A monoclonal antibody 2B7 for recognizing Epstein-Barr virus gp42 protein and its application

Abstract

The present invention belongs to the field of antibody technology and discloses a monoclonal antibody 2B7 that recognizes the Epstein-Barr virus gp42 protein and its application. The monoclonal antibody or its antigen-binding fragment has good binding activity with the gp42 protein and has a high affinity for the gp42 protein (KD(M)=2.09E-12), can significantly inhibit EBV infection of epithelial cells and B cells, and can be used to detect the presence or level of gp42 protein in a sample, detect EBV, diagnose diseases caused by EBV infection, prevent EBV infection, and / or treat and / or prevent diseases caused by EBV infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibodies, and in particular relates to a monoclonal antibody 2B7 for recognizing Epstein-Barr virus gp42 protein and its application. Background Art

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

[0003] EBV is most commonly transmitted as a latent infection, with over 90% of EBV-infected individuals remaining latently infected for life. Under certain conditions, the virus can be activated, leading to cancer. Current research has linked human EBV to malignancies such as nasopharyngeal carcinoma, infectious mononucleosis, Hodgkin's and non-Hodgkin's lymphomas, Burkitt's lymphoma, and epithelial cell cancers, including gastric cancer. EBV initially replicates in the oropharynx, where it grows and multiplies within B lymphocytes and oral epithelial cells. It then infects these cells, which then enter the bloodstream in large numbers, causing systemic infection. When the immune system is weakened, latent EBV can reactivate, leading to recurrent infection.

[0004] Currently, there is no effective vaccine against EBV, and diseases caused by EBV infection lack specific treatments. Infectious mononucleosis is mostly treated with antiviral drugs such as acyclovir. While these drugs can alleviate symptoms to some extent, they cannot eliminate EBV in B lymphocytes or the epithelium of the throat. Treatment for EBV-associated tumors primarily involves chemotherapy and radiotherapy, but these are less effective for patients with metastatic or recurrent disease.

[0005] Monoclonal antibodies can be mass-produced, and their high affinity and specificity for binding to antigens significantly reduce adverse reactions during clinical use. These antibody molecules can also be engineered to increase their antiviral potency. Antibodies, with their specificity and flexibility, are a promising approach for treating infectious diseases. However, to date, no monoclonal antibodies targeting the EBV envelope glycoprotein have been commercially available. Therefore, developing anti-EBV monoclonal antibodies would provide more effective prevention and treatment options for EBV-related diseases. Summary of the Invention

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

[0007] The second aspect of the present invention aims to provide a recombinant protein.

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

[0009] The fourth aspect of the present invention aims to provide a conjugate.

[0010] 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.

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

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

[0013] The eighth aspect of the present invention aims to provide a method for preparing the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

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

[0015] In a first aspect of the present invention, there is provided an anti-EBV gp42 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain and a light chain:

[0016] The heavy chain comprises:

[0017] a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 14;

[0018] The light chain comprises:

[0019] A light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO: 29.

[0020] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively, and the CDRs are defined according to the Kabat definition scheme.

[0021] Preferably, the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1 and CDR-L3 are shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 33 and SEQ ID NO: 32, respectively, the amino acid sequence of the CDR-L2 is: NDN, and the CDRs are defined according to the IMGT definition scheme.

[0022] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 17, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively, and the CDRs are defined according to the Chothia definition scheme.

[0023] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 respectively, and the CDRs are defined according to the Contact definition scheme.

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

[0025] a1) SEQ ID NO. 14; or

[0026] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids to SEQ ID NO. 14 and having the same function as the protein shown in SEQ ID NO. 14; or

[0027] a3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% identical to SEQ ID NO. 14 and has the same function as the protein shown in SEQ ID NO. 14;

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

[0029] b1) SEQ ID NO. 29; or

[0030] b2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO. 29 and having the same function as the protein shown in SEQ ID NO. 29; or

[0031] b3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% identical to SEQ ID NO. 29 and has the same function as the protein shown in SEQ ID NO. 29.

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

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

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

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

[0036] c1) an amino acid sequence consisting of amino acids 154 to 483 of SEQ ID NO: 13; or

[0037] c2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in the amino acid sequence described in c1) and having the same function as the protein of the amino acid sequence described in c1); or

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

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

[0040] d1) an amino acid sequence consisting of amino acids 130 to 235 of SEQ ID NO: 28; or

[0041] d2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in the amino acid sequence described in d1) and having the same function as the protein of the amino acid sequence described in d1); or

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

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

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

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

[0046] e1) an amino acid sequence consisting of amino acids 1 to 19 of SEQ ID NO: 13; or

[0047] e2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids to the amino acid sequence described in e1) and having the same function as the protein of the amino acid sequence described in e1); or

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

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

[0050] f1) an amino acid sequence consisting of amino acids 1 to 19 of SEQ ID NO: 28; or

[0051] f2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in the amino acid sequence described in f1) and having the same function as the protein of the amino acid sequence described in f1); or

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

[0053] Preferably, the amino acid sequence of EBV gp42 comprises:

[0054] g1) an amino acid sequence consisting of amino acids 1 to 190 of SEQ ID NO: 4; or

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

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

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

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

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

[0060] The third aspect of the present invention provides a 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 h1) to h16):

[0061] h1) 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;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0079] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain of the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention comprises:

[0080] a211) the nucleotide sequence shown in SEQ ID NO: 26; or

[0081] a212) a nucleotide sequence in which one or more nucleotides are substituted and / or deleted and / or added to SEQ ID NO: 26, and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 26; or

[0082] a213) a nucleotide sequence having 80%, 85% or 90% or greater homology to SEQ ID NO: 26, and having the same function as the nucleic acid molecule represented by SEQ ID NO: 26;

[0083] The nucleotide sequence encoding the nucleic acid molecule encoding the light chain of the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention comprises:

[0084] a221) the nucleotide sequence shown in SEQ ID NO: 27; or

[0085] a222) a nucleotide sequence in which one or more nucleotides are substituted and / or deleted and / or added to SEQ ID NO: 27, and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 27; or

[0086] a223) has 80%, 85% or 90% or more homology with SEQ ID NO: 27, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 27.

[0087] 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;

[0088] 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.

[0089] Preferably, the detectable label is selected from radioisotopes, fluorescent substances, chemiluminescent substances, colored substances, or any combination thereof.

[0090] 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.

[0091] 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;

[0092] The product comprises at least one of a drug, a reagent, a detection plate, a test kit, and a detection chip.

[0093] Preferably, the drug has at least one of the functions i1) to i2):

[0094] i1) Prevention of EBV infection;

[0095] i2) Treating and / or preventing diseases caused by EB virus infection.

[0096] Preferably, the reagent, detection plate, detection chip or kit has at least one of the functions j1) to j3):

[0097] j1) detecting the presence or level of gp42 protein in the sample;

[0098] j2) Detection of Epstein-Barr virus;

[0099] j3) Diagnosis of diseases caused by EBV infection.

[0100] 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.

[0101] Preferably, the medicament comprises a vaccine.

[0102] A sixth aspect of the present invention provides a product comprising at least one of k1) to k3):

[0103] k1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0104] k2) the recombinant protein according to the second aspect of the present invention;

[0105] k3) the conjugate according to the fourth aspect of the invention;

[0106] The product comprises at least one of a reagent, a detection plate, a test kit, and a detection chip.

[0107] Preferably, the product has at least one of the functions j1) to j3):

[0108] j1) detecting the presence or level of gp42 protein in the sample;

[0109] j2) Detection of Epstein-Barr virus;

[0110] j3) Diagnosis of diseases caused by EBV infection.

[0111] 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.

[0112] The seventh aspect of the present invention provides a drug comprising at least one of 11) to 14):

[0113] 11) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0114] 12) The recombinant protein according to the second aspect of the present invention;

[0115] l3) the biomaterial according to the third aspect of the invention;

[0116] 14) The conjugate according to the fourth aspect of the present invention.

[0117] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0118] Preferably, the drug has at least one of the functions i1) to i2):

[0119] i1) Prevention of EBV infection;

[0120] i2) Treating and / or preventing diseases caused by EB virus infection.

[0121] 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.

[0122] Preferably, the medicament comprises a vaccine.

[0123] Preferably, a vaccine comprises at least one of 11) to 14) and an adjuvant:

[0124] 11) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0125] 12) The recombinant protein according to the second aspect of the present invention;

[0126] l3) the biomaterial according to the third aspect of the invention;

[0127] 14) The conjugate according to the fourth aspect of the present invention.

[0128] The eighth aspect of the present invention is to provide a method for preparing the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention or the recombinant protein of the second aspect of the present invention, which is obtained by culturing the transgenic cell line, microorganism or virus of the third aspect of the present invention.

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

[0130] The present invention provides an anti-EBV gp42 monoclonal antibody or an antigen-binding fragment thereof. The monoclonal antibody or the antigen-binding fragment thereof has good binding activity with the gp42 protein and has a high affinity for the gp42 protein (KD(M)=2.09E-12), can significantly inhibit EBV infection of epithelial cells and B cells, and can be used to detect the presence or level of gp42 protein in a sample, detect EBV, diagnose diseases caused by EBV infection, prevent EBV infection, and / or treat and / or prevent diseases caused by EBV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] Figure 1 This is the affinity test result of monoclonal antibody 2B7 and gp42 protein.

[0132] Figure 2 This is an ELISA assay result of the binding activity of monoclonal antibody 2B7 to gp42 protein.

[0133] Figure 3This figure shows the results of monoclonal antibody 2B7 blocking EBV infection of epithelial cells.

[0134] Figure 4 This figure shows the results of monoclonal antibody 2B7 blocking EBV infection of B cells. DETAILED DESCRIPTION

[0135] The present invention is further described in detail below through specific examples.

[0136] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0137] 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.

[0138] Example 1 Preparation of Monoclonal Antibodies (mAbs) Against Epstein-Barr Virus gp42 Protein (EBV gp42)

[0139] 1.1 Preparation of EBV gp42 recombinant protein

[0140] The gp42 protein plays an important role in the process of EBV invading epithelial cells and B cells. In the examples of the present invention, the inventors selected gp42 as a bait protein to screen specific antibodies.

[0141] The selected gp42 original sequence includes a KOZAK sequence, a CD5 signal peptide, a gp42 protein (34-223aa), a His tag, and a stop codon; specifically, 5'--3' (SEQ ID NO: 1); wherein, the sequence composed of nucleotides 1 to 6 in SEQ ID NO: 1 is a KOZAK sequence, and the sequence composed of nucleotides 7 to 78 in SEQ ID NO: 1 is a CD5 signal peptide sequence; the sequence composed of nucleotides 79 to 648 in SEQ ID NO: 1 is a gp42 protein (34-223aa) sequence, the sequence composed of nucleotides 649 to 666 in SEQ ID NO: 1 is a His tag sequence, and the sequence composed of nucleotides 667 to 669 in SEQ ID NO: 1 is a stop codon.

[0142] The above gp42 original sequence was ligated into the mammalian cell expression vector pcDNA3.1+ (Invitrogen) as follows:

[0143] (1) Amplification of gp42 protein gene:

[0144] Use 50 μL of NEB The PCR amplification reaction system (Table 1) was used to amplify the gp42 original sequence, wherein the upstream primer was: 5'-GTGTGATCAGATATCGCGGCCGCATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCTGCCTCGGAGGAGGGCGGGTGGCAGCC-3' (SEQ ID NO: 2); the downstream primer was: 5'-ACTAGAAGGCACAGCAGATCTTTAGTGGTGATGGTGATGATGGCTATTTGATCTTTGACTGACACATAAA-3' (SEQ ID NO: 3).

[0145] The amplified target fragment was analyzed by agarose gel electrophoresis, the band of the correct molecular weight was cut under ultraviolet light, and the PCR product was recovered according to the instructions of the commercially available kit.

[0146] Table 1 PCR amplification reaction system for gp42 original sequence

[0147] Components content 5×Reaction Buffer 10 μL dNTPs 1 μL Upstream primer (10uM) 2.5 μL Downstream primer (10uM) 2.5 μL EBV genomic DNA template 1 μL DNA polymerase 0.5μL High GC Enhancer (Q5 kit from NEB) 10 μL <![CDATA[ddH2O]]> Make up to 50 μL

[0148] (2) Enzyme digestion and ligation of target fragment and vector:

[0149] The eukaryotic expression plasmid pcDNA3.1+ was used as the vector. The target fragment and the vector were digested with Not I and Bgl II (using 50 μL of the enzyme digestion reaction system (Table 2)).

[0150] Table 2 Enzyme digestion reaction system

[0151] Components content 10×CutSmart Buffer 5μL BglII-HF 1 μL NotⅠ-HF 1 μL Target fragment or vector 5 μg <![CDATA[ddH2O]]> Make up to 50 μL

[0152] Perform the enzyme digestion at 37°C for 2–3 hours. Run the digested vector on an agarose gel and then recover it using a gel recovery kit to obtain the linearized vector. Recover the insert directly using a DNA purification kit. After recovery, ligate the fragment using 10 μL of the ligation reaction system (Table 3). Reaction conditions are 37°C for 30 minutes. This yields the ligation product.

[0153] Table 3 Ligation reaction system

[0154] Components content 5×CEⅡ Buffer 2μL CEⅡ 1 μL Linearized vector 100ng Insert 100ng <![CDATA[ddH2O]]> Make up to 10 μL

[0155] (3) Transformation of ligation products and screening of positive clones:

[0156] Add the ligation product to a freshly thawed DH5α competent cell suspension, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, and return to ice for 5 minutes. Add 200 μL of LB medium and slowly shake at 30°C for 40 minutes. Pipette the culture medium onto an ampicillin-resistant LB plate and incubate at 37°C overnight.

[0157] Pick a single clone colony for sequencing verification. Once the sequencing result is correct, the target recombinant plasmid is obtained. Extract the plasmid in large quantities.

[0158] (4) Expression and extraction of recombinant proteins:

[0159] Human renal epithelial cells 293F were cultured to obtain a cell density of 1.5×10 6 The recombinant plasmid obtained in step (3) was transfected using PEI transfection reagent. The specific operation was as follows: 2 mg of recombinant plasmid was diluted with 25 mL of Union-293 culture medium; 6 mL of 1 mg / mL PEI was added to 25 mL of Union-293 culture medium. The plasmid and PEI were thoroughly shaken and mixed, and then added to the 293F cell suspension after standing at room temperature for 20 minutes. After culturing for 5 days, the cell supernatant was collected and centrifuged at 6000 rpm for 1 hour at 4°C. The cell pellet was discarded to obtain the supernatant containing the target protein.

[0160] The target protein was purified by affinity chromatography.

[0161] Because the resulting gp42 recombinant protein carries a 6×His tag at its C-terminus, it can be affinity purified using a nickel column. The supernatant containing the target protein was filtered through 0.65 μm filter paper, passed through the nickel column beads three times for binding, washed three times with 30 mM imidazole, and eluted with 500 mM imidazole. Further purification was performed using gel filtration chromatography. The protein eluted from the nickel column was concentrated to a volume of less than 1 mL using a 3 kD concentrator. Purification was then performed using a Superdex 200 Increase 10 / 300 GL.

[0162] The amino acid sequence of the target protein finally obtained is: GGRVAAAAITWVPKPNVEVWPVDPPPPVNFNKTAEQEYGDKEVKLPHWTPTLHTFQVPQNYTKANCTYCNTREYTFSYKGCCFYFTKKKHTWNGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTFKVYQIFGSHCTYVSKFSTVPVSHHECSFLKPCLCVSQRSNSHHHHHH (SEQ ID NO: 4); wherein, the sequence composed of amino acid residues 1 to 190 in SEQ ID NO: 4 is the gp42 protein sequence (34-223aa), and the sequence composed of amino acid residues 191 to 196 is the His tag sequence.

[0163] 1.2 Construction of phage antibody library

[0164] (1) Total RNA extraction:

[0165] Nasopharyngeal carcinoma (NPC) patient blood was diluted 1:1 with PBS (10 mL blood + 10 mL PBS). Then, 20 mL of the dilution was carefully overlaid on 15 mL of lymphocyte separation medium, maintaining the layered interface. The tube was centrifuged at 2000 rpm for 20 minutes at room temperature. The speed was slowly reduced, and the middle mononuclear cell layer was aspirated into a new 15 mL centrifuge tube. The volume was made up to 15 mL with PBS, and the tube was centrifuged at 300 g for 20 minutes at room temperature. The supernatant was transferred to a new 50 mL centrifuge tube and centrifuged at 300 g for 20 minutes at room temperature. The supernatant was removed and the cell pellets in the 15 mL and 50 mL centrifuge tubes were resuspended with 1 mL of Trizol each (i.e., 2 mL of Trizol was used for every 10 mL of blood).

[0166] Add 4 mL of chloroform to 20 mL of Trizol-derived cell pellet suspension and shake on a shaker for 15 seconds. Allow to stand at room temperature for 5 minutes. Centrifuge at 4000 g for 30 minutes at 4°C. Once layers have formed, carefully transfer the top, clear layer to a new 50 mL centrifuge tube that is free of RNase and DNase. Add a 1:1 volume of isopropanol to each tube, invert several times to mix thoroughly, and allow to stand at room temperature for 10 minutes. Centrifuge at 4000 g for 30 minutes, remove the supernatant, and retain the pellet. Add 1 mL of 75% ethanol to the pellet and transfer it to a 1.5 mL centrifuge tube, flicking the pellet several times to thoroughly dissolve it. Centrifuge at 7500 g for 5 minutes, remove the supernatant, and retain the pellet. Allow the tube to dry at room temperature for 10 minutes, leaving the tube open. Add 400 μL of enzyme-free water and incubate at 55°C for 10 minutes to ensure complete dissolution of the RNA. This will yield total RNA.

[0167] Take 1 μL and use a nucleic acid concentration meter to detect the RNA concentration and A260 / A280 and record them.

[0168] (2) RNA reverse transcription:

[0169] Reverse transcription kit (Promega GoScript TM The total RNA obtained in step (1) was reverse transcribed using a reverse transcription kit.

[0170] The specific operations are:

[0171] The total RNA sample was divided into two portions. One portion used the Oligo dT Primer in the kit as a primer, and the other portion used the Random 6-mers in the kit as a primer. The reaction solution was prepared according to the system shown in Table 4 in a 1.5 mL centrifuge tube. The reaction system was expanded in the same proportion according to the amount of RNA for amplification.

[0172] After the above reaction, the reaction solution was divided into eight PCR tubes and placed in a PCR instrument. Briefly centrifuged, incubated at 70°C for 5 minutes to denature the RNA. After the reaction, the tubes were quickly cooled on ice. Then, the remaining reaction solutions were added to the eight PCR tubes according to the ratios in Table 5. The reaction solutions in Table 5 were mixed thoroughly and briefly centrifuged. The tubes were placed in a PCR instrument and incubated at 45°C for 60 minutes, followed by 75°C for 15 minutes. The reaction was cooled on ice to obtain cDNA after reverse transcription of the total RNA. The cDNA was stored at 4°C.

[0173] Table 4 5 μL reverse transcription reaction system

[0174] Components content Oligo dT Primer(50μM) / Random 6-mers(50μM) 1 μL Total RNA 2 μg <![CDATA[ddH2O]]> Make up to 5 μL

[0175] Table 5 Other reaction liquid ratios

[0176] Components content Table 4 System after reaction 5μL 5×Reaction Buffer 4μL <![CDATA[MgCl2]]> 2uL dNTP mix 1uL RNase inhibitors 0.4μL RTase 1 μL <![CDATA[ddH2O]]> Make up to 20 μL

[0177] (3) PCR amplification:

[0178] Using the cDNA obtained in step (2) as a template, NEB Q5 high-fidelity DNA polymerase was used to amplify the single-chain antibody fragment (ScFv) by two rounds of PCR. The constructed structure was VL-linker-VH (light chain-linker-heavy chain).

[0179] The first round of PCR was performed using cDNA as a template. The reaction system for the first round of PCR is shown in Table 6. The PCR reaction used a three-temperature method: each cycle consisted of denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute, repeated 35 times. The primers used in the first round of PCR included:

[0180] Vλ forward primer: 5′-CCTTTCTATGCGGCCCAGCCGGCCgagctcCAGTCTGTSBTGACGCAGCCGCC-3′ (SEQ ID NO: 5);

[0181] Vλ-linker reverse primer: 5′-GGAAGATCTAGAGGAACCACCTAGGACGGTSASCTTGGTCC-3′ (SEQ ID NO: 6);

[0182] Vκ forward primer: 5′-CCTTTCTATGCGGCCCAGCCGGCCgagctcGACATCCRGDTGACCCAGTCTCC-3′ (SEQ ID NO: 7);

[0183] Vκ-linker reverse primer: 5′-GGAAGATCTAGAGGAACCACCTTTGATTTCCACCTTGGTCC-3′ (SEQ ID NO: 8);

[0184] linker-VH forward primer: 5'-GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGGTGCAGCTGCAGGAGTCSG-3' (SEQ ID NO: 9);

[0185] Linker-VH reverse primer: 5′-CAGTCATTCTCGACTTactagtTGAGGAGACRGTGACCAGGGTG-3′ (SEQ ID NO: 10);

[0186] Among them, the lowercase part in each primer is the restriction site; the Vλ forward primer is used in pair with the Vλ-linker reverse primer, the Vκ forward primer is used in pair with the Vκ-linker reverse primer, and the linker-VH forward primer is used in pair with the linker-VH reverse primer.

[0187] Table 6 First round PCR reaction system

[0188] Components content cDNA 5μL Forward primer (10uM) 2.5 μL Reverse primer (10uM) 2.5 μL dNTP Mix 1 μL 5×Reaction Buffer 10 μL High GC Enhancer 10 μL polymerase 0.5μL <![CDATA[ddH2O]]> Make up to 50 μL

[0189] After the reaction, all PCR products were subjected to 1.5% agarose gel electrophoresis and the target fragments (approximately 320 bp for VL and 350 bp for VH) were recovered. DNA was purified using the NEB DNA GEL purification kit according to the kit instructions. The collected DNA solution, representing the first-round PCR amplification product, was stored at 4°C after concentration measurement.

[0190] The second round of PCR was performed using the product amplified from the first round of PCR as a template. The second round of PCR reaction system is shown in Table 7. The reaction conditions for the second round of PCR were the same as those for the first round of PCR. The primers used in the second round of PCR included: OF: 5'-CCTTTCTATGCGGCCCAGCCGGCCgagctc-3' (SEQ ID NO: 11); OR: 5'-CAGTCATTCTCGACTTactagt-3' (SEQ ID NO: 12).

[0191] Table 7 Second round PCR reaction system

[0192] Components content VH / VL glue recovery products 80ng VH+80ng VL dNTP Mix 1 μL 5×Reaction Buffer 10 μL High GC Enhancer 10 μL Forward primer OF (10uM) 2.5 μL Reverse primer OR (10uM) 2.5 μL polymerase 0.5μL <![CDATA[ddH2O]]> Make up to 50 μL

[0193] After the reaction, the PCR products were subjected to 1.5% agarose gel electrophoresis at 160 V for 20 min. The target band with a fragment size of 750 bp was finally selected from the electrophoresis results. DNA was recovered using the NEB DNA GEL purification kit according to the kit instructions. The collected DNA solution was the second-round PCR amplification product and stored at 4°C after concentration measurement.

[0194] (4) Enzyme ligation of vector and PCR amplification product:

[0195] The second-round PCR product (ScFv fragment) was ligated into the phage plasmid pComb3XSS by enzyme digestion using restriction endonucleases SpeI and SacI, thereby constructing a phage plasmid library containing the amplified target fragment (ScFv fragment).

[0196] The enzyme digestion systems for the pComb3XSS vector and the second-round PCR amplification product (amplified target fragment (ScFv fragment)) are shown in Tables 8 and 9, respectively.

[0197] Table 8 Vector enzyme digestion system

[0198]

[0199]

[0200] Table 9 Target fragment (ScFv fragment) enzyme digestion system

[0201] Components content scFv 5 μg SpeI 5μL SacI 5μL CutSmart 10×buffer 25 μL <![CDATA[ddH2O]]> Make up to 250 μL

[0202] Reaction conditions: Incubate at 37°C for 2 hours, then at 80°C for 3 minutes. After digestion, run the vector digestion product on a gel and recover it using the NEB DNA purification kit (do not expose to UV light). ScFv fragment digestion products do not require gel running and can be recovered directly.

[0203] After enzymatic digestion and purification, measure the concentration of the recovered product. Then, ligate the digested vector and ScFv digested product using the ligation reaction system shown in Table 10. Incubate the ligation system at 37°C overnight (16-24 hours). After ligation, recover the ligation product using the NEB DNA purification and recovery kit. Measure and record the concentration of the recovered ligation product and store at 4°C.

[0204] Table 10 Ligation reaction system

[0205] Components content 10×T4 reaction buffer 2μL ScFv fragment (after enzyme digestion) 68ng Vector (after enzyme digestion) 100ng T4 ligase 1 μL <![CDATA[ddH2O]]> Make up to 20 μL

[0206] 1.3 Construction of bacterial library:

[0207] (1) Preparation of TG1 E. coli competent cells:

[0208] Streak a single colony of the TG1 strain onto 2×YT solid medium and incubate overnight at 37°C. Pick a single colony from the single colony plate and transfer it to 10 mL of 2×YT medium. Incubate overnight at 37°C at 220 rpm. Inoculate the bacteria into 100 mL of 2×YT medium at a 1:100 dilution. Incubate at 37°C at 250 rpm for 40 minutes. Measure the OD value and then measure it every 20 minutes until the OD600 reaches 0.3–0.35. Collect the culture suspension and centrifuge at 3200g for 10 minutes at 0–4°C. Discard the supernatant, place on ice, and resuspend in 40 mL of pre-chilled ddH2O. Centrifuge at 3200g for 10 minutes at 0–4°C. Discard the supernatant, place on ice, and resuspend in 1 mL of pre-chilled ddH2O. Transfer the suspension to a 1.5 mL pre-chilled EP tube and centrifuge at 10,000g for 30 seconds at 4°C. Repeat this process. The supernatant was discarded, and the cell suspension was placed on ice. 400 μL of pre-cooled ddH 2 O was added to resuspend the cell suspension to obtain a TG1 E. coli competent cell suspension.

[0209] Take the Comb3XSS recombinant plasmid containing the ScFv fragment obtained in the above embodiment, and use the electroporation method to construct an E. coli library. The specific method is: use a pre-cooled gun tip to add 100ng of the Comb3XSS recombinant plasmid containing the ScFv fragment to 50μL of the above-mentioned TG1 E. coli competent cell suspension, blow gently, transfer it to a pre-cooled 1mm electric transfer cup, confirm that the mixture is at the bottom of the electric transfer cup and there are no bubbles, set 1800V, 1mm spacing, and perform electroporation. Immediately after completion, add 1000μL of 37℃ SOC culture medium, take the mixed solution out of the electric transfer cup, and shake at 37℃, 180rpm for 90min to recover. Use 2×YT liquid culture medium to perform 10-fold gradient dilution, and dilute a total of 6 gradients (10 dilutions each). 1 , 10 2 , 10 3 , 10 4, 10 5 , 10 6 5 μL of each gradient was evenly added dropwise to 2×YT-GA solid medium, allowed to dry, and incubated overnight at 37°C. The number of colonies on the gradient dilution plates was counted, and the ligation efficiency was calculated. The ligation efficiency formula is:

[0210] E (pfu / ug) = N × D × 10;

[0211] Where, E is the competent efficiency (unit: pfu / ug), D is the dilution factor, and N is the number of monoclonal colonies on the plate at the corresponding dilution factor.

[0212] Repeat 100 electroporation reactions as described above. Spread the recovered bacterial suspension evenly onto 100 2×YT-GA 245mm square plates. After drying, invert and incubate at 37°C overnight. Remove the overnight incubated square plates and add 6 mL of 2×YT liquid medium to the surface of each plate. Gently scrape the colonies from the 100 square plates with a spreading rod and collect the bacterial suspension into a 50 mL centrifuge tube. Add glycerol to a final concentration of 20% to create the bacterial library.

[0213] Take 10 μL of bacterial solution and add it to 990 μL of 2×YT liquid medium and measure OD600 using NanoDrop. Calculate and record the total OD600 of the bacterial library.

[0214] T OD600 =M OD600 ×100;

[0215] Among them, T OD600 is the total bacterial OD600, M OD600 is the measured OD600.

[0216] 1.4 Phage library construction:

[0217] Take an appropriate amount of the bacterial library from the above into a 1.5 mL EP tube. The calculation formula for the bacterial liquid volume is:

[0218]

[0219] Wherein, V is the volume of the transferred bacterial solution (unit: μL), and OD600 is the total OD600 of the constructed bacterial library.

[0220] Transfer the cells to 100 mL of 2×YT-GA liquid medium to an initial OD600 of 0.1. Incubate in a shaker at 37°C, 250 rpm, until the OD600 reaches 0.5–0.55. Add helper phage M13K07 to a bacterial:phage ratio of 1:20, calculated using the following formula:

[0221]

[0222] Where V is the volume of helper phage added (unit: mL), T helper-phage is the titer of the helper phage used, and OD600 is the OD600 value of the bacterial solution.

[0223] Incubate the cells in a shaker at 37°C, 220 rpm for 30 min. Centrifuge at 3200 g for 5 min to collect the TG1 bacteria. Remove the supernatant and resuspend the pellet in 100 mL of 2×YT-AK liquid medium. Incubate the cells in a shaker at 30°C, 250 rpm, overnight.

[0224] Transfer the overnight culture to a new 50 mL centrifuge tube and centrifuge at 4000 g for 30 minutes at 4°C. Remove the supernatant and add 1 / 4 volume of 4°C pre-cooled 20% PEG / 2.5M NaCl, mix thoroughly, and place on ice for 30 minutes. Centrifuge at 4000 rpm at 4°C for 30 minutes, discard the supernatant, and invert on paper for 2 minutes. Add 1 mL of PBS to resuspend the pellet and centrifuge at 12000 rpm at 4°C for 20 minutes. Remove the supernatant and add 1 / 4 volume of pre-cooled 20% PEG / 2.5M NaCl solution, mix thoroughly, and place on ice for 10 minutes. Centrifuge at 12000 rpm at 4°C for 10 minutes, discard the supernatant, and resuspend the pellet in 1 mL of PBS. Centrifuge at 12000 rpm at 4°C for 2 minutes. Remove the supernatant (this is the phage library), add glycerol to a final concentration of 20%, and store at -80°C.

[0225] The titer of the phage library was tested as follows: the phage library was added to 10 mL of 2×YT liquid culture medium and cultured at 37°C and 250 rpm for about 45 to 60 minutes until the OD600 was 0.5 to 0.55. 10 μL of the cultured phage library was diluted 10-fold (a total of 13 dilution gradients). 90 μL of untreated TG1 bacterial solution was added to each dilution gradient, mixed well, and incubated at 37°C for 20 minutes. Then 5 μL of each dilution gradient was added dropwise to 2×YT-GA solid culture medium, dried, and cultured overnight at 37°C. The phage particles were counted and the number of phage particles per milliliter of phage solution was calculated according to the phage library titer formula.

[0226] T (pfu / ml) = N × D × 400;

[0227] Wherein, T is the phage titer (unit: pfu / mL), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0228] 1.5 Screening of gp42 protein-specific antibodies

[0229] Antibodies that specifically bind to gp42 protein were screened using antigen solid phase adsorption screening method.

[0230] The specific operations are as follows:

[0231] Dissolve 50 μg of the gp42 protein obtained above in 2 mL of PBS and coat the immunotubes overnight at 4°C (positive tubes). Simultaneously, coat the negative control protein (50 μg of BSA) as a negative control. Discard the coated protein, rinse three times with 2 mL of PBS, and then block with 2 mL of 3% BSA (dissolved in PBST) at room temperature for 2 hours. Discard the blocking solution, take 100 μL of the obtained phage library, dilute it with 2 mL of PBS, and incubate it in the immunotubes for 1 hour. Discard the solution, wash five times with 2 mL of PBST, each for 5 minutes. Then, wash five more times with 2 mL of PBS, each for 5 minutes. During the washes, transfer 1 mL of the overnight TG1 saturated bacterial suspension to 100 mL of 2×YT liquid medium and incubate at 37°C at 150 rpm until OD600 = 0.5 (approximately 1.5 hours). Discard the wash solution, and elute with 1 mL of 0.1 mg / mL Trypsin at room temperature for 30 minutes. Add 1 mL of the eluate to 10 mL of cultured TG1 bacterial suspension (OD = 0.5) and infect at 37°C, 150 rpm, for 30 minutes. Centrifuge at 4°C, 3000 g, for 10 minutes. Resuspend the pellet in 1 mL of 2×YT liquid medium. Evenly spread 1 mL of the resuspended product from the positive immunofluorescence tube onto a 245 mm x 245 mm 2×YT solid culture dish and incubate at 37°C overnight (16-20 hours). Measure the phage titer of the positive and negative control tubes using the same method as described above.

[0232] Based on the phage titer comparison results, add 5 mL of 2×YT liquid medium to the dish with the highest phage titer after overnight culture. Use a spreader to scrape off all colonies, and prepare a daughter phage library according to the method described in the previous example. Repeat this process until the antibody with the highest affinity for gp42 (compared to the negative control) is screened.

[0233] The rotation speed during antigen coating, blocking, incubation, washing and elution processes was 15 rpm unless otherwise specified.

[0234] The highest affinity antibodies were detected using ELISA.

[0235] Take 10 μL of the culture medium containing the antibody with the highest affinity for gp42 and dilute it with 1 mL of 2×YT liquid medium. Streak the colonies onto 2×YT-GA solid culture plates and incubate overnight at 37°C for 16–20 hours. Pick 192 single colonies and transfer them to a 96-well plate (200 μL of 2×YT-GA per well) and incubate at 37°C until saturation occurs. Transfer 2 μL of the saturated culture medium to a new 96-well plate (200 μL of 2×YT-A per well) to an initial OD of approximately 0.03. Incubate at 37°C for 2.5–3 hours until the OD is approximately 0.5. Add 0.1 μL of the aforementioned helper phage to each well and infect at 37°C for 30 minutes. Add 0.2 μL of Kana (kanamycin) to each well and incubate overnight at 30°C. Coat the ELISA plate with protein (- / +) overnight. Centrifuge the 96-well plate containing the overnight incubation at 3400 g for 5 minutes at 4°C. Discard the plate contents and wash once with 350 μL of PBS. Block with 350 μL of 3% BSA (PBST) at 37°C for 1 hour. Discard the blocking solution, wash once with 350 μL of PBST, and tap the plate to remove the liquid. Add 140 μL of 3% BSA (PBST) to each well, followed by 60 μL of the above-mentioned phage library-expressed antibody (the highest affinity antibody, serving as the primary antibody) and incubate at 37°C for 1 hour. Discard the primary antibody solution, wash five times with 350 μL of PBST, and tap the plate. Add 100 μL of M13 Antibody (HRP) as a secondary antibody (1:8000 ratio relative to the blocking solution), incubate at 37°C for 1 hour, discard the secondary antibody solution, wash five times with 350 μL of PBST, and tap the plate. 100 μL TMB was incubated in the dark for 2-3 minutes, and the reaction was terminated with 100 μL dilute hydrochloric acid (concentrated hydrochloric acid: water = 1:12). The OD450 and OD630 values ​​of the ELISA plate were read. In this example, the best antibody obtained by the inventors was named 2B7.

[0236] 1.6 Expression and purification of monoclonal antibodies

[0237] By linking the antibody heavy chain variable region upstream to a CMV fragment and downstream to the human IgG1 constant region and a ployA fragment, a complete heavy chain fragment can be expressed. Conversely, by linking the antibody light chain variable region upstream to a CMV fragment and downstream to the light chain kappa / lambda constant region and a ployA fragment, a complete light chain fragment can be expressed. Antibody expression can be achieved by co-transfecting plasmids carrying the full-length heavy and light chain sequences into 293T cells, and the antibodies can be purified using protein A beads.

[0238] The 2B7 full-length heavy chain has a total of 483 amino acid residues (excluding *), specifically:

[0239]

[0240] The underlined portion of the sequence represents the amino acid sequence of the heavy chain variable region (SEQ ID NO: 14). The underlined and bolded portions represent the amino acid sequences of the three complementary regions of the heavy chain variable region, CDR-H1 (SEQ ID NO: 15), CDR-H2 (SEQ ID NO: 16), and CDR-H3 (SEQ ID NO: 17), respectively, according to the Kabat definition. The italicized portion represents the signal peptide. Amino acids 154 to 483 in SEQ ID NO: 13 represent the heavy chain constant region. * indicates a stop codon.

[0241] CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region using other CDR definition schemes are shown in Table 11.

[0242] Table 11 CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region using other CDR definition schemes

[0243]

[0244] The 2B7 full-length heavy chain coding gene consists of 1452 bases, specifically:

[0245]

[0246] The underlined portion of the sequence represents the nucleotide sequence of the heavy chain variable region. The underlined and bolded portions represent the nucleotide sequences of the three complementary regions of the heavy chain variable region, CDR-H1, CDR-H2, and CDR-H3 (Kabat definition). 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.

[0247] The 2B7 full-length light chain consists of 235 amino acid residues (excluding *), specifically:

[0248]

[0249] The underlined portion of the sequence represents the amino acid sequence of the light chain variable region (SEQ ID NO: 29). The underlined and bolded portions represent the amino acid sequences of the three complementarity determining regions (CDR-L1) (SEQ ID NO: 30), CDR-L2 (SEQ ID NO: 31), and CDR-L3 (SEQ ID NO: 32) in the light chain variable region (Kabat definition). The italicized portion represents the signal peptide. Amino acids 130 to 235 in SEQ ID NO: 28 represent the heavy chain constant region. * indicates a stop codon.

[0250] CDR-H1, CDR-H2, and CDR-H3 in the light chain variable region using other CDR definition schemes are shown in Table 12.

[0251] Table 12 CDR-H1, CDR-H2, and CDR-H3 in the light chain variable region using other CDR definition schemes

[0252]

[0253] The gene encoding the 2B7 full-length light chain contains 708 bases, specifically:

[0254]

[0255] The underlined portion of the sequence represents the nucleotide sequence of the light chain variable region. The underlined and bolded portions represent the nucleotide sequences of the three complementary regions of the light chain variable region: CDR-L1, CDR-L2, and CDR-L3. 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.

[0256] Effect embodiment

[0257] 1. The affinity of antibody 2B7 was determined using biofilm interferometry (BLI).

[0258] BLI can be performed according to conventional methods in the art. In this embodiment, the specific operation is as follows: a biosensor (Sartorius, Germany) SA probe) is immersed in a buffer solution (a mixture of KB buffer, 0.1% BSA and 0.02% Tween 20) for equilibrium. It is then taken out and immersed in a solution containing 5μg / mL gp42-Biotin (biotin-labeled gp42 protein). The gp42 antigen in the solution will bind to the surface of the SA (streptavidin) biological probe, increasing the thickness of the surface film. The biosensor with a known concentration of solidified antigen is then immersed in a buffer solution as a baseline. By immersing the solidified biosensor with a known concentration of antigen in a sample solution containing 7.8-500nM 2B7 antibody for about 80 seconds, the specific binding between antigen and antibody will lead to an increase in the thickness of the film layer. The biosensor bound to the 2B7 antibody is immersed in a buffer solution for about 300 seconds for dissociation. The antibody to be tested (2B7 antibody) will fall off the surface of the biosensor, resulting in a decrease in the thickness of the film layer. By real-time monitoring of the biosensor biofilm thickness during the experiment, the kinetic constant of the sample to be tested (2B7 antibody) can be obtained. The results are shown in Figure 1 As shown: KD (M) of 2B7 antibody = 2.09E-12, indicating that 2B7 antibody has extremely high affinity with gp42 antigen.

[0259] 2.2B7 Antibody Related Applications

[0260] Based on the high affinity of the 2B7 antibody, it can be effectively used in related detection of gp42 antigen, such as in qualitative or quantitative detection such as ELISA, and can also be used for clinical purposes related to the prevention and treatment of EBV infection.

[0261] 2.12B7 antibody for ELISA detection of gp42 antigen

[0262] (1) Dilute gp42 antigen to 1 μg / mL with PBS buffer. Transfer 100 μL of gp42 antigen solution to the ELISA plate wells and incubate at 4°C overnight.

[0263] (2) Remove the gp42 antigen solution, wash with PBS three times, and block with PBS solution containing 3% BSA at room temperature for 30 minutes.

[0264] (3) Dilute the 2B7 antibody or 2G4 control antibody (the antibody has been disclosed in the literature: Molecular characterization of the monoclonal antibodies composing ZMAb: a protective cocktail against Ebola virus.) to 0.5 μg / mL with PBS solution containing 3% BSA, add 200 μL of the diluted antibody or BSA control solution to each well, and incubate at room temperature for 2 hours; the 2G4 control antibody is an antibody against Ebola virus, and the BSA control is a PBS solution containing 3% BSA.

[0265] (4) Remove the liquid from the wells and wash with PBST four times. Add 100 μL of 1:10000 diluted anti-human IgG-HRP antibody (Abc am) to each well and incubate at room temperature for 1 hour.

[0266] (5) Remove the liquid from the wells and wash four times with PBST. Add 100 μL of TMB solution to each well for color development. After about 20 seconds, terminate the color development with ten-fold diluted hydrochloric acid. Read the absorbance of the solution at a wavelength of 450 nm.

[0267] The results are as follows Figure 2 As shown, the 2B7 antibody has extremely high binding activity with the gp42 antigen.

[0268] Neutralizing activity of 3.2B7 antibody

[0269] (1) Preparation of EBV virus:

[0270] 1) CNE2 cells infected with EBV-GFP (the virus is 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 cultured in RPMI1640 + 5% FBS in a 37°C incubator (5% CO2). When the cells reached 90% density (10 cm dish), they were induced by adding TPA at a final concentration of 20 ng / mL and NaB (sodium butyrate) at a final concentration of 2.5 mM. The medium was changed after 12 hours.

[0271] 2) 48-72 hours after the medium exchange, collect the culture supernatant to isolate and purify the virus. Directly aspirate the supernatant, centrifuge, and filter through a 0.45 μm filter. After concentration, resuspend in serum-free RPMI1640 and use immediately for infection or store at -80°C.

[0272] (2) Neutralization activity assay of 2B7 monoclonal antibody in epithelial cells

[0273] 1) Plate 1x10 per well in a 96-well plate. 6 NOK epithelial cells were cultured and 100 μL of DMEM medium containing 10% FBS was added to each well.

[0274] 2) The next day, adjust the 2B7 monoclonal antibody from the previous example to a concentration of 2 mg / mL. Add 60 μL of DMEM medium to each well of a new 96-well plate, and add 120 μL of 12.5 μg / mL 2B7 antibody diluted in DMEM to the first well (do not add RPMI1640 medium to the first well).

[0275] 3) After 2-fold gradient dilution (gradient dilution will aspirate 60uL from the first well and add it to the second well, and so on, aspirate 60uL from the last well and discard it, and the final volume of each well is 60uL), add 60uL of virus diluent to each well (the virus is diluted with DMEM medium, the titer is about 4*10 6 After incubation at 37°C for 2 hours, the cells were added to the NOK cells plated the day before and cultured in a 37°C incubator for 48 hours before detection.

[0276] 4) NOK cells were trypsinized to prepare a cell suspension. The infection rate was measured by flow cytometry. The inhibitory rate (neutralization efficiency, %) of the antibody in the NOK epithelial cell infection model was calculated by measuring the reduction in the number of GFP-positive cells in the antibody-treated group compared to the infection control group (addition of an equal volume of DMEM). The IC50 of the 2B7 monoclonal antibody was calculated and plotted using Prism. 2G4 antibody was used as a negative control antibody. 2G4 antibody is a control antibody against Ebola virus.

[0277] The results are as follows Figure 3 As shown: The IC50 of monoclonal antibody 2B7 in the epithelial cell infection model is 0.090ug / mL, and the control antibody has no neutralizing activity; monoclonal antibody 2B7 can significantly inhibit EBV infection of epithelial cells.

[0278] (3) Detection of B cell neutralizing activity of 2B7 monoclonal antibody

[0279] 1) Take the 2B7 monoclonal antibody from the above example and adjust the concentration to 2 mg / mL. Add 60 μL of RPMI1640 medium to each well of a new 96-well plate. Add 90 μL of 500 μg / mL 2B7 antibody diluted in RPMI1640 to the first well (do not add RPMI1640 medium to the first well).

[0280] 2) After 3-fold gradient dilution (gradient dilution will aspirate 30uL from the first well and add it to the second well, and so on, aspirate 30uL from the last well and discard it, the final volume of each well is 60uL); add 60uL of virus diluent to each well (the virus is diluted with DMEM medium, the titer is about 4*10 6 / mL), and then 1*10 6 Raji cells were cultured in a 37°C incubator for 48 hours before testing.

[0281] 3) Raji cells were aspirated to prepare a cell suspension. The infection rate was determined by flow cytometry. The inhibitory rate (neutralization efficiency, %) of the antibody in the Raji B cell infection model was calculated by measuring the reduction in the number of GFP-positive cells in the antibody-treated group compared to the infection control group (addition of an equal volume of RPM I1640). The IC50 of the 2B7 monoclonal antibody was calculated and plotted using Prism. The 2G4 antibody was used as a negative control.

[0282] The results are as follows Figure 4 As shown: The IC50 of monoclonal antibody 2B7 in the B cell infection model is 2.646ug / mL, and the control antibody has no neutralizing activity; monoclonal antibody 2B7 can significantly inhibit EBV infection of B cells.

[0283] 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. An anti-EBV gp42 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain and a light chain: The heavy chain comprises: a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 14; The light chain comprises: a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 29; The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively, and the CDRs are defined according to the Kabat definition scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 are shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 33, and SEQ ID NO: 32, respectively; the amino acid sequence of CDR-L2 is: NDN; and the CDRs are defined according to the IMGT definition scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 17, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively, and the CDRs are defined according to the Chothia definition scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively. The CDRs are defined based on the Contact definition scheme.

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: a1) SEQ ID NO. 14; or a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids to SEQ ID NO. 14 and having the same function as the protein shown in SEQ ID NO. 14; or a3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% identical to SEQ ID NO. 14 and has the same function as the protein shown in SEQ ID NO. 14; The amino acid sequence of the light chain variable region comprises: b1) SEQ ID NO.29; or b2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO. 29 and having the same function as the protein shown in SEQ ID NO. 29; or b3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% identical to SEQ ID NO. 29 and has the same function as the protein shown in SEQ ID NO.

29.

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 heavy chain further comprises a heavy chain constant region; and / or The light chain also comprises a light chain constant region.

5. The monoclonal antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The amino acid sequence of the heavy chain constant region comprises: c1) an amino acid sequence consisting of amino acids 154 to 483 of SEQ ID NO: 13; or c2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in the amino acid sequence described in c1) and having the same function as the protein of the amino acid sequence described in c1); or c3) An amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in c1) and has the same function as the protein of the amino acid sequence described in c1).

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein: The amino acid sequence of the light chain constant region comprises: d1) an amino acid sequence consisting of amino acids 130 to 235 of SEQ ID NO: 28; or d2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in the amino acid sequence described in d1) and having the same function as the protein of the amino acid sequence described in d1); or d3) An amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in d1) and has the same function as the protein of the amino acid sequence described in d1).

7. A recombinant protein comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; and It consists of a tag sequence that facilitates expression and / or purification.

8. A biomaterial related to the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the recombinant protein according to claim 7, comprising at least one of h1) to h16): h1) a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the recombinant protein according to claim 7; h2) an expression cassette comprising the nucleic acid molecule described in h1); h3) a vector comprising the nucleic acid molecule described in h1); h4) a vector comprising the expression cassette described in h2); h5) a transgenic cell line comprising the nucleic acid molecule described in h1); h6) a transgenic cell line comprising the expression cassette described in h2); h7) a transgenic cell line comprising the vector described in h3); h8) a transgenic cell line comprising the vector described in h4); h9) a microorganism comprising the nucleic acid molecule described in h1); h10) a microorganism comprising the expression cassette described in h2); h11) a microorganism comprising the vector described in h3); h12) a microorganism comprising the vector described in h4); h13) A virus comprising the nucleic acid molecule described in h1); h14) A virus comprising the expression cassette described in h2); h15) A virus comprising the vector described in h3); h16) A virus comprising the vector described in h4).

9. A conjugate comprising: at least one of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and the recombinant protein according to claim 7; and a coupling portion, wherein the coupling portion is at least one of a detectable label and a radionuclide.

10. 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 6; (2) The recombinant protein according to claim 7; (3) The biomaterial according to claim 8; (4) The conjugate according to claim 9; The product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip; The drug has the function of treating and / or preventing diseases caused by EB virus infection: The reagent, detection plate, detection chip or kit has at least one function among j1) to j3): j1) detecting the presence or level of gp42 protein in the sample; j2) Detection of Epstein-Barr virus; j3) Diagnosis of diseases caused by EBV infection.

11. A product comprising at least one of k1) to k3): k1) the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; k2) the recombinant protein according to claim 7; k3) the conjugate according to claim 9; The product comprises at least one of a reagent, a detection plate, a test kit, and a detection chip.

12. A drug comprising at least one of (11) to (14): (11) The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; (12) The recombinant protein according to claim 7; (13) The biomaterial according to claim 8; (l4) The conjugate according to claim 9.

13. The drug according to claim 12, characterized in that: The drug further comprises a pharmaceutically acceptable carrier.

14. A vaccine comprising at least one of (11) to (14) and an adjuvant: (11) The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; (12) The recombinant protein according to claim 7; (13) The biomaterial according to claim 8; (l4) The conjugate according to claim 9.

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