A monoclonal antibody for recognizing EB virus gL protein and its application

Abstract

The present invention belongs to the field of antibody technology and discloses a monoclonal antibody that recognizes the gL protein of Epstein-Barr virus and its application. The monoclonal antibody or its antigen-binding fragment has good binding activity with the gL protein and / or the gHgL protein, has a high affinity for the gL protein and / or the gHgL protein, can specifically recognize the natural gL protein and / or the gHgL protein expressed on the cell surface, can significantly inhibit EBV infection of B cells and epithelial cells, and effectively inhibits cell membrane fusion. It can be used to detect the presence or level of the gL protein and / or the gHgL 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 gL protein and its application. 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) GFSLSTYW (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) IGGSGST (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) ARDSGAGVRFRF (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) ENIGSR (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) RAS; or

[0035] a2) RAS 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 RAS; or

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

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

[0038] a1) QCTYGVSITINYGND (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)QQVKESGGRLVTPGTPLTLTCTASGFSLSTYWMSWVRQAPGKGLEYIGVIGGSGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDSGAGVRFRFWGPGTLVTVSS(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)DLVMTQTPASVEAGVGGTVTINCQASENIGSRLAWYQQKPGQPPKLLIYRASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQCTYGVSITINYGNDFGGGTEVVVK(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 represented by 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 gL protein and / or gHgL protein.

[0060] Preferably, the gHgL protein comprises gH protein and gL protein.

[0061] Preferably, the monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that recognizes and / or targets the gL protein of Epstein-Barr virus.

[0062] Preferably, the monoclonal antibody or antigen-binding fragment thereof recognizes aa 104 to 123 of the gL protein.

[0063] Preferably, the key sites for the monoclonal antibody or antigen-binding fragment thereof to recognize the gL protein are amino acids 106, 108, 109, 110, 112, 114, 116, and 119 of the gL protein.

[0064] 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

[0065] Optional tag sequence to facilitate expression and / or purification.

[0066] Preferably, the tag sequence is at least one selected from the following group: 6×His tag, GGGS sequence, FLAG tag.

[0067] 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):

[0068] 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;

[0069] b2) an expression cassette comprising the nucleic acid molecule described in b1);

[0070] b3) a vector comprising the nucleic acid molecule described in b1);

[0071] b4) a vector comprising the expression cassette described in b2);

[0072] b5) a transgenic cell line comprising the nucleic acid molecule described in b1);

[0073] b6) a transgenic cell line comprising the expression cassette described in b2);

[0074] b7) a transgenic cell line comprising the vector described in b3);

[0075] b8) A transgenic cell line comprising the vector described in b4).

[0076] Preferably, the transgenic cell line comprises no reproductive material.

[0077] 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;

[0078] 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:

[0079] a1) SEQ ID NO. 16; or

[0080] a2) a nucleotide sequence obtained by substituting and / or deleting and / or adding one or more nucleotides of SEQ ID NO. 16 and having the same function as the nucleic acid molecule shown in SEQ ID NO. 16; or

[0081] a3) a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% identical to SEQ ID NO. 16 and has the same function as the nucleic acid molecule represented by SEQ ID NO. 16;

[0082] 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:

[0083] a1) SEQ ID NO. 17; or

[0084] a2) a nucleotide sequence obtained by substituting and / or deleting and / or adding one or more nucleotides of SEQ ID NO. 17 and having the same function as the nucleic acid molecule shown in SEQ ID NO. 17; or

[0085] a3) a nucleotide sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology to SEQ ID NO. 17 and has the same function as the nucleic acid molecule shown in SEQ ID NO. 17.

[0086] 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;

[0087] 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:

[0088] a1) SEQ ID NO. 18; or

[0089] a2) a nucleotide sequence obtained by substituting and / or deleting and / or adding one or more nucleotides of SEQ ID NO. 18 and having the same function as the nucleic acid molecule shown in SEQ ID NO. 18; or

[0090] a3) a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% identical to SEQ ID NO. 18 and has the same function as the nucleic acid molecule represented by SEQ ID NO. 18;

[0091] 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:

[0092] a1) SEQ ID NO. 19; or

[0093] a2) a nucleotide sequence obtained by substituting and / or deleting and / or adding one or more nucleotides in SEQ ID NO. 19 and having the same function as the nucleic acid molecule shown in SEQ ID NO. 19; or

[0094] a3) a nucleotide sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology to SEQ ID NO. 19 and has the same function as the nucleic acid molecule shown in SEQ ID NO. 19.

[0095] 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;

[0096] 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.

[0097] Preferably, the detectable label is selected from radioisotopes, fluorescent substances, chemiluminescent substances, colored substances, or any combination thereof.

[0098] 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.

[0099] 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;

[0100] The product comprises at least one of a drug, a reagent, a test plate, and a test kit.

[0101] Preferably, the drug has at least one of the functions c1) to c2):

[0102] c1) Prevention of EBV infection;

[0103] c2) Treating and / or preventing diseases caused by EB virus infection.

[0104] Preferably, the reagent, detection plate or kit has at least one of the functions d1) to d3):

[0105] d1) detecting the presence or level of gL protein and / or gHgL protein in the sample;

[0106] d2) Detection of Epstein-Barr virus;

[0107] d3) Diagnosis of diseases caused by EBV infection.

[0108] 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.

[0109] Preferably, the gHgL protein comprises gH protein and gL protein.

[0110] A sixth aspect of the present invention provides a product comprising at least one of e1) to e3):

[0111] e1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0112] e2) the recombinant protein according to the second aspect of the present invention;

[0113] e3) the conjugate according to the fourth aspect of the present invention;

[0114] The product comprises at least one of a reagent, a detection plate, and a test kit.

[0115] Preferably, the product has at least one of the functions d1) to d3):

[0116] d1) detecting the presence or level of gL protein and / or gHgL protein in the sample;

[0117] d2) Detection of Epstein-Barr virus;

[0118] d3) Diagnosis of diseases caused by EBV infection.

[0119] 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.

[0120] Preferably, the gHgL protein comprises gH protein and gL protein.

[0121] A seventh aspect of the present invention provides a drug comprising at least one of f1) to f4):

[0122] f1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0123] f2) the recombinant protein according to the second aspect of the present invention;

[0124] f3) the biomaterial according to the third aspect of the invention;

[0125] f4) The conjugate according to the fourth aspect of the present invention.

[0126] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0127] Preferably, the drug has at least one of the functions c1) to c2):

[0128] c1) Prevention of EBV infection;

[0129] c2) Treating and / or preventing diseases caused by EB virus infection.

[0130] 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.

[0131] 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.

[0132] The beneficial effects of the present invention are:

[0133] 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 gL protein and / or gHgL protein, has high affinity for gL protein and / or gHgL protein, can specifically recognize natural gL protein and / or gHgL protein expressed on the cell surface, can significantly inhibit EBV infection of B cells and epithelial cells, and effectively inhibit cell membrane fusion. It can be used to detect the presence or level of gL protein and / or gHgL 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

[0134] Figure 1 This is a graph showing the results of an ELISA assay of the binding activity of the monoclonal antibody 10E4 to the gHgL protein in Example 2.

[0135] Figure 2 This is a graph showing the results of Western Blot detection of gHgL using monoclonal antibody 10E in Example 2.

[0136] Figure 3 This is a graph showing the affinity constant test results between the monoclonal antibody 10E4 and the gHgL protein in Example 3.

[0137] Figure 4 Flow cytometric graphs of the monoclonal antibody 10E4 in Example 4 detecting overexpressed Epstein-Barr virus gHgL protein or gL protein in cells: wherein A is a flow cytometric graph of the monoclonal antibody 10E4 detecting overexpressed Epstein-Barr virus gHgL protein in cells; B is a flow cytometric graph of the monoclonal antibody 10E4 detecting overexpressed Epstein-Barr virus gL protein in cells.

[0138] Figure 5 This is a graph showing the results of detecting gHgL protein expression in EBV-positive cell lines using monoclonal antibody 10E4 in Example 5.

[0139] Figure 6 These are the results of monoclonal antibody 10E4 blocking EBV infection of B cells or epithelial cells in Example 6: A is the result of monoclonal antibody 10E4 blocking EBV infection of B cells; B is the result of monoclonal antibody 10E4 blocking EBV infection of epithelial cells.

[0140] Figure 7 This is a diagram showing the blocking results of the monoclonal antibody 10E4 in the cell fusion model in Example 7.

[0141] Figure 8 This is a diagram showing the epitope identification results of the monoclonal antibody 10E4 based on the reaction with the biotin-labeled polypeptide in Example 8: wherein A is a diagram showing the amino acid sequence of the biotin-labeled polypeptide; and B is a diagram showing the reaction results of the monoclonal antibody 10E4 and the biotin-labeled polypeptide.

[0142] Figure 9 This is a graph showing the results of Western Blot reaction between the monoclonal antibody 10E4 and the gHgL protein with alanine point mutation in Example 9.

[0143] Figure 10 This is a graph showing the ELISA reaction results between the monoclonal antibody 10E4 and the gHgL protein with alanine point mutation in Example 9.

[0144] Figure 11 This is a diagram showing the location of the epitope recognized by monoclonal antibody 10E4 in Example 9 in the 3D structure of the gL protein.

[0145] Figure 12 This is a diagram showing the location of the epitope recognized by monoclonal antibody 10E4 in the 3D structure of the gHgL / gp42 / E1D1 complex in Example 9.

[0146] Figure 13 This is a diagram showing the location of the epitope recognized by monoclonal antibody 10E4 in Example 9 in the 3D structure of the gHgL / EphA2 LBD complex. DETAILED DESCRIPTION

[0147] The present invention is further described in detail below through specific examples.

[0148] 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.

[0149] 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.

[0150] Example 1 Preparation of rabbit monoclonal antibody (mAb) against EB virus gHgL protein

[0151] Rabbits were subcutaneously immunized with Freund's adjuvant mixed with antigen, with each 14-day immunization cycle. Rabbit blood was collected before booster immunization, and serum was separated for subsequent titer testing. Whole blood was collected from rabbits and separated using Ficoll (dextran) density gradient centrifugation to obtain PBMCs. PBMCs were stained with non-B cell marker dyes and labeled with B cell IgG marker fluorescent antibodies and fluorescently labeled antigens. Flow cytometry was used to negatively select non-B cell marker-positive cell populations and positively select IgG-positive and antigen-fluorescent-positive cell populations, which were separated into 96-well plates. Rabbit B cell RNA in the positive wells was extracted and reverse transcribed to obtain cDNA, and specific primers were used to fish for the light and heavy chain variable region genes of rabbit monoclonal antibodies. The rabbit monoclonal antibody genes were constructed into light and heavy chain expression plasmid vectors, respectively, and co-transfected into the 293 cell line for preliminary transfection expression identification. Positive wells with antigen-specific binding were selected for further large-scale expression and functional evaluation to prepare a relatively complete gHgL-specific antibody library.

[0152] 1.1 Preparation of protein antigen: With reference to the complete gene sequence of EB virus M81 strain (KF373730.1), the C-terminus of the gL protein extracellular domain sequence (corresponding to aa 24-137 of the protein encoded by the viral BKRF2 gene) was connected to the gH protein extracellular domain sequence (corresponding to aa 19-678 of the protein encoded by the viral BXLF2 gene) through a flexible amino acid sequence. The N-terminus of the gL protein was connected to a signal peptide coding sequence, and the C-terminus of the gH protein was connected to a polyhistidine polypeptide (6×His) for affinity chromatography purification. The above sequences were constructed into a suitable eukaryotic expression vector, and the successfully constructed recombinant plasmid was transfected into 293F cells for expression and purification. Finally, the full-length gHgL extracellular domain protein was obtained (the amino acid sequence of the gHgL protein is shown in SEQ ID NO.12, which includes, in sequence: a signal peptide sequence (MPMGSLQPLATLYLLGMLVASCLG, SEQ ID NO.6), a gL protein (SEQ ID 24 to 137 of the amino acid sequence shown in SEQ ID NO.1 (MRAVGVFLATCLVTIFVLPTWGNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCANGLNVVSFFISILKRSSSALTSHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGG), a linker sequence (GGGGSGGGGSGGGGS, SEQ ID NO.14), and a gH protein (SEQ ID NO.13 () from position 19 to position 678 of the amino acid sequence shown in), tag protein (HHHHHH, SEQ ID NO.15).

[0153] 1.2 Immunization of experimental rabbits:

[0154] Ten-week-old female New Zealand rabbits were purchased from the Songlian Experimental Animal Farm in Songjiang District, Shanghai. Standard in vivo immunization was used. For details, see Ed Harlow et al., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, 1988. The procedure is briefly as follows:

[0155] 500 μg of the EBV gHgL protein purified in step 1.1 above was mixed with equal volumes of Freund's complete adjuvant (CFA) (purchased from SIGMA-ALDRICH, Catalog No. F5881) to emulsify into 2 mL and injected into the rabbits at multiple sites on the neck and back. On days 14 and 28 after the first immunization, 500 μg of the EBV gHgL protein purified in step 1.1 above was mixed with equal volumes of Freund's incomplete adjuvant (IFA) (purchased from SIGMA-ALDRICH, Catalog No. F5506) to emulsify into 2 mL and injected into the rabbits at multiple sites on the neck and back. On day 42 after the first immunization, whole blood was collected from the rabbits' central ear artery, and serum was separated for gHgL antibody titer determination.

[0156] 1.3 Preparation of rabbit peripheral blood mononuclear cells (PBMC)

[0157] 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 Ficoll solution 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 800×g 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.

[0158] 1.4 Screening of specific B cells against EBV gHgL

[0159] Resuspend the 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 gHgL 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 live / dead aqua, 5 μL CD4-FITC, 5 μL CD8-FITC, 5 μL T lymphocytes-FITC, 5 μL IgM-RPE, 1 μL IgG-BV421, 1 μL streptavidin-conjugated APC, and 100 μL PBS. Incubate at 4°C in the dark for 30 minutes to select B cells.

[0160] 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 binding to gHgL were sorted into the corresponding U-bottom plate at a standard of 1 cell / well.

[0161] 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.

[0162] The cloning vector was digested with enzymes; the heavy chain expression plasmid had EcoR I / BamH I cleavage sites, while the light chain expression plasmid had Nhe I / Sal I cleavage 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 to generate IgK and IgH plasmids, respectively. pRVRCH contained a nucleic acid sequence encoding the heavy chain constant region of a rabbit monoclonal antibody, while 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: CAGCAGGTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTACCTACTGGATGAGCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAATACATTGGAGTCATTGGTGGTAGTGGTAGCACATACTACGCGAGCTGGGCGAAGGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAGATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGACAGTGGTGCTGGTGTAAGATTTAGATTCTGGGGCCCTGGCACCCTGGTCACCGTCTCTTCA (SEQ ID NO.16); The nucleic acid sequence of the light chain variable region is: GACCTCGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGGTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTGAGAACATTGGTAGTAGATTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTG GAATCTGGGGTCCCATCGCGCTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGCACTTATGGTGTTAGTATTACTATTAATTATGGTAATGATTTCGGCGGAGGGACCGAGGTGGTCGTCAAA(SEQ ID NO.17); The nucleic acid sequence of the heavy chain constant region is: GGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA(SEQ ID NO.18); The nucleic acid sequence of the light chain constant region is: GGTGATCCAGTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCA TCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACGTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG(SEQ ID NO.19). .

[0163] After the expression vector was constructed, HEK293T cells were transiently transfected with the liposome method to express the rabbit monoclonal antibody against the EBV-gHgL protein. 12 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: 0.2 μg IgH plasmid and 0.2 μg IgK plasmid were added to 10 μL Opti-MEM; Tube B: 0.4 μL Novozymes was added to 10 μL Opti-MEM. 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 10E4 with good reactivity with gHgL protein was obtained, whose heavy chain variable region is: QQVKESGGRLVTPGTPLTLTCTASGFSLSTYWMSWVRQAPGKGLEYIGVIGGSGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDSGAGVRFRFWGPGTLVTVSS (SEQ ID NO.8), the light chain variable region is: DLVMTQTPASVEAGVGGTVTINCQASENIGSRLAWYQQKPGQPPKLLIYRASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQCTYGVSITINYGNDFGGGTEVVVK (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 its CDR region sequence was analyzed by IMGT database (http: / / www.imgt.org / ), among which, the CDR1 of the heavy chain variable region is: GFSLSTYW (SEQ ID NO.2), CDR2 is: IGGSGST (SEQ ID NO. 3), CDR3 is: ARDSGAGVRFRF (SEQ ID NO. 4); CDR1 of the light chain variable region is: ENIGSR (SEQ ID NO. 5), CDR2 is RAS, and CDR3 is: QCTYGVSITINYGND (SEQ ID NO. 7).

[0164] 1.5 Preparation of rabbit monoclonal antibody against EBV gHgL protein

[0165] To express the 10E4 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, cell viability >95%, take 400mL of cells and place them in a new cell culture flask as a transfection system. Tube A: Add 600μg of plasmids expressing 10E4 light and heavy chains (IgH plasmid and IgK plasmid) to 20mL of suspension cell culture medium and shake to mix; Tube B: Add 1.2mg of PEI transfection reagent to 20mL of suspension cell culture medium and shake to mix. Add the solution in tube B to tube A, shake to mix and incubate at room temperature for 15 minutes. Then add the mixed liquid to 400mL of cell culture system and place it in a cell shaker at 100rpm, 37℃, 5% CO2 for 6 days to express the antibody. After the culture is completed, collect the cell supernatant and incubate at 4℃, 4000rpm for 10 minutes.

[0166] 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 10E4 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.

[0167] Example 2 ELISA determination of the binding activity of monoclonal antibody 10E4 to gHgL protein

[0168] 2.1 Preparation of reaction plates

[0169] The full-length gHgL extracellular domain protein was diluted with 50 mM CB buffer (NaHCO₃ / Na₂CO₃ buffer, final concentration 50 mM, pH 9.6) to prepare a coating solution with a final gHgL protein concentration of 2 μg / mL. 100 μL of coating solution was added to each well of a 96-well microtiter plate and coated at 37°C for 2 h. The plate was washed once with PBST (20 mM PB7.4, 150 mM NaCl, 0.1% Tween₂O). Then, 200 μL of blocking solution (20 mM Na₂HPO₄ / NaH₂PO₄ buffer, pH 7.4, containing 20% ​​calf serum and 1% casein) was added to each well and blocked at 37°C for 2 h. The blocking solution was discarded, and the plate was dried and stored in aluminum foil bags at 2-8°C until ready for use.

[0170] 2.2 ELISA assay for the reactivity of monoclonal antibody 10E4 with gHgL protein

[0171] Monoclonal antibody 10E4 obtained in Example 1 (using 9E1 as a control antibody, which is disclosed 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 a starting concentration of 1 μg / mL, for a total of 20 dilutions. 100 μL of the diluted sample was added to each well of the gHgL protein-coated ELISA plate from step 2.1 above, and the plate was 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), and 100 μL of HRP-labeled goat anti-rabbit IgG reaction solution (Abeam, Catalog No. ab6721) 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 Biological Pharmaceutical Co., Ltd.) (50 μL) was added to each well and incubated at 37°C for 15 minutes. After the color development step, stop solution (purchased from Beijing Wantai Biological Pharmaceutical 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.

[0172] The results are as follows Figure 1 As shown, the EC of rabbit monoclonal antibody 10E4 and gHgL protein 50 The value was 33.49 ng / mL, and the results showed that monoclonal antibody 10E4 had good binding activity to the purified gHgL protein.

[0173] 2.3 Western Blot Detection of Rabbit Monoclonal Antibody 10E4 Against gHgL Protein

[0174] The gHgL protein sample was added to reducing buffer and heated at 100°C for 10 min to prepare the sample, and then subjected to SDS-PAGE protein electrophoresis.

[0175] Transfer: Use a transfer apparatus to transfer the proteins on the protein gel to the nitrocellulose membrane.

[0176] Blocking: Rinse the membrane with ultrapure water, add commercial blocking solution-1 (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.), and incubate at room temperature for 2 h.

[0177] Primary antibody incubation: Discard the blocking solution, dilute the monoclonal antibody 10E4 to 1 μg / mL with commercial enzyme diluent ED-13 (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.), add it to the membrane, and incubate it on a shaker at room temperature for 1 hour.

[0178] Secondary antibody incubation: Use PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20) to wash away unbound primary antibody on the membrane, wash the membrane three times, 5 minutes each time, add diluted HRP-labeled goat anti-rabbit IgG reaction solution (purchased from Abcam, cat. no. ab6721), and incubate on a shaker at room temperature for 1 hour.

[0179] Wash with PBST (20mM PB7.4, 150mM NaCl, 0.1% Tween20) three times, 5 minutes each time. Add an appropriate amount of chemiluminescent substrate mixture, cover the surface of the nitrocellulose membrane, and image and take pictures on a chemiluminescent imager. The results are as follows Figure 2 shown.

[0180] from Figure 2 The results showed that the rabbit monoclonal antibody 10E4 can be used for Western blot detection of gHgL protein.

[0181] Example 3 Detection of affinity constant between monoclonal antibody 10E4 and gHgL protein

[0182] The binding kinetics of the monoclonal antibody 10E4 and the antigen gHgL were analyzed using the Biacore 8K system (Cytiva). All steps were performed in PBS buffer. The monoclonal antibody was captured at a dilution of 1 μg / mL using the company's Protein A chip. EBV-gHgL protein was used as the detection antigen in a gradient of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM. The assay was performed according to the following protocol: capture 60 s, analysis 120 s, dissociation 240 s, and regeneration 60 s. The equilibrium dissociation constant of the antibody was calculated using the instrument's accompanying data acquisition and analysis software.

[0183] The results are as follows Figure 3 As shown, the equilibrium dissociation constant (K D ) was 3.53 nM, indicating that the rabbit monoclonal antibody 10E4 had a high affinity binding ability to gHgL protein.

[0184] Example 4 Detection of Overexpressed EB Virus gHgL Protein in Cells Using Monoclonal Antibody 10E4 (Flow Cytometry)

[0185] (1) 293T cells were seeded into a 10 cm cell culture plate and transfected when the cell confluence reached 60-80%.

[0186] (2) Tube A: Add 30 μg of the eukaryotic expression plasmid containing the full-length gene of EBV gL, gH protein, or the full-length gene of gL (pCAGGS-gH (gH accession number: BAU51590.1; vector: pCAGGS), pCAGGS-gL (gL accession number: BAU51568.1, vector: pCAGGS)) to 2 mL of Opti-MEM;

[0187] (3) Tube B: Add 60 μL of Novozymes to 2 mL of Opti-MEM. TransfectionReagent;

[0188] (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;

[0189] (5) Add the plasmid-transfection reagent complex dropwise to the cells and culture them in a cell culture incubator;

[0190] (6) 48 h after transfection, the cells were digested with trypsin and 5×10 5The cells were placed in Eppendorf tubes, centrifuged at 450 × g for 5 min, washed once with 1 mL of PBS, centrifuged at 450 × g for 5 min, 10E4 (9E1 was used as a control antibody, which has been disclosed in the literature: Rabbit Monoclonal Antibody Specifically Recognizing a Linear Epitope in the RBD of SARS-CoV-2 Spike Protein) was diluted to 10 μg / mL with PBS, 50 μL was added to each tube, incubated at 4°C for 30 min, and washed three times with 1 mL of PBS per tube;

[0191] (7) BV421-labeled goat anti-rabbit IgG secondary antibody was diluted 1:500 with 2% BSA in PBS, 50 μL was added to each tube, and incubated at 4°C for 30 min. 1 mL of PBS was added to each tube and washed three times;

[0192] (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.

[0193] The results are as follows Figure 4 As shown, monoclonal antibody 10E4 had a significant reaction to 293T cells transfected with gHgL protein or gL protein full-length gene plasmid, while the control antibody had no reaction, indicating that monoclonal antibody 10E4 can specifically recognize natural gHgL protein or gL protein expressed on the cell surface.

[0194] Example 5 Detection of gHgL protein expression in EBV-positive cell lines using monoclonal antibody 10E4

[0195] Akata-EBV-GFP cells (disclosed in the literature: An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus) were washed once with 1 mL of PBS, blocked with anti-human FcR block (BioLegend) at 4°C for 30 min, and then treated with Fixation Buffer / Permeabilization Wash Buffer. 10E4 (9E1 was used as a control antibody, which was disclosed in the literature: Rabbit Monoclonal Antibody Specifically Recognizing a Linear Epitope in the RBD of SARS-CoV-2 Spike Protein) was diluted to 10 μg / mL with PBS, 50 μL was added to each tube, incubated at 4°C for 30 min, and washed three times with 1 mL of PBS. BV421-labeled goat anti-rabbit IgG secondary antibody was diluted 1:500 in 2% BSA in PBS. 50 μL was added to each tube and incubated at 4°C for 30 min. Each tube was washed three times with 1 mL of PBS. The cell pellet was resuspended in 500 μL of PBS and the proportion of BV421-positive cells was determined using a BD flow cytometer, the LSRFortessa X-20.

[0196] The results are as follows Figure 5 As shown, 10E4 can bind to the gHgL protein in EBV-positive cell lines. The above results indicate that 10E4 can recognize the native gHgL protein in EBV-positive cell lines.

[0197] Example 6 Analysis of the Neutralizing Ability of Monoclonal Antibody 10E4 in a Virus Infection Model

[0198] EBV B cell neutralization model:

[0199] Take monoclonal antibody 10E4 and irrelevant antibody VRC01, dilute them with RPMI1640 serum-free medium, start with 100 μg / mL as the starting concentration, and dilute them 3-fold. Dilute 8 gradients. Take 20 μL of diluted antibody in a 96-well cell plate, add 20 μL of virus dilution carrying GFP green fluorescent protein gene produced by CNE2 cells (the virus dilution has been disclosed in AnAntibody Targeting the Fusion Mach inery Neutralizes Dual-Tropic Infectionand Defines a Site of Vulnerability on Epstein-Barr Virus), mix well, and incubate in a 37 ° C incubator for 2 hours. Resuspend 1×10 6 Akata cells were cultured, 160 μL of cell suspension was added to the above virus and antibody mixture, and the cells were incubated in a 37°C incubator for 48 h. The virus infection rate of Akata cells was detected using a BD flow cytometer LSRFortessaX-20. The reduction in the number of GFP-positive cells in the antibody-treated group was calculated compared with the infection control group (adding an equal volume of RPMI1640 serum-free medium). The inhibition rate of the antibody in the B cell infection model (neutralization efficiency, %) was calculated.

[0200] EBV Neutralization Model in Epithelial Cells:

[0201] HNE1 cells were plated in 96-well plates in 160 μL of 10% FBS DMEM medium at a standard density of 5,000 cells / well and incubated at 37°C for 24 hours. After the cells adhered, monoclonal antibody 10E4 and an unrelated antibody, VRC01, were diluted in serum-free DMEM medium, starting at 100 μg / mL and serially diluted 2-fold over 12 steps. Twenty μL of each diluted antibody concentration was mixed thoroughly with 20 μL of EBV suspension produced in Akata cells (virus dilutions are disclosed in the literature: An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus). The mixture was incubated at 37°C for 3 hours. The antibody-virus mixture was then added to the 96-well plate containing HNE1 cells and incubated at 37°C. After 48 hours, HNE1 cells in 96-well plates were digested, and the proportion of HNE1 cells expressing GFP green fluorescent protein was detected using BD flow cytometer LSRFortess aX-20. The reduction ratio (%) of the number of GFP-positive cells in the antibody-treated group was calculated compared with the infection control group (addition of an equal volume of RPMI1640 serum-free medium), and the inhibition rate (neutralization efficiency, %) of the antibody in the epithelial cell infection model was calculated.

[0202] The results are as follows Figure 6 As shown, the IC of monoclonal antibody 10E4 in B cell and epithelial cell infection models 50 The concentrations of monoclonal antibody 10E4 were 15.15 μg / mL and 0.73 μg / mL, respectively. This indicates that monoclonal antibody 10E4 can neutralize viral infection in both B cell infection model and epithelial cell infection model.

[0203] Example 7 Analysis of the blocking ability of monoclonal antibody 10E4 in a cell fusion model

[0204] 293T cells were seeded into 10 cm cell culture plates 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, and gL (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), and pCAGSS-T7 (T7 accession number: CP053597.1, vector: pCAGGS). The specific construction method is referenced in the literature: Fusion of epithelial cells by Epstein–Barr virus proteins is triggered by binding of viral glycoproteins gHgL to Panel B: 293T cells were transfected with 10 μg of a eukaryotic expression plasmid containing the Luciferase gene controlled by a T7 promoter (pT7EMCLuc (Luciferase accession number: M15077.1, vector: pCAG) using PEI as the transfection reagent. For the specific construction method, refer to the reference: Fusion of epithelial cells by Epstein–Barr virus proteins is triggered by binding of viral glycoproteins gHgL to integrins αvβ6 or αvβ8).

[0205] 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 (10E4, 1 reported monoclonal antibody AMMO1 (Immunity, 2018, 48: 799-811.e9), control monoclonal antibody VRC01, and no Ab group added with equal amount of solvent) were added. The cells were incubated in a 37°C incubator for 30 minutes and then transferred to a 24-well plate. The cells in plate B were digested in advance and 2×10 5 cells and cultured at 37°C for 24 h.

[0206] 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 well (No Ab group) was calculated to determine the blocking efficiency (%) of the antibody in the cell fusion model.

[0207] The results are as follows Figure 7 As shown, the results showed that the monoclonal antibody 10E4 had a concentration-dependent blocking effect on the cell fusion model, indicating that it had a specific blocking effect on cell membrane fusion, and under the same concentration conditions, the membrane fusion inhibition effect of 10E4 was comparable to that of AMMO1.

[0208] Example 8 Epitope Identification Results of Monoclonal Antibody 10E4

[0209] The sequence of aa 24-137 region of gL protein was synthesized into a peptide chain with a length of 20 aa per peptide chain and a sequence overlap of 10 aa between adjacent peptides, resulting in a total of 11 peptide chains (e.g. Figure 8 The peptide chain was labeled with biotin at its N-terminus. Peptide synthesis and biotin labeling were completed by GenScript.

[0210] Dilute the polypeptide chain in 50mM CB buffer (NaHCO₃ / Na₂CO₃ buffer, final concentration 50mM, pH 9.6) to prepare a coating solution with a final concentration of 2μg / mL. Add 100μL of coating solution to each well of a 96-well streptomycin-precoated ELISA plate and incubate at 37°C for 2 hours. Wash once with PBST (20mM PB7.4, 150mM NaCl, 0.1% Tween₂O). Then, add 200μL of blocking solution (20mM Na₂HPO₄ / NaH₂PO₄ buffer, pH 7.4, containing 20% ​​calf serum and 1% casein) to each well and incubate at 37°C for 2 hours. Discard the blocking solution, dry the plate, and store in aluminum foil bags at 2-8°C until ready for use.

[0211] The monoclonal antibody 10E4 obtained in Example 1 was diluted to 1 μg / mL in 20 mM PBS buffer. 100 μL of the diluted antibody was added to each well of the gL polypeptide-coated ELISA plate and the gHgL protease-coated plate described above, and the plate was incubated at 37°C for 30 minutes. The plate was washed five times with PBST (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), and 100 μL of HRP-labeled goat anti-rabbit IgG reaction solution (Abeam, Catalog No. ab6721) was added to each well and incubated at 37°C for 30 minutes. 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.) was added to each well and incubated at 37°C for 15 min. After the color development step, 50 μL of stop solution (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) was added to each well of the plate, and the OD450 / 630 values ​​of each well were measured on a microplate reader.

[0212] The results are as follows Figure 8 As shown, the results showed that the monoclonal antibody 10E4 reacted with the P9 peptide and the positive control gHgL protein, that is, the monoclonal antibody 10E4 recognized 104 to 123 aa.

[0213] Example 9 Identification of key sites of the epitope recognized by monoclonal antibody 10E4

[0214] 9.1 Construction of point mutant protein of HgL extracellular domain

[0215] In order to identify the key sites recognized by rabbit neutralizing antibodies on gHgL protein, point mutation cloning, expression and evaluation of gHgL protein extracellular domain were carried out.

[0216] Primers were designed according to the instructions of the Mut Express II Fast Mutagenesis Kit V2 (purchased from Novazom) and point mutation cloning was performed to mutate the amino acids at the corresponding sites in the extracellular domain of the gHgL protein to alanine (gHgL-L104A indicates a gHgL protein in which the 104th amino acid in the gHgL protein (sequence shown in SEQ ID NO. 12) was mutated to alanine; gHgL-R105A indicates a gHgL protein in which the 105th amino acid in the gHgL protein was mutated to alanine; gHgL-E106A indicates a gHgL protein in which the 106th amino acid in the gHgL protein was mutated to alanine; gHgL-L107A indicates a gHgL protein in which the 107th amino acid in the gHgL protein was mutated to alanine; gHgL-L108A indicates a gHgL protein in which the 108th amino acid in the gHgL protein was mutated to alanine). gHgL protein with amino acid 109 mutated to alanine; gHgL-T109A represents a gHgL protein with amino acid 109 mutated to alanine; gHgL-T110A represents a gHgL protein with amino acid 110 mutated to alanine; gHgL-L111A represents a gHgL protein with amino acid 111 mutated to alanine; gHgL-E112A represents a gHgL protein with amino acid 112 mutated to alanine; gHgL-T113A represents a gHgL protein with amino acid 113 mutated to alanine; gHgL protein in which the amino acid at position 113 of the gHgL protein is mutated to alanine; gHgL-L114A indicates a gHgL protein in which the amino acid at position 114 of the gHgL protein is mutated to alanine; gHgL-Y115A indicates a gHgL protein in which the amino acid at position 115 of the gHgL protein is mutated to alanine; gHgL-G116A indicates a gHgL protein in which the amino acid at position 116 of the gHgL protein is mutated to alanine; gHgL-S117A indicates a gHgL protein in which the amino acid at position 117 of the gHgL protein is mutated to alanine gHgL protein; gHgL-F118A represents a gHgL protein in which the 118th amino acid in the gHgL protein is mutated to alanine; gHgL-S119A represents a gHgL protein in which the 119th amino acid in the gHgL protein is mutated to alanine; gHgL-V120A represents a gHgL protein in which the 120th amino acid in the gHgL protein is mutated to alanine; gHgL-E121A represents a gHgL protein in which the 121st amino acid in the gHgL protein is mutated to alanine; the sequence of gHgL-WT is shown in SEQ ID NO.12), and sequencing was used to verify whether the point mutation clone was constructed correctly. The correct point mutation clone was transiently transfected into 293F cells for eukaryotic expression, and the point mutation protein was purified by nickel column.

[0217] 9.2 Western Blot Identification of Key Antibody Recognition Sites

[0218] The wild-type (WT) or point-mutated gHgL protein samples obtained in step 9.1 above were added to reducing buffer and heated at 100°C for 10 min to prepare the samples, and then subjected to SDS-PAGE protein electrophoresis.

[0219] Transfer: Use a transfer apparatus to transfer the proteins on the protein gel to the nitrocellulose membrane.

[0220] Blocking: Rinse the membrane with ultrapure water, add commercial blocking solution (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.), and incubate at room temperature for 2 h.

[0221] Primary antibody incubation: Discard the blocking solution, dilute the monoclonal antibody 10E4 to 1 μg / mL with commercial enzyme diluent ED-13 (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.), add it to the membrane, and incubate it on a shaker at room temperature for 1 hour.

[0222] Add secondary antibody: Wash the membrane with PBST (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20) to remove unbound primary antibody. Wash the membrane three times for 5 min each time. Add diluted HRP-labeled goat anti-rabbit IgG reaction solution (purchased from Abcam, cat. no. ab6721) and incubate on a shaker at room temperature for 1 h.

[0223] Wash the membrane with PBST (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20) to remove any unbound secondary antibody. Wash for 5 minutes each. Add an appropriate amount of chemiluminescent substrate mixture and coat the nitrocellulose membrane. Image and photograph the membrane using a chemiluminescent imager. Record the results.

[0224] Western blot results are as follows Figure 9 As shown, the results show that mutations at some sites affect the antibody's ability to bind to the protein. Seven amino acid sites, 106, 108, 109, 110, 112, 114, and 116, are key sites for the 10E4 antibody. Mutations in any of these sites can cause the monoclonal antibody 10E4 to be unresponsive or have a weaker response to the protein.

[0225] 9.3 ELISA method to identify the reactivity of gHgL monoclonal antibodies with point mutation proteins

[0226] Dilute the wild-type (WT) or point-mutated gHgL protein samples obtained in step 9.1 above with 50 mM CB buffer (NaHCO₃ / Na₂CO₃ buffer, pH 9.6, final concentration 50 mM) to obtain a coating solution with a final 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 block at 37°C for 2 h. Discard the blocking solution. After drying, store in aluminum foil pouches at 2-8°C until ready for use.

[0227] Monoclonal antibody 10E4 obtained in Example 1 was diluted two-fold in 20 mM PBS buffer starting at a starting concentration of 10 μg / mL, for a total of 10 dilutions. 100 μL of the diluted sample was added to each well of a gHgL protein-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), and 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 step, the plate was washed five times with PBST (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), and 50 μL of TMB colorimetric reagent (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) was added to each well and incubated at 37°C for 15 min. After the color development step was completed, 50 μL of stop solution (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) was added to each well of the ELISA plate after the reaction, and the OD450 / 630 value of each well was detected on a microplate reader.

[0228] ELISA OD450 test results are as follows Figure 10 As shown in Figure 1, amino acid positions 106, 108, 109, 110, 112, 114, 116, and 119 are key sites for the 10E4 antibody. Mutation of any amino acid at these sites can cause the monoclonal antibody 10E4 to be unresponsive or have a weaker response to the protein. The position of the epitope recognized by the 10E4 monoclonal antibody in the protein 3D structure is shown in Figure 1. Figure 11 (Drawing with pymol software, PDB ID of EBV gHgL: 3PHF) is shown. Figure 12(Drawing with pymol software, PDB ID of EBV gHgL / gp42 / E1D1: 5T1D) As shown, the epitope recognized by 10E4 is different from that of E1D1. The epitope recognized by 10E4 monoclonal antibody is compared with the epitope binding to EphA2 LBD. Figure 13 (Drawing using pymol software, PDB ID of EBV gHgL / EphA2 LBD: 7CZE) As shown, the epitope recognized by 10E4 is different from the epitope binding to EphA2 LBD.

[0229] 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 gL 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: GFSLSTYW (SEQ ID NO. 2); The amino acid sequence of CDR2 of the heavy chain variable region is: IGGSGST (SEQ ID NO. 3); The amino acid sequence of CDR3 of the heavy chain variable region is: ARDSGAGVRFRF (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: ENIGSR (SEQ ID NO. 5); The amino acid sequence of CDR2 of the light chain variable region is: RAS; The amino acid sequence of CDR3 of the light chain variable region is: QCTYGVSITINYGND (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: QQVKESGGRLVTPGTPLTLTCTASGFSLSTYWMSWVRQAPGKGLEYIGVIGGSGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDSGAGVRFRFWGPGTLVTVSS (SEQ ID NO.8); The amino acid sequence of the light chain variable region comprises: DLVMTQTPASVEAGVGGTVTINCQASENIGSRLAWYQQKPGQPPKLLIYRASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQCTYGVSITINYGNDFGGGTEVVVK (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 gL protein and / or gHgL 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.

Images