5. A biological material related to the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4, wherein the biological material comprises at least one of h1) to h16): h1) a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4; h2) an expression cassette comprising the nucleic acid molecule described in h1); h3) a vector comprising the nucleic acid molecule described in h1); h4) a vector comprising the expression cassette described in h2); h5) a transgenic cell line comprising the nucleic acid molecule described in h1); h6) a transgenic cell line comprising the expression cassette described in h2); h7) a transgenic cell line comprising the vector described in h3); h8) a transgenic cell line comprising the vector described in h4); h9) a microorganism comprising the nucleic acid molecule described in h1); h10) A microorganism comprising the expression cassette described in h2); h11) a microorganism comprising the vector described in h3); h12) a microorganism comprising the vector described in h4); h13) A virus comprising the nucleic acid molecule described in h1); h14) A virus comprising the expression cassette described in h2); h15) A virus comprising the vector described in h3); h16) A virus comprising the vector described in h4).

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

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

8. Use of at least one of (1) to (4) in the preparation of a product; (1) The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (2) The recombinant protein according to claim 4; (3) The biomaterial according to claim 5; (4) The conjugate according to claim 7; The product comprises at least one of a drug, a vaccine, a reagent, a test plate, a test kit, and a test chip.

9. A drug comprising at least one of (11) to (14): (l1) The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (12) The recombinant protein according to claim 4; (13) The biomaterial according to claim 5; (14) The conjugate according to claim 7; Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

10. A kit for detecting Epstein-Barr virus, the kit comprising at least one of (l1) to (l4) and an adjuvant: (l1) the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (12) The recombinant protein according to claim 4; (13) The biomaterial according to claim 5; (14) The conjugate according to claim 7.

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