Bispecific antibody targeting EB (Epstein-Barr) virus and application of bispecific antibody

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a bispecific antibody targeting EB virus and application of the bispecific antibody. The invention provides a bispecific antibody targeting EB virus, the bispecific antibody comprises 9G01, 9G02 and 9G03, IC50 of the 9G01, 9G02 and 9G03 in an RAJI B cell infection model is 4.268 * 10 <-6 > mu g / mL, 0.02 mu g / mL and 0.014 mu g / mL respectively, the bispecific antibody can significantly inhibit EB virus infection on epithelial cells and B cells, the neutralization efficiency is further improved, and the EB virus targeted bispecific antibody can be used for preparing a drug for treating EB virus infection. The probe can be used for detecting EB virus, diagnosing diseases caused by EB virus infection, preventing EB virus infection and / or treating and / or preventing diseases caused by EB virus infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a bispecific antibody targeting EB virus and its application. Background Technology

[0002] Epstein Barr virus (EB virus), also known as human herpesvirus 4, is a DNA oncogenic virus isolated and strained from Burkitt lymphoma cells by Epstein and Barr in 1964. It belongs to the genus Lymphovirus of the subfamily Gammaherpesvirinae and is a DNA oncogenic virus. The viral particle consists of a core protein, capsid, capsid, and envelope. It primarily infects oropharyngeal epithelial cells and B lymphocytes, mainly existing as a latent infection. More than 90% of adults carry the virus throughout their lives.

[0003] Epstein-Barr virus (EBV) initially replicates in the oropharynx, multiplying within B lymphocytes and oral epithelial cells before entering the bloodstream and causing systemic infection. It then remains latent in lymphoid tissues for extended periods. When the body's immune function is weakened, the latent virus is activated, potentially inducing various diseases. Primary infection can lead to infectious mononucleosis, manifesting as fever, pharyngitis, and lymphadenopathy. Persistent or reactivated infection may cause chronic active EBV infection or post-transplant lymphoproliferative disorder, and may even progress to hemophagocytic lymphohistiocytosis syndrome or malignant lymphoma. EBV is also closely associated with malignant tumors such as nasopharyngeal carcinoma, Hodgkin / non-Hodgkin lymphoma, Burkitt lymphoma, and gastric cancer. Currently, there are no effective vaccines or specific treatments for EBV infection. Infectious mononucleosis is often treated with antiviral drugs such as acyclovir to relieve symptoms, but these cannot completely eliminate the virus from cells. Related tumors primarily rely on chemotherapy and radiotherapy, with limited efficacy for metastatic or recurrent cases.

[0004] In recent years, researchers have made significant progress in understanding the interaction between Epstein-Barr virus (EBV) and host cells, as well as its infection pathways. gp350, the most abundant envelope glycoprotein of EBV, is the most abundant protein on the viral surface. It primarily initiates the infection process on B lymphocytes by binding to host cell surface receptors (such as CD21 / CR2). Due to its crucial role in viral invasion, gp350 has become an ideal target for vaccine development and antibody therapy. Gp350 antibodies can specifically recognize and bind to gp350, effectively blocking the binding of EBV to B lymphocytes, thereby preventing viral infection and transmission. EBV surface glycoproteins gH / gL form a high-affinity complex with gp42. gp42 mediates viral transmembrane fusion into B cells by binding to host HLA class II receptors, while entry into epithelial cells is initiated by the direct interaction of gH / gL with integrin receptors or EphA2 receptors. Furthermore, the structural analysis of glycoproteins such as gp350, gH / gL, and gp42, and the application of their monoclonal antibodies, provide a theoretical basis for preventing primary infection and controlling latent viral reactivation.

[0005] Bispecific antibodies, with their unique dual-target synergistic effect, have demonstrated significant advantages in the treatment of infectious diseases and cancer. In infectious diseases, bispecific antibodies can simultaneously and efficiently neutralize multiple key target proteins, effectively overcoming the limitations of single-target neutralization and improving neutralization efficiency. In cancer treatment, bispecific antibodies, through dual blockade, not only significantly enhance therapeutic efficacy but also reduce off-target toxicity. Furthermore, their safety and treatment sustainability have been further optimized through engineered design. Therefore, the development of bispecific antibodies targeting EBV gp350 and gH / gL / gp42 trimers holds promise for providing a more effective new approach to the prevention and treatment of EBV-related diseases. Summary of the Invention

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

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

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

[0009] A fourth aspect of the present invention is to provide a coupling agent.

[0010] The fifth aspect of this invention aims to provide the use of the bispecific 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.

[0011] The sixth aspect of this invention aims to provide a product.

[0012] The seventh aspect of this invention aims to provide a method for preparing a bispecific antibody or an antigen-binding fragment thereof according to the first aspect of this invention.

[0013] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: A first aspect of the present invention provides a bispecific antibody against EB virus or an antigen-binding fragment thereof, said bispecific antibody or antigen-binding fragment comprising: Targeting the first functional region of the gp350 protein, and Secondary protein functional regions that target gH / gL / gp42, gp42, or gB proteins.

[0014] in: The first protein functional region is an immunoglobulin; The functional region of the first protein includes a heavy chain and a light chain; The second protein functional region is a single-chain antibody; The second protein functional region includes a heavy chain and a light chain.

[0015] In some embodiments of the present invention, the first protein functional region targeting the gp350 protein includes a heavy chain and a light chain.

[0016] The second protein functional region includes any one of a1) to a3): a1) Targets the second protein functional region of the gH / gL / gp42 protein complex; a2) Targets the second functional region of the gp42 protein; a3) Targets the second protein functional region of gB protein.

[0017] In some embodiments of the present invention, the heavy chain of the first protein functional region includes a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 81; In some embodiments of the present invention, the light chain of the first protein functional region includes a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, the light chain variable region having the amino acid sequence shown in SEQ ID NO: 84.

[0018] In some embodiments of the present invention, the CDR sequence of the heavy chain variable region of the first protein functional region is shown in any one of b1) to b4): b1) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the first protein functional region are shown in SEQ ID NO: 2, 3 and 4 respectively, wherein the CDR is defined according to the IMGT definition scheme; b2) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the first protein functional region are shown in SEQ ID NO: 10, 11 and 12 respectively, wherein the CDR is defined according to the Kabat definition scheme; b3) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the first protein functional region are shown in SEQ ID NO: 18, 19 and 12 respectively, wherein the CDR is defined according to the Chothia definition scheme; b4) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the first protein functional region are shown in SEQ ID NO: 22, 23 and 24 respectively, wherein the CDR is defined according to the Contact definition scheme; In some embodiments of the present invention, the CDR sequence of the light chain variable region of the first protein functional region is shown in any one of b5) to b8): b5) The amino acid sequences of CDR-L1 and CDR-L3 of the light chain variable region of the first protein functional region are shown in SEQ ID NO: 33 and 34 respectively, and the amino acid sequence of CDR-L2 is GAS. The CDR is defined according to the IMGT definition scheme. b6) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the first protein functional region are shown in SEQ ID NO: 35, 36 and 34 respectively, wherein the CDR is defined according to the Kabat definition scheme; b7) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the first protein functional region are shown in SEQ ID NO: 35, 36 and 34, respectively, wherein the CDR is defined according to the Chothia definition scheme; b8) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the first protein functional region are shown in SEQ ID NO: 37, 38 and 39 respectively, wherein the CDR is defined by the Contact definition scheme.

[0019] In some embodiments of the present invention, the first protein functional region targeting gp350 protein is a monoclonal antibody or its antigen-binding fragment 1A12, as detailed in patent CN202411836537.1.

[0020] In some embodiments of the present invention, the functional region of the second protein is a single-chain antibody; The second protein functional region includes a heavy chain and a light chain.

[0021] a1) The second protein functional region targeting the gH / gL / gp42 protein complex includes: The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 82.

[0022] The light chain variable region includes CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, the light chain variable region having the amino acid sequence shown in SEQ ID NO: 83.

[0023] In some embodiments of the present invention, the CDR sequence of the heavy chain variable region of the second protein functional region is shown in any one of c1) to c4): c1) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region are shown in SEQ ID NO: 5, 6 and 7 respectively, wherein the CDR is defined according to the IMGT definition scheme; c2) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region are shown in SEQ ID NO: 13, 14 and 15, respectively, wherein the CDR is defined according to the Kabat definition scheme; c3) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region are shown in SEQ ID NO: 20, 21 and 15, respectively, wherein the CDR is defined according to the Chothia definition scheme; c4) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region are shown in SEQ ID NO: 25, 26 and 27 respectively, wherein the CDR is defined by the Contact definition scheme.

[0024] In some embodiments of the present invention, the CDR sequence of the light chain variable region of the second protein functional region is shown in any one of c5) to c8): c5) The amino acid sequences of CDR-L1 and CDR-L3 in the light chain variable region of the second protein functional region are shown in SEQ ID NO: 8 and 9, respectively. The amino acid sequence of CDR-L2 is DVS. The CDR is defined according to the IMGT definition scheme. c6) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein functional region are shown in SEQ ID NO: 16, 17 and 9 respectively, wherein the CDR is defined according to the Kabat definition scheme; c7) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein functional region are shown in SEQ ID NO: 16, 17 and 9 respectively, wherein the CDR is defined according to the Chothia definition scheme; c8) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein functional region are shown in SEQ ID NO: 28, 29 and 30, respectively, wherein the CDR is defined using the Contact definition scheme.

[0025] In some embodiments of the present invention, the second protein functional region of the gH / gL / gp42 protein complex is a monoclonal antibody or its antigen-binding fragment 4G12, as detailed in patent CN2023114999477.

[0026] a2) The second protein functional region targeting the gp42 protein includes: The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 85; The light chain variable region includes CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, the light chain variable region having the amino acid sequence shown in SEQ ID NO: 86.

[0027] In some embodiments of the present invention, the CDR sequence of the heavy chain variable region of the second protein functional region is shown in any one of d1) to d4): d1) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region are shown in SEQ ID NO: 42, 43 and 44 respectively, wherein the CDR is defined according to the IMGT definition scheme; d2) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region are shown in SEQ ID NO: 47, 48 and 49, respectively, wherein the CDR is defined according to the Kabat definition scheme; d3) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region are shown in SEQ ID NO: 52, 53 and 49, respectively, wherein the CDR is defined according to the Chothia definition scheme; d4) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region are shown in SEQ ID NO: 54, 55 and 56 respectively, wherein the CDR is defined according to the Contact definition scheme; In some embodiments of the present invention, the CDR sequence of the light chain variable region of the second protein functional region is shown in any one of d5) to d8): d5) The amino acid sequences of CDR-L1 and CDR-L3 of the light chain variable region of the second protein functional region are shown in SEQ ID NO: 45 and 46 respectively, and the amino acid sequence of CDR-L2 is ATS. The CDR is defined according to the IMGT definition scheme. d6) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein functional region are shown in SEQ ID NO: 50, 51 and 46 respectively, wherein the CDR is defined according to the Kabat definition scheme; d7) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein functional region are shown in SEQ ID NO: 50, 51 and 46 respectively, wherein the CDR is defined according to the Chothia definition scheme; d8) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein functional region are shown in SEQ ID NO: 57, 58 and 59 respectively, wherein the CDR is defined by the Contact definition scheme.

[0028] In some embodiments of the present invention, the second protein functional region of the gp42 protein is a monoclonal antibody or its antigen-binding fragment 2C1, as detailed in patent CN2023102619617.

[0029] a3) The second protein functional region targeting the gB protein includes: The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 87; The light chain variable region includes CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, the light chain variable region having the amino acid sequence shown in SEQ ID NO: 88.

[0030] In some embodiments of the present invention, the CDR sequence of the heavy chain variable region of the second protein functional region is shown in any one of e1) to e4): e1) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region are shown in SEQ ID NO: 62, 63 and 64, respectively, wherein the CDR is defined according to the IMGT definition scheme; e2) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region are shown in SEQ ID NO: 67, 68 and 69, respectively, wherein the CDR is defined according to the Kabat definition scheme; e3) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region are shown in SEQ ID NO: 72, 73 and 69, respectively, wherein the CDR is defined according to the Chothia definition scheme; e4) The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region are shown in SEQ ID NO: 74, 75 and 76 respectively, wherein the CDR is defined by the Contact definition scheme.

[0031] In some embodiments of the present invention, the CDR sequence of the light chain variable region of the second protein functional region is shown in any one of e5) to e8): e5) The amino acid sequences of CDR-L1 and CDR-L3 in the light chain variable region of the second protein functional region are shown in SEQ ID NO: 65 and 66 respectively, and the amino acid sequence of CDR-L2 is AAS. The CDR is defined according to the IMGT definition scheme. e6) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein functional region are shown in SEQ ID NO: 70, 71 and 66 respectively, wherein the CDR is defined according to the Kabat definition scheme; e7) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein functional region are shown in SEQ ID NO: 70, 71 and 66 respectively, wherein the CDR is defined according to the Chothia definition scheme; e8) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein functional region are shown in SEQ ID NO: 77, 78 and 79 respectively, wherein the CDR is defined by the Contact definition scheme.

[0032] In some embodiments of the present invention, the second protein functional region of the gp42 protein is a monoclonal antibody or its antigen-binding fragment 4H2, as detailed in patent CN2022108837606.

[0033] In some embodiments of the present invention, the second protein functional region is directly connected to the first protein functional region or connected through a linker fragment; and / or the heavy chain and light chain of the second protein functional region are directly connected or connected through a linker fragment.

[0034] In some embodiments of the present invention, the connection segment includes (GGGGS)n, where n is a positive integer and can be 1, 2, 3, 4, 5, or 6.

[0035] In some embodiments of the present invention, prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al., Proc. Natl. Acad. Sci. USA 90 (1993): 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al., Protein Eng. 8 (1995): 725731, Choi et al., Eur. J. Immunol. 31 (2001): 94-106, Hu et al., Cancer Res. 56 (1996): 3055-3061, Kipriyanov et al., J. Mol. Biol. 293 (1999): 41-56, and Roovers et al., Cancer Immunol. (2001).

[0036] In some embodiments of the present invention, the heavy chain of the first protein functional region is directly connected to the second protein functional region or through a linker fragment.

[0037] In some embodiments of the present invention, the anti-EB virus bispecific antibody or its antigen-binding fragment comprises: 1) 9G01 antibody; The 9G01 antibody consists of a first protein functional region that targets the gp350 protein and a second protein functional region that targets the gH / gL / gp42 protein complex. 2) 9G02 antibody; The 9G02 antibody consists of a first protein functional region that targets the gp350 protein and a second protein functional region that targets the gp42 protein. 3) 9G03 antibody; The 9G03 antibody consists of a first protein functional region that targets the gp350 protein and a second protein functional region that targets the gB protein.

[0038] In some embodiments of the present invention, the full-length heavy and light chain amino acid sequences of the 9G01 antibody are as shown in SEQ ID NO: 1 and 32.

[0039] In some embodiments of the present invention, the full-length heavy and light chain amino acid sequences of the 9G02 antibody are as shown in SEQ ID NO: 41 and 32.

[0040] In some embodiments of the present invention, the full-length heavy and light chain amino acid sequences of the 9G03 antibody are as shown in SEQ ID NO: 61 and 32.

[0041] A second aspect of the invention provides a recombinant protein comprising: a bispecific antibody or an antigen-binding fragment thereof of the first aspect of the invention; and optionally a tag sequence for assisting expression and / or purification.

[0042] In some embodiments of the present invention, the tag sequence is selected from at least one of the following groups: His tag, GGGS sequence, FLAG tag; further, His tag; and even further, 6×His tag.

[0043] A third aspect of the invention provides biological materials related to the bispecific antibody or its antigen-binding fragment of the first aspect of the invention, or the recombinant protein of the second aspect of the invention, said biological material comprising at least one of f1) to f16): f1) A nucleic acid molecule encoding a bispecific antibody or its antigen-binding fragment of any one of claims 1 to 3, or the recombinant protein of claim 4; f2) An expression cassette containing the nucleic acid molecule described in f1); f3) A carrier containing the nucleic acid molecule described in f1); f4) A carrier containing the expression box described in f2); f5) A transgenic cell line containing the nucleic acid molecules described in f1); f6) Transgenic cell lines containing the expression cassette described in f2); f7) A transgenic cell line containing the vector described in f3); f8) A transgenic cell line containing the vector described in f4); f9) Microorganisms containing the nucleic acid molecules described in f1); f10) Microorganisms containing the expression cassette described in f2); f11) Microorganisms containing the carrier described in f3); f12) Microorganisms containing the carrier described in f4); f13) A virus containing the nucleic acid molecule described in f1); f14) is a virus containing the expression box described in f2); f15) is a virus containing the vector described in f3); f16) contains a virus with the vector described in f4).

[0044] In some embodiments of the present invention, the transgenic cell line does not contain propagation material.

[0045] In some embodiments of the present invention, the nucleic acid molecule encoding the bispecific antibody or antigen-binding fragment thereof as described in the first aspect of the present invention comprises a nucleic acid molecule encoding a heavy chain of the bispecific antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, and a nucleic acid molecule encoding a light chain of the bispecific antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.

[0046] In some embodiments of the present invention, the sequences of the nucleic acid molecules encoding the heavy chain and light chain amino acids of the 9G01 antibody are shown as SEQ ID NO: 31 and 40, respectively.

[0047] In some embodiments of the present invention, the sequences of the nucleic acid molecules encoding the heavy chain and light chain amino acids of the 9G02 antibody are shown as SEQ ID NO: 60 and 40, respectively.

[0048] In some embodiments of the present invention, the sequences of the nucleic acid molecules encoding the heavy chain and light chain amino acids of the 9G03 antibody are shown as SEQ ID NO: 80 and 40, respectively.

[0049] A fourth aspect of the present invention provides a conjugate comprising at least one of a bispecific antibody or an antigen-binding fragment thereof from the first aspect of the present invention and a recombinant protein from the second aspect of the present invention; And a coupling portion, the coupling portion comprising at least one of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

[0050] In some embodiments of the present invention, the detectable marker is selected from radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, or any combination thereof.

[0051] In some embodiments of the present invention, the conjugate is selected from: fluorescent substances, chemiluminescent markers, colored substances, radioactive isotopes, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines, antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes, chemotherapeutic agents, or any form of nanoparticles.

[0052] A fifth aspect of the present invention provides the use of the bispecific 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 a product; The product includes at least one of the following: drug, reagent, test plate, reagent kit, and test chip.

[0053] In some embodiments of the present invention, the drug has at least one function among g1) to g2): g1) Prevention of EB virus infection; g2) Treatment and / or prevention of diseases caused by EB virus infection.

[0054] In some embodiments of the present invention, the reagent, detection plate, detection chip, or reagent kit has at least one function among h1) to h3): h1) Detect the presence or level of gH / gL / gp42, gp42, gB or gp350 protein in the sample; h2) Detection of EB virus; h3) Diagnose diseases caused by EB virus infection.

[0055] In some embodiments of the present invention, the disease includes at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disorders, and infectious mononucleosis.

[0056] In some embodiments of the present invention, the drug comprises a vaccine.

[0057] A sixth aspect of the present invention provides a product comprising a bispecific antibody or an antigen-binding fragment thereof of the first aspect, a recombinant protein of the second aspect, a biomaterial of the third aspect, and / or a conjugate of the fourth aspect.

[0058] The product includes at least one of reagents, test plates, reagent kits, and test chips.

[0059] In some embodiments of the present invention, the product has at least one function among h1) to h3): h1) Detect the presence or level of gH / gL / gp42, gp42, gB or gp350 protein in the sample; h2) Detection of EB virus; h3) Diagnose diseases caused by EB virus infection.

[0060] In some embodiments of the present invention, the disease includes at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disorders, and infectious mononucleosis.

[0061] A seventh aspect of the present invention provides a medicament comprising a bispecific antibody or an antigen-binding fragment thereof of the first aspect, a recombinant protein of the second aspect, a biological material of the third aspect, and / or a conjugate of the fourth aspect.

[0062] In some embodiments of the present invention, the medicament further comprises a pharmaceutically acceptable carrier.

[0063] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: propellants, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors.

[0064] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0065] In some embodiments of the present invention, the drug has at least one function among g1) to g2): g1) Prevention of EB virus infection; g2) Treatment and / or prevention of diseases caused by EB virus infection.

[0066] In some embodiments of the present invention, the disease includes at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disorders, and infectious mononucleosis.

[0067] The eighth aspect of the present invention is to provide a method for preparing a bispecific antibody or antigen-binding fragment thereof from the first aspect of the present invention or a recombinant protein from the second aspect of the present invention, obtained by culturing the transgenic cell line, microorganism or virus from the third aspect of the present invention.

[0068] The beneficial effects of this invention are: This invention provides a bispecific antibody targeting Epstein-Barr virus (EBV), comprising 9G01, 9G02, and 9G03, wherein 9G01, 9G02, and 9G03 have an IC50 value in a RAJI B cell infection model. 50 They are 4.268×10 -6 These bispecific antibodies, at concentrations of μg / mL, 0.02 μg / mL, and 0.014 μg / mL, can significantly inhibit EBV infection of epithelial cells and B cells, further improving neutralization efficiency. They can be used to detect EBV, diagnose diseases caused by EBV infection, prevent EBV infection and / or treat and / or prevent diseases caused by EBV infection. Attached Figure Description

[0069] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results of Coomassie Brilliant Blue staining with 9G01 antibody are shown.

[0070] Figure 2 The results of Coomassie Brilliant Blue staining with 9G02 antibody.

[0071] Figure 3 The results of Coomassie brilliant blue staining with 9G03 antibody are shown.

[0072] Figure 4 Results of affinity determination for the 9G01 antibody using biomembrane interference (BLI) technique.

[0073] Figure 5 The results show the neutralization rate of the 9G01 antibody, where: B: 9G01 blocks EBV infection on Raji; C: 9G01 blocks EBV infection on Akata; D: 9G01 blocks EBV infection on Daudi B; and E: 9G01 blocks EBV infection on HEK293.

[0074] Figure 6 The results show the effect of the 9G01 antibody on the body weight of mice.

[0075] Figure 7 The results show the effect of the 9G01 antibody on the survival rate of virus-infected mice.

[0076] Figure 8Results of affinity determination for the 9G02 antibody using biomembrane interference (BLI) technique.

[0077] Figure 9 The results show the neutralization rate of the 9G02 antibody.

[0078] Figure 10 The results show the neutralization rate of the 9G03 antibody.

[0079] Figure 11 The results show the comparison of neutralization rates of antibodies 9G01, 9G02, and 9G03. Detailed Implementation

[0080] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0081] Example 1: Expression and purification of 9G01 antibody Ligating the upstream of the variable region of the heavy chain of gp350 monoclonal antibody 1A12 with a CMV fragment, the downstream of the variable region, the constant region of human IgG1, GGGS, the variable region of the heavy chain of gHgLgp42 monoclonal antibody 4G12, the variable region of the light chain of ghglgp42 monoclonal antibody 4G12, and the polyA fragment allows for the expression of the complete heavy chain fragment. Similarly, ligating the upstream of the variable region of the light chain of gp350 monoclonal antibody 1A12 with a CMV fragment, the downstream of the constant region of the light chain κ / λ, and the polyA fragment allows for the expression of the complete light chain fragment. Co-transfection of 293F cells with plasmids containing the full-length sequences of the antibody heavy and light chains achieves antibody expression, and protein A beads are used for antibody purification.

[0082] 1. Heavy chain sequence of 9G01 antibody The 9G01 full-length heavy chain contains 728 amino acid residues, specifically: MHSSALLCCLVLLTGVRA QVQLVQSGAEVKKPGASMKVSCKASGYTFAGYYIHWVRQAPGQGLEWMG SINPNSGKATYAQRFHGRVTLTREMSINTAYMELTRLTSDDTAIYYCARDATYSTSFSPRWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS QVQLVQSGAEVKKPGQSLRISCQGSGHSFADYWINWVRQKPGEGLE WMGRVDPSDSHATYSPSFRGHVTFSTDKSISTAYLQWSSLRASDTAMYFCARSYYDSRTGSVSPYYFDYWGQGTLV TVSS GGGGSGGGGSGGGGS QSVLIQPRSVSGSPGQSVTISCTGTSTDVGNYNYVSWYQQHPGTAPRFLIYDVSNRP SGVPDRFSGSKSGNTASLTISGLQADDEADYYCCSYAGKSIVVCGGGTQLTVL (SEQ ID NO: 1).

[0083] The underlined portions in the sequence are, in order, the amino acid sequences of the 1A12 heavy chain variable region (SEQ ID NO: 81), the 4G12 heavy chain variable region (SEQ ID NO: 82), and the 4G12 light chain variable region (SEQ ID NO: 83), and the italicized portions are the signal peptides.

[0084] According to different CDR definition schemes, the CDR sequences of the amino acid sequences of the 1A12 heavy chain variable region, the 4G12 heavy chain variable region, and the 4G12 light chain variable region are shown in Table 1.

[0085] Table 1

[0086] The 9G01 full-length heavy chain contains 2187 bases, specifically: ATG CACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC C AGGTGCAGCTGG TGCAGAGTGGAGCAGAGGTGAAAAAGCCCGGCGCCTCCATGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCGC CGGCTACTACATCCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTCCATCAACCCCAACTCC GGCAAGGCCACCTACGCCCAGAGGTTCCACGGCAGGGTGACCCTGACCAGGGAGATGTCCATCAACACCGCCTACA TGGAGCTGACCAGGCTGACCTCCGACGACACCGCCATCTACTACTGCGCCAGGGACGCCACCTACTCCACCTCCTT CTCCCCCAGGTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCCAGG TGCAGCTGGTGCAGAGTGGAGCAGAGGTGAAAAAGCCCGGCCAGTCCCTGAGGATCTCCTGCCAGGGCTCCGGCCA CTCCTTCGCCGACTACTGGATCAACTGGGTGAGGCAGAAGCCCGGCGAGGGCCTGGAGTGGATGGGCAGGGTGGAC CCCTCCGACTCCCACGCCACCTACTCCCCCTCCTTCAGGGGCCACGTGACCTTCTCCACCGACAAGTCCATCTCCA CCGCCTACCTGCAGTGGTCCTCCCTGAGGGCCTCCGACACCGCCATGTACTTCTGCGCCAGGTCCTACTACGACTC CAGGACCGGCTCCGTGTCCCCCTACTACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC GGTGGTGGCGGATCAGGTGGGGGAGGCTCTGGTGGAGGCGGTAGT CAGTCTGTCCTGATCCAGCCCAGGTCCGTCTCCGGAT CTCCAGGACAGTCCGTCACCATCTCCTGCACAGGCACCTCCACCGACGTCGGAAACTACAACTACGTCTCCTGGTA CCAGCAGCACCCCGGTACAGCACCAAGGTTCCTGATCTATGACGTGTCCAACCGCCCCTCCGGCGTGCCTGATAGA TTCTCTGGCTCCAAGAGCGGCAACACCGCCTCTCTGACTATCTCCGGCCTGCAGGCTGACGACGAGGCTGATTATT ACTGCTGCAGTTACGCCGGAAAATCCATTGTGGTGTGTGGCGGGGGCACCCAGCTGACAGTGCTG TGA (SEQ ID NO: 31).

[0087] In this sequence, the underlined portions represent the nucleotide sequences of the 1A12 heavy chain variable region, the 4G12 heavy chain variable region, and the 4G12 light chain variable region, respectively. The first three bases at the 5' end and the last three bases at the 3' end are the start codon and stop codon, respectively. The italicized portion is the signal peptide.

[0088] 2. Light chain sequence of 9G01 antibody The 9G01 light chain consists of 232 amino acid residues, specifically: MHSSALLCCLVLLTGVRA EIVLTQSPGTLSLSPGERATLSCRASQSISSNSLAWYQQRPGQAPRLLI YGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGPGTKVDIK GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 32).

[0089] In this sequence, the underlined portion is the amino acid sequence of the light chain variable region (SEQ ID NO: 84), and the italicized portion is the signal peptide sequence.

[0090] According to different CDR definition schemes, the CDR sequences of the amino acid sequence of the light chain variable region of 9G01 are shown in Table 2.

[0091] Table 2

[0092] The gene encoding the full-length light chain of 9G01 consists of 699 bases, specifically: ATG CACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC GAAATCGTGCTGA CCCAGTCCCCCGGAACCCTGTCTCTGTCTCCTGGAGAGAGGGCCACCCTGTCTTGTAGGGCTAGTCAGTCCATCAG CTCCAACTCCCTGGCATGGTACCAGCAGAGGCCCGGACAGGCTCCTAGACTGCTGATCTATGGCGCCAGCAGCAGG GCAACAGGAATCCCTGACAGGTTCAGTGGCTCCGGCTCCGGAACAGACTTCACCCTGACTATCTCAAGGCTGGAAC CCGAGGATTTCGCCGTGTACTACTGCCAGCAGTACGGGAGCAGTCCCAGGACATTCGGACCCGGAACCAAGGTGGA TATCAAAGGTCAGCCCAAGGCTGCCCTCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGG AGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGGAGCTACAGTTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGA (SEQ ID NO: 40)。

[0093] The underlined portion of the sequence represents the nucleotide sequence of the light chain variable region. The first three bases at the 5' end and the last three bases at the 3' end are the start and stop codons, respectively. The italicized portion represents the signal peptide.

[0094] 3. Coomassie brilliant blue staining to identify the expression of light and heavy chains of 9G01. The specific operating steps are as follows: After preparing a 10% separating gel, mix well and quickly add 7 mL to the gel mold, then add 2 mL of anhydrous ethanol for sealing. After the lower layer of separating gel solidifies, discard the anhydrous ethanol and allow it to evaporate. Prepare a stacking gel, add 2 mL to the top layer of the separating gel, and quickly insert the comb. Perform protein quantification using the BCA protein quantification kit. Add 10 μg of 4C1 protein sample to 5×SDS loading buffer and incubate at 95°C for 10 min. After the gel dries, place the mold in the electrophoresis tank, add electrophoresis buffer, and remove the comb. Add the processed protein sample to the sample wells and add the marker. First, concentrate the protein using 80 V low voltage, then change the voltage to 120 V for protein separation. The electrophoresis time is determined based on the marker position. Place the gel in Coomassie Brilliant Blue staining solution (Novizan) and shake on a shaker until bands appear. Discard the developing solution, add double-distilled water, and wash the gel on a shaker several times until the gel background is clean.

[0095] The results are as follows Figure 1 As shown, the 76 kDa band is a heavy chain, and the 23 kDa band is a light chain.

[0096] Example 2: Expression and purification of 9G02 antibody Ligating the upstream of the variable region of the heavy chain of gp350 monoclonal antibody 1A12 with the CMV fragment, downstream of the CMV fragment, the constant region of human IgG1, GGGS, the variable region of the heavy chain of gp42 monoclonal antibody 2C1, the variable region of the light chain of gp42 monoclonal antibody 2C1, and the polyA fragment allows for the expression of the complete heavy chain fragment. Similarly, ligating the upstream of the variable region of the light chain of gp350 monoclonal antibody 1A12 with the CMV fragment, downstream of the constant region of the light chain κ / λ, and the polyA fragment allows for the expression of the complete light chain fragment. Co-transfection of 293F cells with plasmids containing the full-length sequences of the antibody heavy and light chains achieves antibody expression, and protein A beads are used for antibody purification.

[0097] 1. Heavy chain sequence of 9G02 antibody The 9G02 full-length heavy chain contains 720 amino acid residues, specifically: MHSSALLCCLVLLTGVRA QVQLVQSGAEVKKPGASMKVSCKASGYTFAGYYIHWVRQAPGQGLEWMG SINPNSGKATYAQRFHGRVTLTREMSINTAYMELTRLTSDDTAIYYCARDATYSTSFSPRWFDPWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS QVQLVQSGAEVKKPGSSVKVSCRASGGTFDTYAISWVRQAPGHGLE WMGGIIPVSGTANYAQKFQGRVTITADESTGTAYMDLSSLRSEDTAVYYCARVPDYGTNTPFDYWGQGTLVTVSS GGGGSGGGGSGGGGS DIRLTQSPSSLPASVGDRVTITCRASQDIATYLAWYQQKPGRAPNLLIYATSTLQSGVPPRF SGSRSGTDFTLTISSLQPEDFATYYCQQLRTYPITFGQGTRLEIK (SEQ ID NO: 41).

[0098] The underlined portions in the sequence are, in order, the amino acid sequences of the 1A12 heavy chain variable region (SEQ ID NO: 81), the 2C1 heavy chain variable region (SEQ ID NO: 85), and the 2C1 light chain variable region (SEQ ID NO: 86), and the italicized portions are the signal peptides.

[0099] According to different CDR definition schemes, the CDR sequences of the amino acid sequences of the 1A12 heavy chain variable region, the 2C1 heavy chain variable region, and the 2C1 light chain variable region are shown in Table 3.

[0100] Table 3

[0101] The 9G02 full-length heavy chain contains 2163 bases, specifically: ATG CACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC CAGGTGCAGCTGG TGCAGAGTGGAGCAGAGGTGAAAAAGCCCGGCGCCTCCATGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCGC CGGCTACTACATCCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTCCATCAACCCCAACTCC GGCAAGGCCACCTACGCCCAGAGGTTCCACGGCAGGGTGACCCTGACCAGGGAGATGTCCATCAACACCGCCTACA TGGAGCTGACCAGGCTGACCTCCGACGACACCGCCATCTACTACTGCGCCAGGGACGCCACCTACTCCACCTCCTT CTCCCCCAGGTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCCAGG TGCAGCTGGTGCAGAGTGGAGCAGAGGTGAAAAAGCCCGGCTCCTCCGTGAAGGTGTCCTGCAGGGCCTCCGGCGG CACCTTCGACACCTACGCCATCTCCTGGGTGAGGCAGGCCCCCGGCCACGGCCTGGAGTGGATGGGCGGCATCATC CCCGTGTCCGGCACCGCCAACTACGCCCAGAAGTTCCAGGGCAGGGTGACCATCACCGCCGACGAGTCCACCGGCA CCGCCTACATGGACCTGTCCTCCCTGAGGTCCGAGGACACCGCCGTGTACTACTGCGCCAGGGTGCCCGACTACGG CACCAACACCCCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC GGTGGTGGCGGATCAGGTGGGGGAGGCTCTGGTGGAGGCGGTAGT GATATCAGGCTGACCCAGTCCCCCTCCTCCCTGCCTGCTTCTGTGGGAGATA GGGTGACCATTACCTGCAGGGCCTCCCAGGATATTGCCACCTACCTGGCTTGGTACCAGCAGAAGCCCGGCAGAGC TCCTAACCTGCTGATCTACGCCACCTCTACCCTGCAGTCCGGCGTGCCTCCTAGATTTTCCGGCTCCAGGAGCGGC ACCGACTTCACACTGACCATCTCCTCCCTGCAGCCCGAGGATTTCGCCACTTACTACTGCCAGCAGCTGAGAACCT ACCCCATCACCTTCGGCCAGGGCACAAGGCTGGAAATCAAG TGA (SEQ ID NO: 60).

[0102] In this sequence, the underlined portions represent the nucleotide sequences of the 1A12 heavy chain variable region, the 2C1 heavy chain variable region, and the 2C1 light chain variable region, respectively. The first three bases at the 5' end and the last three bases at the 3' end are the start codon and the stop codon, respectively. The italicized portion is the signal peptide.

[0103] 2. Light chain sequence of 9G02 antibody The light chain sequence of antibody 9G02 is the same as that of antibody 9G01.

[0104] 3. Coomassie brilliant blue staining to identify the expression of light and heavy chains of 9G02. The method is the same as in Example 1.

[0105] The results are as follows Figure 2 As shown, the 76 kDa band is a heavy chain, and the 23 kDa band is a light chain.

[0106] Example 3 Heavy chain sequence of 9G02 antibody Ligating the upstream of the variable region of the heavy chain of gp350 monoclonal antibody 1A12 with the CMV fragment, downstream of the CMV fragment, the constant region of human IgG1, GGGS, the variable region of the heavy chain of gB monoclonal antibody 4H2, the variable region of the light chain of gB monoclonal antibody 4H2, and the polyA fragment allows for the expression of the complete heavy chain fragment. Similarly, ligating the upstream of the variable region of the light chain of gp350 monoclonal antibody 1A12 with the CMV fragment, downstream of the constant region of the light chain κ / λ, and the polyA fragment allows for the expression of the complete light chain fragment. Co-transfection of 293F cells with plasmids containing the full-length sequences of the antibody heavy and light chains achieves antibody expression, and protein A beads are used for antibody purification.

[0107] 1. Heavy chain sequence of 9G03 antibody The 9G03 full-length heavy chain contains 722 amino acid residues, specifically: MHSSALLCCLVLLTGVRA QVQLVQSGAEVKKPGASMKVSCKASGYTFAGYYIHWVRQAPGQGLEWMG SINPNSGKATYAQRFHGRVTLTREMSINTAYMELTRLTSDDTAIYYCARDATYSTSFSPRWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS EVQLLESGGGLVQPGRSLRLSCATSGFTFDDYAMHWVRQAPGKGLE WVSGISWNAENIAYADSVKGRFTISRDNAKNSLFLHMNSLRAEDTAFYYCARDRAHYFGSGSYFDSSGQGTLVTVS S GGGGSGGGGSGGGGS EIVLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK (SEQ ID NO: 61).

[0108] The underlined portions in the sequence are, in order, the amino acid sequences of the 1A12 heavy chain variable region (SEQ ID NO: 81), the 4H2 heavy chain variable region (SEQ ID NO: 87), and the 4H2 light chain variable region (SEQ ID NO: 88), and the italicized portions are the signal peptides.

[0109] According to different CDR definition schemes, the CDR sequences of the amino acid sequences of the 1A12 heavy chain variable region, the 4H2 heavy chain variable region, and the 4H2 light chain variable region are shown in Table 4.

[0110] Table 4

[0111] The 9G03 full-length heavy chain contains 2169 bases, specifically: ATG CACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC CAGGTGCAGCTGG TGCAGAGTGGAGCAGAGGTGAAAAAGCCCGGCGCCTCCATGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCGC CGGCTACTACATCCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTCCATCAACCCCAACTCC GGCAAGGCCACCTACGCCCAGAGGTTCCACGGCAGGGTGACCCTGACCAGGGAGATGTCCATCAACACCGCCTACA TGGAGCTGACCAGGCTGACCTCCGACGACACCGCCATCTACTACTGCGCCAGGGACGCCACCTACTCCACCTCCTT CTCCCCCAGGTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC GAAG TGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGAAGATCCCTGAGGCTGAGCTGCGCAACAAGCGGATT TACCTTTGATGATTATGCTATGCATTGGGTGAGGCAGGCCCCCGGAAAAGGACTGGAATGGGTGAGCGGAATTAGT TGGAACGCCGAGAACATCGCCTACGCCGATAGCGTGAAGGGCAGGTTTACCATTAGCAGGGACAACGCTAAGAACT CCCTGTTCCTGCACATGAACAGCCTGAGGGCCGAGGACACCGCTTTCTATTATTGCGCCCGGGACAGAGCCCACTA TTTCGGAAGCGGCTCCTACTTCGACAGCTCCGGACAGGGAACCCTGGTGACTGTCTCCTCC GGTGGTGGCGGATCAGGTGGGGGAGGCTCTGGTGGAGGCGGTAGT GAAATCGTGCTGACCCAGTCCCCCTCCTCCCTGTCTGCTAGTGTGG GAGACAGGGTGACCATCACCTGCAGGGCTAGCCAGTCCATCAGCTCCTACCTGAACTGGTACCAGCAGAAGCCCGG CAAAGCCCCAAAACTGCTGATTTACGCCGCCTCCTCCCTGCAGAGCGGAGTGCCTTCTCGATTCTCCGGTTCCGGC TCCGGAACCGATTTCACCCTGACTATTCTCCTCCCTGCAGCCCGAGGACTTCGCAACATACTACTGCCAGCAGTCCT ACTCCACACCCTTCACCTTCGGCCCCGGTACAAAAGTCGACATCAAA TGA (SEQ ID NO: 80).

[0112] In this sequence, the underlined portions represent the nucleotide sequences of the 1A12 heavy chain variable region, the 4H2 heavy chain variable region, and the 4H2 light chain variable region, respectively. The first three bases at the 5' end and the last three bases at the 3' end are the start codon and stop codon, respectively. The italicized portion is the signal peptide.

[0113] 2. Light chain sequence of 9G03 antibody The light chain sequence of antibody 9G03 is the same as that of antibody 9G01.

[0114] 3. Coomassie brilliant blue staining to identify the expression of light and heavy chains of 9G03. The method is the same as in Example 1.

[0115] The results are as follows Figure 3 As shown, the 76 kDa band is a heavy chain, and the 23 kDa band is a light chain.

[0116] Example 1: Effect Test of 9G01 1. Determining the affinity of 9G01 using biomembrane interferometry (BLI): BLI can be performed according to conventional methods in the art. In this embodiment, the specific operation is as follows: the biosensor (Sartorius SA probe from Germany) is immersed in a buffer solution (a mixture of KB buffer and 0.02% Tween 20) for equilibration. Then it is removed and immersed in a solution containing 10 μg / mL gH / gL / gp42 complex. A solution of Biotin (i.e., biotin-labeled gH / gL / gp42 complex protein) and a solution containing 10 μg / mL gp350-biotin (i.e., biotin-labeled gp350 protein) were used. The gH / gL / gp42 complex protein and gp350 protein in the solution bound to the surface of the SA (streptavidin) bioprobe, increasing the thickness of its surface membrane. Then, a biosensor with a known concentration of cured antigen was immersed in buffer as a baseline. The biosensor with the cured known concentration of antigen was immersed in a sample solution containing 15.6 nM–500 nM 9G01 antibody for approximately 60–120 seconds. Due to the antigen… The specific binding between antibodies leads to an increase in membrane thickness. Immersing the biosensor bound to the 9G01 antibody in buffer for approximately 180 seconds causes dissociation, resulting in the detachment of the test antibody (9G01 antibody) from the biosensor surface and a reduction in membrane thickness. By monitoring the biosensor membrane thickness in real time during the experiment, the kinetic constants of the test sample (9G01 antibody) can be obtained.

[0117] The results are as follows Figure 4 As shown: the dissociation equilibrium constants between the 9G01 antibody and the two glycoproteins are 7.715E. The values ​​of 11 and 4.977E-9 indicate that the 9G01 antibody has extremely high affinity for both the gH / gL / gp42 complex antigen and the gp350 protein.

[0118] 2. Neutralizing activity of 9G01 antibody EBV preparation: EBV was infected by culturing in RPMI 1640 + 10% FBS at 37°C (5% CO2). GFP in CNE2 cells (the virus has been documented in the literature: An Antibody Targeting the Fusion Machinery Neutralizes Dual) Tropic Infection and Defines a Site of Vulnerability onEpstein (Disclosed in Barr Virus) When the cells reached 90% density (10 cm dish), TPA (phorbol ester) and NaB (sodium butyrate) at a final concentration of 20 ng / mL were added to induce toxin production. After 12 hours, the culture medium was replaced with fresh RPMI 1640 medium containing 10% FBS.

[0119] The culture medium was collected 48 hours after the medium change, filtered through a 0.45 μm filter, and concentrated by high-speed centrifugation. After concentration, it was resuspended in serum-free RPMI 1640 and used immediately for infection or aliquoted and stored at -80 degrees Celsius for long-term storage.

[0120] Detection of 9G01 neutralizing activity in B cells and epithelial cells: The 9G01 antibody from the above examples was adjusted to a concentration of 2 mg / mL. 150 μL of RPMI 1640 medium was added to each well of a new 96-well plate. The first well contained 200 μL of 100 μg / mL 9G01 antibody diluted with RPMI 1640. After a 4-fold serial dilution (50 μL was aspirated from the first well and added to the second well, and so on, until 50 μL was aspirated from the last well and discarded, resulting in a final volume of 150 μL per well), 50 μL of virus dilution buffer (the virus was diluted with RPMI 1640 medium to a titer of approximately 4 × 10⁻⁶) was added to each well. 6 After incubating at 37°C for 2 hours, 100 μL of the solution was added to each well of a 96-well plate containing either 10,000 RAJI cells per well or 1,000 HEK293 cells per well (100 μL RPMI 1640 medium, cell concentration 1×10⁻⁶). 5 Cells / ml). After incubation at 37°C for 48 hours, the cells were analyzed. Infection rates of RAJI and HEK293 cells were detected by flow cytometry. The inhibition rate (neutralization rate %) of the antibody in the RAJI and HEK293 cell infection models was calculated by detecting the reduction in the number of GFP-positive cells in the antibody-treated group compared to the infection control group (which received an equal volume of RPMI 1640). The IC50 of the 9G01 antibody was calculated using Prism. 50 2G4 antibody was used as a negative control antibody (2G4 antibody is a control antibody against Ebola virus, Howell KA, Brannan JM, Bryan C, et al. Cooperativity Enables Non-neutralizing Antibodies to Neutralize Ebolavirus. Cell Rep. 2017;19 (2):413-424. doi:10.1016 / j.celrep.2017.03.049).

[0121] The results are as follows Figure 5 As shown, the IC50 of 9G01 in the RAJI B cell infection model 50 4.268×10 -6 μg / mL, IC50 in the HEK293 cell infection model 50The concentration was 0.0003 μg / mL, while the control group antibody showed no neutralizing activity. The dual-target antibody 9G01 significantly inhibited EBV infection of B cells and epithelial cells.

[0122] 3. The role of 9G01 in blocking viral infection in vivo In this implementation case, a humanized mouse model was used as the test subject to test the effect of the 9G01 antibody in blocking EB virus infection and replication.

[0123] The specific steps are as follows: 24 hours before intravenous injection of EB virus, administer an intraperitoneal injection of 9G01 antibody (25 mg / kg), followed by an intravenous injection of 5×10 oz. 5 Epstein-Barr virus (EBV) at a concentration of GRU / mL. Mice were intraperitoneally injected with 9G01 (25 mg / kg) on ​​days 2, 7, 14, and 21 following EBV injection. Mouse weight and survival status were recorded weekly. The experiment ended when the weight of mice in the PBS group dropped to 30% of their original body weight (or when most mice died).

[0124] The results are as follows Figure 6 and Figure 7 As shown.

[0125] The results showed that the body weight of mice injected with the 9G01 antibody did not decrease within 50 days after EB virus challenge, and no mice died. In contrast, the body weight of humanized mice in the PBS group decreased significantly, by more than 20%, and mice began to die from day 25 onwards.

[0126] Example 2: Effect Test of 9G02 1. Determining the affinity of 9GO2 using biomembrane interferometry (BLI). The method is the same as in Example 1.

[0127] The results are as follows Figure 8 As shown, the dissociation equilibrium constants between the 9G02 antibody and the two glycoproteins are 8.516E. The values ​​of 11 and 3.308E-9 indicate that the 9G02 antibody has extremely high affinity for both the gp42 complex antigen and the gp350 protein.

[0128] 2. Neutralizing activity of 9G02 antibody The method is the same as in Example 1.

[0129] The results are as follows Figure 9 As shown, the IC50 of 9G02 in the RAJI B cell infection model 50 At a concentration of 0.02 μg / mL, the dual-target antibody 9G02 can significantly inhibit EB virus infection of B cells.

[0130] Example 3: Effect Test of 9G03 1. Neutralizing activity of 9G03 antibody The method is the same as in Example 1.

[0131] The results are as follows Figure 10 As shown, the IC50 of 9G03 in the RAJI B cell infection model 50 The concentration was 0.014 μg / mL. The dual-target antibody 9G03 significantly inhibited EBV infection of B cells.

[0132] Example 4: Comparison of neutralization effects of 9G01, 9G02, and 9G03 The operation steps are the same as in Example 1.

[0133] The results are as follows Figure 11 As shown, in the RAJI B cell infection model, the IC50 of 9G01 was... 50 The antibody with the lowest value was the most effective, followed by 9G02 and 9G03.

[0134] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A bispecific antibody or antigen-binding fragment thereof against Epstein-Barr virus, characterized in that: the bispecific antibody or antigen-binding fragment thereof comprises: a first protein functional region targeting a gp350 protein, and a second protein functional region targeting a gH / gL / gp42, gp42 or gB protein; wherein: the first protein functional region is an immunoglobulin; the first protein functional region comprises a heavy chain and a light chain; the heavy chain of the first protein functional region comprises: a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, the heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 81; the light chain of the first protein functional region comprises: a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, the light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 84; the second protein functional region is a single-chain antibody; the second protein functional region comprises a heavy chain and a light chain; the second protein functional region comprises any one of a1) to a3): a1) a second protein functional region targeting a gH / gL / gp42 protein complex, comprising: a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, the heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 82; a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, the light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 83; a2) a second protein functional region targeting a gp42 protein, comprising: a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, the heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 85; a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, the light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 86; a3) a second protein functional region targeting a gB protein, comprising: a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, the heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 87; a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, the light chain variable region having an amino acid sequence as set forth in SEQ ID NO:

88. 2.The bispecific antibody or antigen-binding fragment thereof of claim 1, characterized in that: the CDR sequences of the heavy chain variable region of the first protein functional region are as set forth in any one of b1) to b4): b1) the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the first protein functional region are as set forth in SEQ ID NOs: 2, 3 and 4 in sequence, the CDRs being defined in accordance with the IMGT definition scheme. b2) the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the first protein functional region have amino acid sequences as set forth, in sequence, in SEQ ID NOs: 10, 11, 12, respectively, the CDRs being defined according to the Kabat definition scheme; b3) the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the first protein functional region have amino acid sequences as set forth, in sequence, in SEQ ID NOs: 18, 19, 12, respectively, the CDRs being defined according to the Chothia definition scheme; b4) the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the first protein functional region have amino acid sequences as set forth, in sequence, in SEQ ID NOs: 22, 23, 24, respectively, the CDRs being defined according to the Contact definition scheme; the CDR sequences of the light chain variable region of the first protein functional region are as set forth in any one of b5) to b8): b5) the CDR-L1 and CDR-L3 of the light chain variable region of the first protein functional region have amino acid sequences as set forth, in sequence, in SEQ ID NOs: 33, 34, respectively, and the CDR-L2 has an amino acid sequence of GAS, the CDRs being defined according to the IMGT definition scheme; b6) the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the first protein functional region have amino acid sequences as set forth, in sequence, in SEQ ID NOs: 35, 36, 34, respectively, the CDRs being defined according to the Kabat definition scheme; b7) the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the first protein functional region have amino acid sequences as set forth, in sequence, in SEQ ID NOs: 35, 36, 34, respectively, the CDRs being defined according to the Chothia definition scheme; b8) the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the first protein functional region have amino acid sequences as set forth, in sequence, in SEQ ID NOs: 37, 38, 39, respectively, the CDRs being defined according to the Contact definition scheme.

3. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein: a1) the second protein functional region targeting the gH / gL / gp42 protein complex has: the CDR sequences of the heavy chain variable region of the second protein functional region are as set forth in any one of c1) to c4): c1) the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region have amino acid sequences as set forth, in sequence, in SEQ ID NOs: 5, 6, 7, respectively, the CDRs being defined according to the IMGT definition scheme; c2) the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein functional region have amino acid sequences as set forth, in sequence, in SEQ ID NOs: 13, 14, 15, respectively, the CDRs being defined according to the Kabat definition scheme; c3) the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein domain have amino acid sequences of SEQ ID NOs: 20, 21, 15, respectively, the CDRs being defined according to the Chothia definition scheme; c4) the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein domain have amino acid sequences of SEQ ID NOs: 25, 26, 27, respectively, the CDRs being defined according to the Contact definition scheme; the CDR sequences of the light chain variable region of the second protein domain are as in any one of c5) to c8): c5) the CDR-L1 and CDR-L3 of the light chain variable region of the second protein domain have amino acid sequences of SEQ ID NOs: 8, 9, respectively, and the CDR-L2 has an amino acid sequence of DVS, the CDRs being defined according to the IMGT definition scheme; c6) the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein domain have amino acid sequences of SEQ ID NOs: 16, 17, 9, respectively, the CDRs being defined according to the Kabat definition scheme; c7) the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein domain have amino acid sequences of SEQ ID NOs: 16, 17, 9, respectively, the CDRs being defined according to the Chothia definition scheme; c8) the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein domain have amino acid sequences of SEQ ID NOs: 28, 29, 30, respectively, the CDRs being defined according to the Contact definition scheme; a2) in the second protein domain targeting the gp42 protein: the CDR sequences of the heavy chain variable region of the second protein domain are as in any one of d1) to d4): d1) the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein domain have amino acid sequences of SEQ ID NOs: 42, 43, 44, respectively, the CDRs being defined according to the IMGT definition scheme; d2) the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein domain have amino acid sequences of SEQ ID NOs: 47, 48, 49, respectively, the CDRs being defined according to the Kabat definition scheme; d3) the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein domain have amino acid sequences of SEQ ID NOs: 52, 53, 49, respectively, the CDRs being defined according to the Chothia definition scheme; d4) the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein domain have amino acid sequences of SEQ ID NOs: 54, 55, 56, respectively, the CDRs being defined according to the Contact definition scheme; the CDR sequences of the light chain variable region of the second protein domain are as set forth in any one of d5) to d8): d5) the amino acid sequences of CDR-L1 and CDR-L3 of the light chain variable region of the second protein domain are as set forth in SEQ ID NO: 45, 46, respectively, and the amino acid sequence of CDR-L2 is ATS, the CDRs being defined according to the IMGT definition scheme; d6) the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein domain are as set forth in SEQ ID NO: 50, 51, 46, respectively, the CDRs being defined according to the Kabat definition scheme; d7) the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein domain are as set forth in SEQ ID NO: 50, 51, 46, respectively, the CDRs being defined according to the Chothia definition scheme; d8) the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein domain are as set forth in SEQ ID NO: 57, 58, 59, respectively, the CDRs being defined according to the Contact definition scheme; a3) in the second protein domain targeting the gB protein, the CDR sequences of the heavy chain variable region of the second protein domain are as set forth in any one of e1 ) to e4): e1 ) the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein domain are as set forth in SEQ ID NO: 62, 63, 64, respectively, the CDRs being defined according to the IMGT definition scheme; e2) the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein domain are as set forth in SEQ ID NO: 67, 68, 69, respectively, the CDRs being defined according to the Kabat definition scheme; e3) the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein domain are as set forth in SEQ ID NO: 72, 73, 69, respectively, the CDRs being defined according to the Chothia definition scheme; e4) the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region of the second protein domain are as set forth in SEQ ID NO: 74, 75, 76, respectively, the CDRs being defined according to the Contact definition scheme; the CDR sequences of the light chain variable region of the second protein domain are as set forth in any one of e5) to e8): e5) the amino acid sequences of CDR-L1 and CDR-L3 of the light chain variable region of the second protein domain are as set forth in SEQ ID NO: 65, 66, respectively, and the amino acid sequence of CDR-L2 is AAS, the CDRs being defined according to the IMGT definition scheme; e6) the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein functional region have amino acid sequences as set forth in SEQ ID NOs: 70, 71, 66, respectively, the CDRs being defined according to the Kabat definition scheme; e7) the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein functional region have amino acid sequences as set forth in SEQ ID NOs: 70, 71, 66, respectively, the CDRs being defined according to the Chothia definition scheme; e8) the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the second protein functional region have amino acid sequences as set forth in SEQ ID NOs: 77, 78, 79, respectively, the CDRs being defined according to the Contact definition scheme.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein: the second protein functional region is directly linked to the first protein functional region or linked via a linker; and / or the heavy chain and the light chain of the second protein functional region are directly linked or linked via a linker.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein: the linker comprises (GGGGS)n, n being a positive integer.

6. A recombinant protein comprising: the bispecific antibody or antigen-binding fragment thereof of any one of claims 1-5; and optionally a tag sequence facilitating expression and / or purification.

7. A biological material associated with the bispecific antibody or antigen-binding fragment thereof of any one of claims 1-5, or the recombinant protein of claim 6, the biological material comprising at least one of: f1) a nucleic acid molecule encoding the bispecific antibody or antigen-binding fragment thereof of any one of claims 1-3, or the recombinant protein of claim 4; f2) an expression cassette comprising the nucleic acid molecule of f1); f3) a vector comprising the nucleic acid molecule of f1); f4) a vector comprising the expression cassette of f2); f5) a transgenic cell line comprising the nucleic acid molecule of f1); f6) a transgenic cell line comprising the expression cassette of f2); f7) a transgenic cell line comprising the vector of f3); f8) a transgenic cell line comprising the vector of f4); f9) a microorganism comprising the nucleic acid molecule of f1); f10) a microorganism comprising the expression cassette of f2); f11) a microorganism comprising the vector of f3); f12) a microorganism comprising the vector of f4); f13) a virus comprising the nucleic acid molecule of f1); f14) a virus comprising the expression cassette of f2); f15) a virus comprising the vector of f3); f16) a virus comprising the vector of f4).

8. A conjugate comprising: at least one of the bispecific antibody or antigen-binding fragment thereof of any one of claims 1-5 and the recombinant protein of claim 6; and a conjugating moiety, the conjugating moiety comprising at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme. ​ 9. Use of the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the recombinant protein according to claim 6, the biomaterial according to claim 7 and / or the conjugate according to claim 8 in the manufacture of a product; Preferably, the product comprises at least one of a drug, a reagent, a detection plate, a kit, a detection chip; Preferably, the drug has at least one of g1) to g2) functions: g1) preventing EB virus infection; g2) treating and / or preventing a disease caused by EB virus infection; Preferably, the reagent, the detection plate, the kit, the detection chip has at least one of h1) to h3) functions: h1) detecting the presence or level of gH / gL / gp42, gp42, gB or gp350 protein in a sample; h2) detecting EB virus; h3) diagnosing a disease caused by EB virus infection.

10. A product comprising the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the recombinant protein according to claim 6 and / or the conjugate according to claim 8; Preferably, the product comprises at least one of a drug, a reagent, a detection plate, a kit, a detection chip; Preferably, the drug comprises a pharmaceutically acceptable excipient.

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