A monoclonal antibody for recognizing Epstein-Barr virus gp42 protein and its application

Abstract

The present invention belongs to the field of antibody technology and discloses a monoclonal antibody that recognizes Epstein-Barr virus gp42 protein and its application. The monoclonal antibody or its antigen-binding fragment has good binding activity with gp42 protein, has high affinity for gp42 protein, can specifically recognize natural gp42 protein expressed on the cell surface, can significantly inhibit EBV infection of B cells, and effectively inhibits cell membrane fusion. It can be used to detect the presence or level of gp42 protein in a sample, detect Epstein-Barr virus, diagnose diseases caused by Epstein-Barr virus infection, prevent Epstein-Barr virus infection, and / or treat and / or prevent diseases caused by Epstein-Barr virus infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibodies, and in particular relates to a monoclonal antibody for recognizing Epstein-Barr virus gp42 protein and an application thereof. Background Art

[0002] Epstein-Barr virus (EBV) was first successfully cultured and isolated from Burkitt lymphoma cells by Epstein and Barr in 1964. EBV belongs to the gamma herpesvirus subtype and is the first human oncogenic virus discovered. EBV infection is highly prevalent in the human population, with reports suggesting that over 95% of adults worldwide carry EBV. In children and adolescents, EBV infection often causes infectious mononucleosis. Latent EBV infection is associated with the development of various human lymphoid and epithelial tumors, such as Hodgkin lymphoma, Burkitt lymphoma, and NK / T cell lymphomas. Epithelial tumors include nasopharyngeal carcinoma and approximately 10% of gastric cancers. The risk of EBV-related tumors is significantly increased in immunosuppressed individuals, such as organ transplant recipients and those with HIV. Globally, approximately 200,000 new cases of EBV-related cancers are reported annually. The U.S. National Institutes of Health (NIH) officially listed EBV on its 14th list of carcinogens in 2016.

[0003] Currently, there is no effective vaccine against EBV, and specific treatments for diseases caused by EBV infection are lacking. Infectious mononucleosis is often treated with antiviral drugs such as acyclovir, which can alleviate symptoms to some extent but cannot eliminate EBV from B lymphocytes or the epithelium of the throat. Treatments for EBV-associated tumors primarily rely on chemotherapy and radiotherapy, but these are less effective for patients with recurrence or metastasis.

[0004] Monoclonal antibodies can be mass-produced, and their high affinity and specificity for binding to antigens significantly reduce adverse reactions during clinical use. Furthermore, antibody molecules can be modified to enhance their antiviral efficacy. Due to their specificity and flexibility, antibodies are a promising tool for treating infectious diseases. However, to date, there are no commercially available monoclonal antibodies targeting the EBV envelope glycoprotein. Therefore, the development of anti-EBV monoclonal antibodies would provide more effective prevention and treatment options for EBV-related diseases. Summary of the Invention

[0005] The first aspect of the present invention aims to provide a monoclonal antibody or an antigen-binding fragment thereof.

[0006] The second aspect of the present invention aims to provide a recombinant protein.

[0007] The third aspect of the present invention aims to provide biological materials related to the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the recombinant protein according to the second aspect.

[0008] The fourth aspect of the present invention aims to provide a conjugate comprising the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0009] The purpose of the fifth 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 fourth aspect in the preparation of products.

[0010] The sixth aspect of the present invention aims to provide a kit comprising the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention and / or the conjugate according to the sixth aspect of the present invention.

[0011] The seventh aspect of the present invention aims to provide a drug comprising the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention and / or the conjugate according to the sixth aspect of the present invention.

[0012] The eighth aspect of the present invention aims to provide a method for preparing the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] In a first aspect of the present invention, a monoclonal antibody or an antigen-binding fragment thereof is provided, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region;

[0015] The heavy chain variable region comprises CDR1, CDR2, and CDR3;

[0016] The amino acid sequence of CDR1 of the heavy chain variable region is:

[0017] a1) GFSLSTYA (SEQ ID NO. 2); or

[0018] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO. 2 and having the same function as the protein shown in SEQ ID NO. 2; or

[0019] a3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% identical to SEQ ID NO. 2 and has the same function as the protein represented by SEQ ID NO. 2;

[0020] The amino acid sequence of CDR2 of the heavy chain variable region is:

[0021] a1) ISASDTT (SEQ ID NO. 3); or

[0022] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO. 3 and having the same function as the protein shown in SEQ ID NO. 3; or

[0023] a3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% identical to SEQ ID NO. 3 and has the same function as the protein represented by SEQ ID NO. 3;

[0024] The amino acid sequence of CDR3 of the heavy chain variable region is:

[0025] a1)ARFSAYESNRDYFDTFDP(SEQ ID NO.4); or

[0026] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO. 4 and having the same function as the protein shown in SEQ ID NO. 4; or

[0027] a3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% identical to SEQ ID NO. 4 and has the same function as the protein represented by SEQ ID NO. 4;

[0028] The light chain variable region comprises CDR1, CDR2, and CDR3;

[0029] The amino acid sequence of CDR1 of the light chain variable region is:

[0030] a1) QNIYRD (SEQ ID NO. 5); or

[0031] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO. 5 and having the same function as the protein shown in SEQ ID NO. 5; or

[0032] a3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% identical to SEQ ID NO. 5 and has the same function as the protein represented by SEQ ID NO. 5;

[0033] The amino acid sequence of CDR2 of the light chain variable region is:

[0034] a1) GAS; or

[0035] a2) GAS is subjected to one or more amino acid substitutions and / or deletions and / or additions and has an amino acid sequence having the same function as the protein having the sequence of GAS; or

[0036] a3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% homologous to GAS and has the same function as a protein having the sequence of GAS;

[0037] The amino acid sequence of CDR3 of the light chain variable region is:

[0038] a1) QCSAYGSGYAAHA (SEQ ID NO. 7); or

[0039] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO. 7 and having the same function as the protein shown in SEQ ID NO. 7; or

[0040] a3) an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology to SEQ ID NO. 7 and has the same function as the protein shown in SEQ ID NO. 7.

[0041] Preferably, the amino acid sequence of the heavy chain variable region comprises:

[0042] a1)QPVEESGGRLVTPGTPLTLTCTVSGFSLSTYAMSWVRQAPGKGLEWIGTISASDTTYFANWTKGRFTISKASTTVDLKITSPTTEDTATFFCARFSAYESNRDYFDTFDPWGPGTLVTVSS(SEQ ID NO.8); or

[0043] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO. 8 and having the same function as the protein shown in SEQ ID NO. 8; or

[0044] a3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% identical to SEQ ID NO. 8 and has the same function as the protein represented by SEQ ID NO. 8;

[0045] The amino acid sequence of the light chain variable region comprises:

[0046] a1)DVVMTQTPSPVSAAVGGTVTIKCQASQNIYRDLAWYQQNPGQPPKLLIYGASNLASGVPSRFSGSGSGTEYILTISDLECADAATYYCQCSAYGSGYAAHAFGGGTKVDIK(SEQ ID NO.9); or

[0047] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO. 9 and having the same function as the protein shown in SEQ ID NO. 9; or

[0048] a3) an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology to SEQ ID NO. 9 and has the same function as the protein shown in SEQ ID NO. 9.

[0049] 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, bispecific antibody, and multispecific antibody.

[0050] Preferably, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region.

[0051] Preferably, the amino acid sequence of the heavy chain constant region comprises:

[0052] a1)GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRT ARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO.10); or

[0053] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO. 10 and having the same function as the protein shown in SEQ ID NO. 10; or

[0054] a3) an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology to SEQ ID NO. 10 and has the same function as the protein represented by SEQ ID NO. 10.

[0055] Preferably, the amino acid sequence of the light chain constant region comprises:

[0056] a1) GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO.11); or

[0057] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO. 11 and having the same function as the protein shown in SEQ ID NO. 11; or

[0058] a3) an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology to SEQ ID NO. 11 and has the same function as the protein shown in SEQ ID NO. 11.

[0059] Preferably, the monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that recognizes and / or targets gp42 protein.

[0060] Preferably, the monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that recognizes and / or targets the gp42 protein of Epstein-Barr virus.

[0061] Preferably, the monoclonal antibody or antigen-binding fragment thereof recognizes aa 109 to aa 114 and / or aa 145 to aa 151 of the gp42 protein.

[0062] Preferably, the key sites of the gp42 protein recognized by the monoclonal antibody or its antigen-binding fragment are amino acids 109, 111, 146, 147, 152, 155, and 222 of the gp42 protein.

[0063] 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

[0064] Optional tag sequence to facilitate expression and / or purification.

[0065] Preferably, the tag sequence is at least one selected from the following group: 6×His tag, GGGS sequence, FLAG tag.

[0066] The third aspect of the present invention provides a biomaterial related to the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the recombinant protein according to the second aspect of the present invention, wherein the biomaterial comprises at least one of b1) to b8):

[0067] b1) a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the recombinant protein according to the second aspect;

[0068] b2) an expression cassette comprising the nucleic acid molecule described in b1);

[0069] b3) a vector comprising the nucleic acid molecule described in b1);

[0070] b4) a vector comprising the expression cassette described in b2);

[0071] b5) a transgenic cell line comprising the nucleic acid molecule described in b1);

[0072] b6) a transgenic cell line comprising the expression cassette described in b2);

[0073] b7) a transgenic cell line comprising the vector described in b3);

[0074] b8) A transgenic cell line comprising the vector described in b4).

[0075] Preferably, the transgenic cell line comprises no reproductive material.

[0076] Preferably, the nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention and a nucleic acid molecule encoding the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention;

[0077] The nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention comprises:

[0078] a1) SEQ ID NO. 12; or

[0079] a2) a nucleotide sequence obtained by substituting and / or deleting and / or adding one or more nucleotides of SEQ ID NO. 12 and having the same function as the nucleic acid molecule shown in SEQ ID NO. 12; or

[0080] a3) a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% identical to SEQ ID NO. 12 and has the same function as the nucleic acid molecule represented by SEQ ID NO. 12;

[0081] The nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention comprises:

[0082] a1) SEQ ID NO. 13; or

[0083] a2) a nucleotide sequence obtained by substituting and / or deleting and / or adding one or more nucleotides of SEQ ID NO. 13 and having the same function as the nucleic acid molecule shown in SEQ ID NO. 13; or

[0084] a3) a nucleotide sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology to SEQ ID NO. 13 and has the same function as the nucleic acid molecule shown in SEQ ID NO. 13.

[0085] Preferably, the nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention further comprises a nucleic acid molecule encoding the heavy chain constant region of the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention and a nucleic acid molecule encoding the light chain constant region of the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0086] The nucleotide sequence of the nucleic acid molecule encoding the heavy chain constant region of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention comprises:

[0087] a1) SEQ ID NO. 14; or

[0088] a2) a nucleotide sequence obtained by substituting and / or deleting and / or adding one or more nucleotides of SEQ ID NO. 14 and having the same function as the nucleic acid molecule shown in SEQ ID NO. 14; or

[0089] a3) a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% identical to SEQ ID NO. 14 and has the same function as the nucleic acid molecule represented by SEQ ID NO. 14;

[0090] The nucleotide sequence of the nucleic acid molecule encoding the light chain constant region of the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention comprises:

[0091] a1) SEQ ID NO. 6; or

[0092] a2) a nucleotide sequence obtained by substituting and / or deleting and / or adding one or more nucleotides in SEQ ID NO. 6 and having the same function as the nucleic acid molecule shown in SEQ ID NO. 6; or

[0093] a3) a nucleotide sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology to SEQ ID NO. 6 and has the same function as the nucleic acid molecule shown in SEQ ID NO. 6.

[0094] The fourth aspect of the present invention provides a conjugate comprising: the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention and at least one of the recombinant protein according to the second aspect of the present invention;

[0095] 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.

[0096] Preferably, the detectable label is selected from radioisotopes, fluorescent substances, chemiluminescent substances, colored substances, or any combination thereof.

[0097] Preferably, the conjugate is selected from the group consisting of 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.

[0098] The fifth aspect of the present invention provides 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 fourth aspect in preparing a product;

[0099] The product comprises at least one of a drug, a reagent, a test plate, and a test kit.

[0100] Preferably, the drug has at least one of the functions c1) to c2):

[0101] c1) Prevention of EBV infection;

[0102] c2) Treating and / or preventing diseases caused by EB virus infection.

[0103] Preferably, the reagent, detection plate or kit has at least one of the functions d1) to d3):

[0104] d1) detecting the presence or level of gp42 protein in the sample;

[0105] d2) Detection of Epstein-Barr virus;

[0106] d3) Diagnosis of diseases caused by EBV infection.

[0107] Preferably, the disease includes at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T cell lymphoma, lymphoproliferative disease, and infectious mononucleosis.

[0108] A sixth aspect of the present invention provides a product comprising at least one of e1) to e3):

[0109] e1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0110] e2) the recombinant protein according to the second aspect of the present invention;

[0111] e3) the conjugate according to the fourth aspect of the present invention;

[0112] The product comprises at least one of a reagent, a detection plate, and a test kit.

[0113] Preferably, the product has at least one of the functions d1) to d3):

[0114] d1) detecting the presence or level of gp42 protein in the sample;

[0115] d2) Detection of Epstein-Barr virus;

[0116] d3) Diagnosis of diseases caused by EBV infection.

[0117] Preferably, the disease includes at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T cell lymphoma, lymphoproliferative disease, and infectious mononucleosis.

[0118] A seventh aspect of the present invention provides a drug comprising at least one of f1) to f4):

[0119] f1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0120] f2) the recombinant protein according to the second aspect of the present invention;

[0121] f3) the biomaterial according to the third aspect of the invention;

[0122] f4) The conjugate according to the fourth aspect of the present invention.

[0123] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0124] Preferably, the drug has at least one of the functions c1) to c2):

[0125] c1) Prevention of EBV infection;

[0126] c2) Treating and / or preventing diseases caused by EB virus infection.

[0127] Preferably, the disease includes at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T cell lymphoma, lymphoproliferative disease, and infectious mononucleosis.

[0128] The eighth aspect of the present invention is 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 of the present invention, which is obtained by culturing the transgenic cell line of the third aspect of the present invention.

[0129] The beneficial effects of the present invention are:

[0130] The present invention provides a monoclonal antibody or an antigen-binding fragment thereof. The monoclonal antibody or the antigen-binding fragment thereof has good binding activity with the gp42 protein, has a high affinity for the gp42 protein, can specifically recognize the natural gp42 protein expressed on the cell surface, can significantly inhibit EBV infection of B cells, and effectively inhibit cell membrane fusion. It can be used to detect the presence or level of gp42 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

[0131] Figure 1 This is a graph showing the results of an ELISA assay of the binding activity of the monoclonal antibody 5E3 to the gp42 protein in Example 2.

[0132] Figure 2 This is a graph showing the affinity SPR test results between the monoclonal antibody 5E3 and the gp42 protein in Example 3.

[0133] Figure 3 This is a flow cytometric graph of the monoclonal antibody 5E3 in Example 4 detecting EB virus gp42 protein overexpressed in cells.

[0134] Figure 4 This is an immunofluorescence image of the monoclonal antibody 5E3 in Example 5 detecting the EB virus gp42 protein overexpressed in cells.

[0135] Figure 5 This is a graph showing the results of the monoclonal antibody 5E3 blocking EBV infection of B cells in Example 6.

[0136] Figure 6 This is a diagram showing the blocking results of the monoclonal antibody 5E3 in the cell fusion model in Example 7.

[0137] Figure 7 This is a diagram showing the location of the epitope recognized by the monoclonal antibody 5E3 in Example 8 in the 3D structure of the gp42 protein.

[0138] Figure 8 This is a diagram showing the results of identifying key amino acids in the epitope recognized by monoclonal antibody 5E3 in Example 9. DETAILED DESCRIPTION

[0139] The present invention is further described in detail below through specific examples.

[0140] It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0141] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in these examples were commercially available unless otherwise specified.

[0142] Example 1 Preparation of rabbit monoclonal antibody (mAb) against Epstein-Barr virus gp42 protein

[0143] 1.1 Preparation of protein antigens

[0144] With reference to the complete gene sequence of EB virus M81 strain (Genbank ID: KF373730.1), the N-terminus of the gp42 protein extramembrane region sequence (corresponding to aa34-223 of the viral BZLF2 gene) was connected to the signal peptide coding sequence, and the C-terminus was connected to a polyhistidine polypeptide (6×His) that facilitates affinity chromatography purification. The above sequence was constructed into a suitable eukaryotic expression vector, and the successfully constructed recombinant plasmid was transfected into 293F cells for expression and purification. Finally, the gp42 protein (MVSFKQVRVPLFTAIALVIVLLLAYFLPPRVRGGGRVAAA) was obtained. AITWVPKPNVEVWPVDPPPPVNFNKTAEQEYGDKEVKLPHWTTPTLHTFQVPQNYTKANCTYCNTREYTFSYKGCCFYFTKKKHTWNGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTFKVYQIFGSHCTYVSKFSTVPVSHHECSFLKPCLCVSQRSNS, SEQ ID NO. 1).

[0145] 1.2 Immunization of experimental New Zealand rabbits

[0146] Ten-week-old female New Zealand rabbits were purchased from the Songlian Experimental Animal Farm in Songjiang District, Shanghai, and immunized using a standard in vivo immunization protocol. For details, see Ed Harlow et al., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, 1988. The procedure is briefly described as follows:

[0147] Purified Epstein-Barr virus gp42 protein (500 μg) was mixed with an equal volume of Freund's complete adjuvant (CFA) to make an emulsified volume of 2 mL and injected into the rabbits at multiple points on the neck and back. Booster immunizations were performed on days 14 and 28 after the initial immunization with an equal volume of Epstein-Barr virus gp42 protein (500 μg) mixed with an equal volume of Freund's incomplete adjuvant (IFA) to make an emulsified volume of 2 mL and injected into the rabbits at multiple points on the neck and back.

[0148] 1.3 Preparation of rabbit peripheral blood mononuclear cells (PBMC):

[0149] Rabbit whole blood was diluted in a 1:1 ratio using serum-free RPMI1640 medium, and peripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll reagent as follows: 1.5 times the volume of rabbit whole blood was added to the bottom of the centrifuge tube, and then the rabbit whole blood dilution was slowly added. Centrifugation was performed at 800g for 30 minutes at 4°C, and the speed was slowly increased and decreased. After centrifugation, the suspended cells at the junction of Ficoll and RPMI1640 medium were collected as PBMCs. PBMC cells were collected by a second centrifugation at 1500rpm for 5 minutes at 4°C.

[0150] 1.4 Screening of specific B cells against EB virus gp42 protein:

[0151] Resuspend isolated rabbit PBMCs in 100 μL of sterile PBS. Add the appropriate amount of protein, based on the standard addition of 1 μg of biotinylated gp42 protein per 3 mL of rabbit whole blood PBMCs. Gently pipette to mix thoroughly and incubate at 4°C for 30 minutes. Centrifuge at 1500 rpm for 3 minutes at 4°C, discard the supernatant, and resuspend the cells in PBS, washing two to three times. Add 100 μL of the following staining system to each tube: 1 μL of live / dead aqua, 5 μL of CD4-FITC, 5 μL of CD8-FITC, 5 μL of T lymphocytes-FITC, 5 μL of IgM-RPE, 1 μL of IgG-BV421, 1 μL of streptavidin-conjugated APC, and 100 μL of PBS. Incubate at 4°C in the dark for 30 minutes to select B cells.

[0152] Single B cell lysate was prepared using the SuperScript III Reverse Transcriptase kit (Invitrogen) according to the manufacturer's instructions and added to a 96-well U-bottom plate. B cells specifically bound to gp42 were sorted into the corresponding U-bottom plate at a standard of 1 cell / well.

[0153] PCR products of paired heavy and light chains were obtained using nested PCR, recovered using a nucleic acid recovery kit, and sequenced. Sequencing results were compared with the IMGT database (http: / / www.imgt.org / ) to determine if the obtained gene was an antibody gene, whether the gene was complete, and whether it could successfully encode the antibody, and to determine the family to which the antibody gene (V region and J region) belonged.

[0154] The cloning vector was digested with enzymes. The heavy chain expression plasmid vector had EcoR I / BamH I restriction sites, and the light chain expression plasmid vector had Nhe I / Sal I restriction sites. The light and heavy chain variable region genes were constructed into the corresponding eukaryotic expression vectors pRVRCH and pRVRCL using the Gibson assembly method. pRVRCH contained a nucleic acid sequence encoding the heavy chain constant region of a rabbit monoclonal antibody, and pRVRCL contained a nucleic acid sequence encoding the light chain constant region of a rabbit monoclonal antibody. The nucleic acid sequence of the heavy chain variable region was: CAGCCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTACCTATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGA AGGGGCTGGAATGGATCGGAACTATTAGTGCTAGTGATACTACATACTTCGCGAACTGGACGAAAGGCCGATTCACCATCTCCAAGGCCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTTTTTCTGTGCCAGATTTAGCGCATATGAGAGTAACCGTGATTATTTTGATACTTTTGATCCCTGGGGCCCAGGCACTCTGGTCACCGTCTCTTCA (SEQ ID NO.12); The nucleic acid sequence of the light chain variable region is: GATGTCGTGATGACCCAGACTCCATCCCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAATATTTACAGGGATTTAGCCTGGTATCAGCAGAACCCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCAAT CTGGCATCTGGGGTCCCCTCGCGGTTCAGCGGCAGTGGATCTGGGACAGAGTATATTCTTACCATCAGTGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGTAGTGCTTATGGTAGTGGTTATGCTGCGCATGCTTTCGGCGGAGGGACCAAGGTGGACATCAAA(SEQ ID NO.13); The nucleic acid sequence of the heavy chain constant region is: GGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAA(SEQ IDNO.14); The nucleic acid sequence of the light chain constant region is: GGTGATCCAGTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGC ATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACGTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGT(SEQ ID NO.6). .

[0155] After the antibody light and heavy chain genes were successfully constructed into the expression vector, HEK293T cells were transiently transfected by liposome method to express rabbit monoclonal antibodies against gp42 protein. 12 to 24 hours before transfection, the cells were passaged and seeded into 48-well cell culture plates. Transfection began when the cell confluence reached 60% to 80%. Tube A: 0.5 μg IgH plasmid and 0.5 μg IgK plasmid (corresponding to pRVRCH and pRVRCL above, respectively) were added to 25 μL Opti-MEM. Tube B: 1 μL Novozymes was added to 25 μL Opti-MEM. 2000 Transfection Reagent; tubes A and B were gently mixed and allowed to stand at room temperature for 5 minutes, then the diluted plasmid was added dropwise to the diluted transfection reagent, gently mixed, and incubated at room temperature for 10 minutes; the plasmid-transfection reagent complex was added dropwise to the cells and cultured in a cell culture incubator; after 48 hours, the cell supernatant was collected and centrifuged at 3000 rpm for 5 minutes at 4°C, the supernatant was removed, the cell debris precipitate was discarded, and the monoclonal antibody 5E3 with good reactivity with gp42 protein was obtained, whose heavy chain variable region is: QPVEESGGRLVTPGTPLTLTCTVSGFSLSTYAMSWVRQAPGKGLEWIGTISASDTTYFANWTKGRFTISKASTTVDLKITSPTTEDTATFFCARFSAYESNRDYFDTFDPWGPGTLVTVSS (SEQID NO.8), the light chain variable region is: DVVMTQTPSPVSAAVGGTVTIKCQASQNIYRDLAWYQQNPGQPPKLLIYGASNLASGVPSRFSGSGSGTEYILTISDLECADAATYYCQCSAYGSGYAAHAFGGGTKVDIK(SEQ ID NO.9), the heavy chain constant region is: GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINN EQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO.10), the light chain constant region is: GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO.11); and after checking the IMGT database (http: / / www.imgt.org / ) to analyze its CDR region sequence. Among them, the CDR1 of the heavy chain variable region is: GFSLSTYA (SEQ ID NO.2), CDR2 is: ISASDTT (SEQ ID NO.3), and CDR3 is: ARFSAYESNRDYFDTFDP (SEQ ID NO.4); the CDR1 of the light chain variable region is: QNIYRD (SEQ ID NO.5), CDR2 is GAS, and CDR3 is: QCSAYGSGYAAHA (SEQ ID NO.7).

[0156] 1.5 Preparation of rabbit monoclonal antibody against EBV gp42 protein

[0157] To express 5E3 monoclonal antibody in large quantities, prepare suspension cells 293F in the logarithmic growth phase and culture them in a cell shaker at 100 rpm, 37°C, 5% CO2 until the density reaches 1.5×10 6 / mL, with a cell viability >95%, place 400mL of cells in a new cell culture flask to create a transfection system. Tube A: Add 600μg of plasmids expressing the 10E4 light and heavy chains to 20mL of suspension cell culture medium and vortex to mix thoroughly. Tube B: Add 1.2mg of PEI transfection reagent to 20mL of suspension cell culture medium and vortex to mix thoroughly. Add the solution in Tube B to Tube A, vortex to mix thoroughly, and incubate at room temperature for 15 minutes. The mixture is then added to 400mL of cell culture medium and placed in a shaker at 100rpm, 37°C, 5% CO2, for antibody expression for 6 days. After incubation, collect the cell supernatant and incubate at 4000rpm at 4°C for 10 minutes.

[0158] Filter the cell supernatant through a 0.22 μm filter. Open the AKTA instrument and rinse channels A and B with Solution A (200 mM sodium phosphate dodecahydrate) and Solution B (100 mM citric acid monohydrate), respectively. Install the Protein A column. Equilibrate the Protein A column with Solution A at a flow rate of 8 mL / min for at least 15 minutes. Once the UV value, pH, and conductivity measured by the instrument are stable, load the sample. Load the sample at a flow rate of 6-10 mL / min. The UV value will rise, indicating the peak of the breakthrough. Continue washing the column with Solution A and collect a sample from the breakthrough peak for analysis. Once the pH stabilizes, inject Solution B at a flow rate of 6-10 mL / min. The pH will decrease and the UV value will rise, indicating the elution peak. The antibody is primarily present in the elution peak. Collect a sample from the elution peak for analysis. Equilibrate the column with Solution A, then fill the channels and Protein A column with 20% ethanol. Remove the column and store at 4°C. The collected flowthrough and elution peak samples were identified by SDS-PAGE. The purified monoclonal antibody 5E3 was dialyzed overnight against 20 mM PBS buffer and the concentration was determined by UV spectrometry or BCA assay. The aliquots were then dispensed into 1.5 mL tubes and stored at -20°C until use.

[0159] Example 2 ELISA determination of the binding activity of monoclonal antibody 5E3 to gp42 protein

[0160] 2.1 Preparation of reaction plates

[0161] Dilute gp42 protein in 50 mM CB buffer (NaHCO₃ / Na₂CO₃ buffer, pH 9.6, final concentration 50 mM) to a final coating concentration of 2 μg / mL. Add 100 μL of coating solution to each well of a 96-well microtiter plate and incubate at 37°C for 2 h. Wash once with PBST (20 mM PB7.4, 150 mM NaCl, 0.1% Tween₂O). Then, add 200 μL of blocking solution (20 mM Na₂HPO₄ / NaH₂PO₄ buffer, pH 7.4, containing 20% ​​calf serum and 1% casein) to each well and incubate at 37°C for 2 h. Discard the blocking solution. After drying, store in aluminum foil bags at 2-8°C until ready for use.

[0162] 2.2 ELISA detection of the reactivity of monoclonal antibody 5E3 with gp42 protein

[0163] Monoclonal antibody 5E3 (9E1, a control antibody previously described in the literature: Rabbit Monoclonal Antibody, Specifically Recognizing a Linear Epitope in the RBD of SARS-CoV-2 Spike Protein) was diluted 2-fold in 20 mM PBS buffer starting at 100 ng / mL, for a total of 20 dilutions. 100 μL of the diluted sample was added to each well of a gp42-coated ELISA plate and incubated at 37°C for 30 min. The plate was washed five times with PBST (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20). 100 μL of HRP-labeled goat anti-rabbit IgG reaction solution was added to each well and incubated at 37°C for 30 min. After the enzyme labeling reaction step, the plate was washed five times with PBST (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20). TMB colorimetric reagent (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) (50 μL) was added to each well and incubated at 37°C for 15 min. After the color development step, stop solution (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) (50 μL) was added to each well of the plate, and the OD450 / 630 values ​​of each well were measured on a microplate reader.

[0164] The results are as follows Figure 1 As shown, the EC of monoclonal antibody 5E3 and gp42 protein 50 The value was 25.27 ng / mL, indicating that monoclonal antibody 5E3 had good binding activity to the purified gp42 protein.

[0165] Example 3 SPR Detection of Affinity between Monoclonal Antibody 5E3 and gp42 Protein

[0166] The binding kinetics of monoclonal antibody 5E3 and antigen were analyzed using the Biacore 8K system (Cytiva). All steps were performed in PBS buffer. The monoclonal antibody was captured at a dilution of 5 μg / mL using the company's Protein A chip. The gp42 protein was used as the detection antigen. The antigen was diluted to seven concentrations: 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM. The assay was performed according to the following protocol: capture for 60 s, analysis for 120 s, dissociation for 120 s, and regeneration for 60 s. The equilibrium dissociation constant of the antibody was calculated using the instrument's accompanying data acquisition and analysis software.

[0167] The results are as follows Figure 2As shown, the equilibrium dissociation constant K of monoclonal antibody 5E3 for gp42 protein D It is 0.0431 nM, indicating that monoclonal antibody 5E3 has a high affinity for gp42 protein.

[0168] Example 4 Detection of Overexpressed EB Virus gp42 Protein in Cells Using Monoclonal Antibody 5E3 (Flow Cytometry)

[0169] (1) 293T cells were seeded into a 10 cm cell culture plate and transfected when the cell confluence reached 60-80%.

[0170] (2) Tube A: Add 30 μg of the eukaryotic expression plasmid containing the full-length gp42 protein gene (pCDH-gp42 (gp42 accession number: AGZ95182.1, vector: pCDH)) to 2 mL of Opti-MEM;

[0171] (3) Tube B: Add 60 μL of Novozymes to 2 mL of Opti-MEM. 2000TransfectionReagent;

[0172] (4) Gently mix tubes A and B separately, let them stand at room temperature for 5 minutes, then add the diluted plasmid dropwise to the diluted transfection reagent, mix gently, and incubate at room temperature for 10 minutes;

[0173] (5) Add the plasmid-transfection reagent complex dropwise to the cells and culture them in a cell culture incubator;

[0174] (6) 48 h after transfection, cells were digested with trypsin and the cells were divided into 5 × 10 5 Add 3 μg of monoclonal antibody 5E3 (9E1 was used as a control antibody, which has been published in the literature: Rabbit Monoclonal Antibody Specifically Recognizing a Linear Epitope in the RBD of SARS-CoV-2 Spike Protein) to each cell / tube and make up to 100 μL with PBS. Incubate at 4°C for 30 min. Wash the cells by adding 1 mL of PBS buffer to each tube, centrifuge at 1500 rpm for 5 min, and discard the supernatant to retain the cells. Wash three times in total.

[0175] (7) Add 2 μL of BV421 fluorescent dye-labeled goat anti-rabbit IgG secondary antibody to each tube of sample, incubate at 4°C in the dark for 30 min, add 1 mL of PBS buffer to each tube to wash the cells, centrifuge at 1500 rpm for 5 min at 4°C, discard the supernatant and retain the cells, and wash three times in total;

[0176] (8) Resuspend the cells in 1 mL of PBS and use a BD flow cytometer (LSRFortessaX-20) to detect the proportion of BV421-positive cells.

[0177] The results are as follows Figure 3 As shown, monoclonal antibody 5E3 had a significant reaction to 293T cells transfected with the full-length gp42 protein gene plasmid, while the control antibody had no reaction, indicating that monoclonal antibody 5E3 can specifically recognize the natural gp42 protein expressed on the cell surface.

[0178] Example 5 Detection of Overexpressed EB Virus gp42 Protein in Cells Using Monoclonal Antibody 5E3 (Immunofluorescence)

[0179] (1) 293β5 cells (ATCC) were seeded into 10 cm cell culture plates and transfected when the cell confluence reached 60–80%.

[0180] (2) Tube A: Add 30 μg of mammalian expression plasmid containing the full-length gene of EBV gp42 protein (pCDH-gp42 (gp42 accession number: AGZ95182.1, vector: pCDH)) to 1.5 mL of Opti-MEM;

[0181] (3) Tube B: Add 60 μL of Novozymes to 1.5 mL of Opti-MEM. 2000TransfectionReagent;

[0182] (4) Gently mix tubes A and B separately, let them stand at room temperature for 5 minutes, then add the diluted plasmid dropwise to the diluted transfection reagent, mix gently, and incubate at room temperature for 10 minutes;

[0183] (5) Add the plasmid-transfection reagent complex dropwise to the cells and culture them in a cell culture incubator;

[0184] (6) 48 h after transfection, cells were digested with trypsin and the cells were 4 The ratio of cells per well was inoculated into 96-well cell culture plates and cultured in a cell culture incubator;

[0185] (7) After 12 h of inoculation into a 96-well cell culture plate, the cell supernatant was discarded, 100 μL of PBS was added to each well for washing once, 100 μL of 4% paraformaldehyde prepared in PBS was added to each well, and the cells were fixed for 15 min in the dark, and 100 μL of 20 mM PBS was added to each well for washing three times;

[0186] (8) Add 100 μL of 3‰ Triton X-100 prepared in PBS to each well, permeabilize the cells for 10 min, and then wash three times with 100 μL of PBS in each well;

[0187] (9) Monoclonal antibody 5E3 (9E1 was used as a control antibody, which has been published in the literature: Rabbit Monoclonal Antibody Specifically Recognizing a Linear Epitope in the RBD of SARS-CoV-2 Spike Protein) was diluted to 1 μg / mL with 2% BSA prepared in PBS, and 100 μL was added to each well of a 96-well cell culture plate. The cells were incubated at room temperature for 30 min, and then washed three times with 100 μL of PBS per well.

[0188] (10) Fluorescent secondary antibody Alexa 488Donkey Anti-Rabbit IgG (H+L) was diluted to 1 μg / mL with 2% BSA in PBS. 100 μL was added to each well of a 96-well cell culture plate and incubated at room temperature for 30 min. The cells were then washed three times with 100 μL of PBS added to each well.

[0189] (11) The nuclear dye DAPI was diluted with 2% BSA in PBS at a ratio of 1:2000, 50 μL was added to each well, incubated at room temperature for 5 min, and 100 μL PBS was added to each well for washing three times;

[0190] (12) Place the 96-well cell culture plate under a fluorescence microscope and observe and photograph the results.

[0191] The results are as follows Figure 4 As shown, the isotype control antibody did not bind to the gp42 expressed on the cells, while the detection results of the monoclonal antibody 5E3 showed that the green fluorescence was distributed in the cell membrane and cytoplasm of 293β5 cells expressing the gp42 protein. The above results indicate that the monoclonal antibody 5E3 has specific binding activity to the gp42 protein expressed on the cells, and 5E3 can also recognize the natural gp42 protein expressed on the cell membrane surface.

[0192] Example 6 Analysis of the Neutralizing Ability of Monoclonal Antibody 5E3 in a Virus Infection Model

[0193] EBV B cell neutralization model:

[0194] Monoclonal antibody 5E3 (9E1 was used as a control antibody, which has been published in the literature: Rabbit Monoclonal Antibody Specifically Recognizing a Linear Epitope in the RBD of SARS-CoV-2 Spike Protein) was diluted in RPMI1640 serum-free medium, starting from 100 μg / mL and diluted 3-fold in 12 steps. 50 μL of the diluted antibody was placed in a 96-well cell plate, and 20 μL of virus dilution solution carrying the GFP green fluorescent protein gene produced by CNE2 cells was added (the virus dilution solution has been published in the literature: An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus). Mix well, place in a 37°C incubator and incubate for 2 hours. Resuspend 1x10 6 Akata cells were cultured, 130 μL of the cell suspension was added to the virus and antibody mixture from the previous step, and the cells were incubated in a 37°C incubator for 48 h. The viral infection rate of the Akata cells was detected using a BD flow cytometer LSRFortessa X-20. The reduction in the number of GFP-positive cells in the antibody-treated group compared with the infection control group (adding an equal volume of RPMI1640) was detected, and the inhibition rate (neutralization efficiency, %) of the antibody in the B cell infection model was calculated.

[0195] The results are as follows Figure 5 As shown, the IC50 of monoclonal antibody 5E3 in the B cell infection model is 0.20 μg / mL, and the control antibody has no neutralizing activity. Monoclonal antibody 5E3 can significantly inhibit EBV infection of B cells.

[0196] Example 7 Analysis of the blocking ability of monoclonal antibody 5E3 in a cell fusion model

[0197] 293T cells were seeded into a 10 cm cell culture plate and transfected when the cell confluence reached 60-80%. Set up plate A: Use PEI as the transfection reagent and prepare 2.5 μg of eukaryotic expression plasmids carrying genes encoding full-length gB, gH, gL, and gp42 (pCAGGS-gB (gB accession number: BAU51603.1; vector: pCAGGS), pCAGGS-gH (gH accession number: BAU51590.1; vector: pCAGGS), pCAGGS-gL (gL accession number: BAU51568.1, vector: pCAGGS), pCAGSS-T7 (T7 accession number: CP053597.1, vector: pCAGGS), and pCDH-gp42 (gp42 accession number: AGZ95182.1, vector: pCDH). For specific construction methods, refer to the literature: Fusion of epithelial cells by Epstein–Barr virus proteins is triggered by binding of viral glycoproteins gHgL to Panel B: PEI was used as the transfection reagent to transfect 293T cells with 10 μg of a eukaryotic expression plasmid containing the Luciferase gene controlled by the T7 promoter (pCAGSS-T7-luc (Luciferase accession number: M15077.1, vector: pCAGGS) and 2.5 μg of a eukaryotic expression plasmid containing the full-length HLA-II gene (α chain accession number: KAI2541864.1, vector: pCDH; β chain accession number: USF94388.1, vector: pCDH). For specific construction methods, refer to the literature: Fusion of epithelial cells by Epstein–Barr virus proteins is triggered by binding of viral glycoproteins gHgL to integrins αvβ6 or αvβ8).

[0198] 24 hours after transfection, the 293T cells in plate A were digested and plated with 2×10 5 The cells were divided into 96-well plates at a ratio of 10 cells / mL and 200 μL of antibodies of different concentrations (5E3, control monoclonal antibody VRC01, blank control group with equal amount of PBS) were added respectively. The cells were incubated in a 37°C incubator for 30 minutes and then transferred to 24-well plates. The cells in plate B were digested in advance and 2×10 5 cells and cultured at 37°C for 24 h.

[0199] 100 μL of firefly luciferase substrate from the Promega Dual-Glo Luciferase Assay System kit was added to a 24-well plate and lysed at 4°C for 20 minutes. 80 μL of cell lysate supernatant was collected from each well for quantitative fluorescence detection. The percentage reduction in fluorescence readings in the antibody-treated group compared to the untreated control wells was calculated to determine the blocking efficiency (%) of the antibody in the cell fusion model.

[0200] The results are as follows Figure 6 As shown, the results showed that monoclonal antibody 5E3 had an inhibitory effect on the cell fusion model, while the control antibody had no inhibitory effect, indicating that monoclonal antibody 5E3 can effectively inhibit cell membrane fusion.

[0201] Example 8 Location of the epitope recognized by monoclonal antibody 5E3 in the 3D structure of gp42 protein (cryo-EM structure analysis)

[0202] Antigen-antibody complexes were prepared and their structures analyzed using cryo-EM. Antibody Fab fragments were prepared first. The antibodies were dialyzed into PBS buffer. Enzyme digestion conditions were explored first. In a 100 μL digestion system, the reagents required for antibody digestion were prepared in advance. 10 μL of 0.5 M EDTA, 3 μL of 1 M cysteine, and an antibody volume to a final concentration of 1 mg / mL were added. Papain was then added at varying mass ratios (papain:antibody = 1:400). The volume was adjusted to 100 μL with PBS and digested in a 37°C water bath for 12 hours. After confirming the digestion conditions by SDS-PAGE, a large number of antibody digestions were performed. After 12 hours of digestion at 37°C, iodoacetamide was added to a final concentration of 20 mM to terminate the digestion reaction. The Fc fragment was adsorbed on a protein A chromatography column, and the Fab fragment was collected in the sample flowthrough. The purified Fab fragment was dialyzed into PBS, concentrated with a concentrator, and then filtered with a 0.22μm filter to remove impurities and stored at -20°C for use. Fab and antigen were mixed in a molar ratio of 1:1 and incubated at 37 degrees for 2 hours. The complex was then purified based on molecular sieves and concentrated to a concentration of more than 3.5mg / ml for cryo-electron microscopy sample preparation and structural analysis. The structural results are shown in Figure 2. Figure 7 As shown, 5E3 was shown to primarily recognize gp42 aa108-112 and aa 146-155.

[0203] Example 9 Identification of epitopes recognized by monoclonal antibody 5E3

[0204] To further identify the key sites recognized by monoclonal antibody 5E3, wild-type gp42 (SEQ ID NO.1) was used as a template to clone, express and evaluate point mutations in the extracellular domain of gp42 protein.

[0205] Primers were designed according to the instructions of the Mut Express II Fast Mutagenesis Kit V2 (purchased from Novozymes), and point mutation cloning was performed to mutate the amino acid at the corresponding site in the extracellular domain of the gp42 protein to arginine (108R indicates a gp42 protein in which the 108th amino acid in the gp42 protein was mutated to arginine; 109R indicates a gp42 protein in which the 109th amino acid in the gp42 protein was mutated to arginine; 110R indicates a gp42 protein in which the 110th amino acid in the gp42 protein was mutated to arginine; 111R indicates a gp42 protein in which the 111th amino acid in the gp42 protein was mutated to arginine; 112R indicates a gp42 protein in which the 112th amino acid in the gp42 protein was mutated to arginine). 2 proteins; 113R indicates a gp42 protein in which the 113th amino acid in the gp42 protein is mutated to arginine; 114R indicates a gp42 protein in which the 114th amino acid in the gp42 protein is mutated to arginine; 115R indicates a gp42 protein in which the 115th amino acid in the gp42 protein is mutated to arginine; 144R indicates a gp42 protein in which the 144th amino acid in the gp42 protein is mutated to arginine; 145R indicates a gp42 protein in which the 145th amino acid in the gp42 protein is mutated to arginine; 146R indicates a gp42 protein in which the 146th amino acid in the gp42 protein is mutated to arginine; 147R indicates a gp42 protein in which the 147th amino acid in the gp42 protein is mutated to arginine. gp42 protein with amino acid position 47 mutated to arginine; 148R indicates a gp42 protein with amino acid position 148 mutated to arginine; 149R indicates a gp42 protein with amino acid position 149 mutated to arginine; 150R indicates a gp42 protein with amino acid position 150 mutated to arginine; 151R indicates a gp42 protein with amino acid position 151 mutated to arginine; 152R indicates a gp42 protein with amino acid position 152 mutated to arginine; 104R indicates a gp42 protein with amino acid position 104 mutated to arginine ; 106R indicates a gp42 protein in which the amino acid at position 106 in the gp42 protein is mutated to arginine; 107R indicates a gp42 protein in which the amino acid at position 107 in the gp42 protein is mutated to arginine; 153R indicates a gp42 protein in which the amino acid at position 153 in the gp42 protein is mutated to arginine; 155R indicates a gp42 protein in which the amino acid at position 155 in the gp42 protein is mutated to arginine; 221R indicates a gp42 protein in which the amino acid at position 221 in the gp42 protein is mutated to arginine; 222R indicates a gp42 protein in which the amino acid at position 222 in the gp42 protein is mutated to arginine), and sequencing was used to verify whether the point mutation clone was constructed correctly.The correct point mutation clones were transiently transfected into 293F cells for eukaryotic expression, and the point mutation proteins were obtained by nickel column purification.

[0206] The amino acids at the antigen-antibody interaction interface were scanned for arginine, and then the binding activity of the mutant protein to the monoclonal antibody 5E3 was detected by ELISA. The results showed that the mutation of amino acids at positions 109, 111, 146, 147, 152, 155, and 222 to arginine affected the binding activity of the 5E3 monoclonal antibody, indicating that amino acids at positions 109, 111, 146, 147, 152, 155, and 222 are key sites for 5E3 antibody recognition ( Figure 8 ).

[0207] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to Epstein-Barr virus gp42 protein, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises CDR1, CDR2, and CDR3; The amino acid sequence of CDR1 of the heavy chain variable region is: GFSLSTYA (SEQ ID NO. 2); The amino acid sequence of CDR2 of the heavy chain variable region is: ISASDTT (SEQ ID NO. 3); The amino acid sequence of CDR3 of the heavy chain variable region is: ARFSAYESNRDYFDTFDP (SEQ ID NO. 4); The light chain variable region comprises CDR1, CDR2, and CDR3; The amino acid sequence of CDR1 of the light chain variable region is: QNIYRD (SEQ ID NO. 5); The amino acid sequence of CDR2 of the light chain variable region is: GAS; The amino acid sequence of CDR3 of the light chain variable region is: QCSAYGSGYAAHA (SEQ ID NO. 7).

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein: The amino acid sequence of the heavy chain variable region comprises: QPVEESGGRLVTPGTPLTLTCTVSGFSLSTYAMSWVRQAPGKGLEWIGTISASDTTYFANWTKGRFTISKASTTVDLKITSPTTEDTATFFCARFSAYESNRDYFDTFDPWGPGTLVTVSS (SEQ ID NO.8); The amino acid sequence of the light chain variable region comprises: DVVMTQTPSPVSAAVGGTVTIKCQASQNIYRDLAWYQQNPGQPPKLLIYGASNLASGVPSRFSGSGSGTEYILTISDLECADAATYYCQCSAYGSGYAAHAFGGGTKVDIK (SEQ ID NO. 9).

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, bispecific antibody, and multispecific antibody.

4. 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 a heavy chain constant region and a light chain constant region.

5. A recombinant protein comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; and It consists of a tag sequence that facilitates expression and / or purification.

6. A biomaterial related to the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the recombinant protein according to claim 5, wherein the biomaterial comprises at least one of b1) to b8): b1) a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the recombinant protein according to claim 5; b2) an expression cassette comprising the nucleic acid molecule described in b1); b3) a vector comprising the nucleic acid molecule described in b1); b4) a vector comprising the expression cassette described in b2); b5) a transgenic cell line comprising the nucleic acid molecule described in b1); b6) a transgenic cell line comprising the expression cassette described in b2); b7) a transgenic cell line comprising the vector described in b3); b8) A transgenic cell line comprising the vector described in b4).

7. A conjugate comprising: at least one of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and the recombinant protein according to claim 5; and a coupling portion, wherein the coupling portion is at least one of a detectable label and a radionuclide.

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 4; (2) The recombinant protein according to claim 5; (3) The biomaterial according to claim 6; (4) The conjugate according to claim 7; The product comprises at least one of a drug, a reagent, a test plate, and a test kit; The drug has the function of treating and / or preventing diseases caused by EB virus infection: The reagent, detection plate or kit has at least one of the functions d1) to d3): d1) detecting the presence or level of gp42 protein in the sample; d2) Detection of Epstein-Barr virus; d3) Diagnosis of diseases caused by EBV infection.

9. A product comprising at least one of e1) to e3): e1) the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; e2) the recombinant protein according to claim 5; e3) the conjugate according to claim 7; The product comprises at least one of a reagent, a detection plate, and a test kit.

10. A drug comprising at least one of (1) to (4): (1) The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; (2) The recombinant protein according to claim 5; (3) The biomaterial according to claim 6; (4) The conjugate according to claim 7.

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