Monoclonal antibody 1B6 recognizing Epstein-Barr virus BARF1 protein and use thereof

Abstract

This invention belongs to the field of antibody technology and discloses a monoclonal antibody 1B6 that recognizes the EBV BARF1 protein and its applications. This monoclonal antibody or its antigen-binding fragment has a high affinity for the BARF1 protein (KD(M) = 3.33E-12), can specifically bind to BARF1 in vivo and in vitro, and can specifically target EBV-positive tumor cells. It is particularly useful for tumor suppression, significantly inhibiting the growth of EBV-positive tumor cells in mice. It can be used to detect the presence or level of BARF1 protein in samples, detect EBV, diagnose diseases caused by EBV infection, prevent and treat EBV-positive tumors, and / or treat and / or prevent diseases caused by EBV infection.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to a monoclonal antibody 1B6 that recognizes the BARF1 protein of EB virus and its applications. Background Technology

[0002] Epstein-Barr virus (EBV) is a human gamma herpesvirus. After primary infection of submucosal B lymphocytes in the nasal / oropharyngeal mucosa, EBV establishes latent infection in memory B cells and persists in the human host for life. Infected cells express limited latent viral proteins, enabling them to maintain viral infection while evading host immune attack.

[0003] EBV infection most commonly occurs latently, with over 90% of EBV-infected individuals remaining latent for life. The virus can be activated and lead to cancer under certain conditions. Although EBV rarely causes disease in most individuals, it is commonly associated with post-transplantation complications in immunosuppressed patients and with AIDS-related lymphoproliferative disorder (PTLD). EBV is also closely related to the development of various cancers, including endemic Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin lymphoma, NK / T cell lymphoma, nasopharyngeal carcinoma, and some gastric carcinomas.

[0004] Currently, there is no effective vaccine against EBV, nor are there any targeted therapies for EBV-related tumors. Treatment for EBV-related tumors primarily involves chemotherapy and radiotherapy, but these are less effective for patients with metastases or recurrences. However, the expression of EBV proteins in malignant cells provides a promising therapeutic target for targeted intervention. Among viral proteins expressed in EBV-related epithelial malignancies, BARF1 is selectively expressed in latently infected epithelial carcinoma, nasopharyngeal carcinoma (NPC), and EBV-positive gastric cancer (EBV-GC). Studies have found that BARF1 protein is expressed as a potential gene only in EBV-positive epithelial tumor cells, and its transcripts are abundantly detected in tissues of NPC and EBV-related GC.

[0005] Monoclonal antibodies can be mass-produced, and their high affinity and specificity for antigens significantly reduce adverse reactions in clinical applications. Antibodies can kill tumor cells by binding to antigens on the cell surface, utilizing complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). These antibody molecules can also be modified to increase their antiviral efficacy. Antibodies, with their specificity and flexibility of use, are a very promising tool in the treatment of infectious diseases and cancer; however, to date, no therapeutic monoclonal antibodies against EBV-positive tumors have been commercialized. Therefore, developing therapeutic monoclonal antibodies against EBV will provide more effective prevention and treatment for EBV-related diseases. Summary of the Invention

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

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

[0008] A third aspect of the present invention aims to provide biological materials related to the monoclonal 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 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 this invention is to provide a reagent kit.

[0012] The seventh aspect of this invention is to provide a medicine.

[0013] The object of 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.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0015] A first aspect of the present invention provides a monoclonal antibody against EBV BARF1 or an antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment comprising a heavy chain and a light chain:

[0016] The heavy chain includes:

[0017] 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: 14;

[0018] The light chain comprises:

[0019] 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: 28.

[0020] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are as shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 30, respectively, and the CDR is defined using the Kabat definition scheme.

[0021] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 are as shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 29, and SEQ ID NO: 30, respectively, and the amino acid sequence of CDR-L2 is GNS. The CDR is defined using 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 as shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 30, respectively, and the CDR is 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 as shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively, and the CDR is defined using 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 of SEQ ID NO. 14 with one or more amino acid substitutions and / or deletions and / or additions that have the same function as the protein shown in SEQ ID NO. 14; or

[0027] a3) has an amino acid sequence that shares 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with 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 includes:

[0029] b1)SEQ ID NO.28; or

[0030] b2) An amino acid sequence of SEQ ID NO. 28 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO. 28; or

[0031] b3) has 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% homology with SEQ ID NO. 28 and has the same function as the protein shown in SEQ ID NO. 28.

[0032] Preferably, the monoclonal antibody or its antigen-binding fragment 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 includes a heavy chain constant region; and / or

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

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

[0036] c1) The amino acid sequence consisting of amino acids 142 to 471 in SEQ ID NO: 13; or

[0037] c2) An amino acid sequence having the same function as the amino acid sequence described in c1) by substitution and / or deletion and / or addition of one or more amino acids; or

[0038] The amino acid sequence described in c3) 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 described in c1).

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

[0040] d1) The amino acid sequence consisting of amino acids 127 to 232 in SEQ ID NO: 27; or

[0041] d2) An amino acid sequence having the same function as the amino acid sequence described in d1) by substitution and / or deletion and / or addition of one or more amino acids; or

[0042] The amino acid sequence described in d3) 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 described in d1).

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

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

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

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

[0047] e2) An amino acid sequence that has undergone substitution and / or deletion and / or addition of one or more amino acids, and has the same function as the amino acid sequence described in e1); or

[0048] The amino acid sequence described in e3) 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 described in e1).

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

[0050] f1) The amino acid sequence consisting of amino acids 1 to 19 in SEQ ID NO: 27; or

[0051] f2) An amino acid sequence that has undergone substitution and / or deletion and / or addition of one or more amino acids, and has the same function as the amino acid sequence described in f1); or

[0052] f3) 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 with the amino acid sequence described in f1).

[0053] Preferably, the amino acid sequence of the EBV BARF1 comprises:

[0054] g1) The amino acid sequence consisting of amino acids 1 to 201 in SEQ ID NO: 4; or

[0055] g2) An amino acid sequence having the same function as the amino acid sequence described in g1) by substitution and / or deletion and / or addition of one or more amino acids; or

[0056] The amino acid sequence described in g3) 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 described in g1).

[0057] A second aspect of the present invention provides a recombinant protein comprising: a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; and optionally a tag sequence for assisting expression and / or purification.

[0058] Preferably, 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.

[0059] A third aspect of the invention provides biological materials related to the monoclonal antibody or its antigen-binding fragment described in the first aspect of the invention, or the recombinant protein described in the second aspect of the invention, said biological material comprising at least one of h1) to h16):

[0060] h1) A nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect;

[0061] h2) contains an expression cassette containing the nucleic acid molecule described in h1);

[0062] h3) A carrier containing the nucleic acid molecule described in h1);

[0063] h4) A carrier containing the expression box described in h2);

[0064] h5) A transgenic cell line containing the nucleic acid molecules described in h1);

[0065] h6) Transgenic cell lines containing the expression cassette described in h2);

[0066] h7) A transgenic cell line containing the vector described in h3);

[0067] h8) A transgenic cell line containing the vector described in h4);

[0068] h9) Microorganisms containing the nucleic acid molecules described in h1);

[0069] h10) contains microorganisms containing the expression cassette described in h2);

[0070] h11) contains microorganisms containing the carrier described in h3);

[0071] h12) contains microorganisms containing the carrier described in h4);

[0072] h13) is a virus containing the nucleic acid molecule described in h1);

[0073] h14) contains the expression cassette described in h2);

[0074] h15) contains a virus with the vector described in h3);

[0075] h16) contains a virus with the vector described in h4).

[0076] Preferably, the transgenic cell line does not contain propagation material.

[0077] Preferably, the nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment as described in the first aspect of the present invention comprises a nucleic acid molecule with a heavy chain encoding the monoclonal antibody or its antigen-binding fragment as described in the first aspect of the present invention and a nucleic acid molecule with a light chain encoding the monoclonal antibody or its antigen-binding fragment as described in the first aspect of the present invention.

[0078] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain of the monoclonal antibody or its antigen-binding fragment of the first aspect of the present invention comprises:

[0079] a211) The nucleotide sequence shown in SEQ ID NO: 26; or

[0080] a212) A nucleotide sequence of SEQ ID NO: 26 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 26; or

[0081] a213) has 80%, 85% or 90% or more homology with SEQ ID NO: 26 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 26;

[0082] The nucleotide sequence of the nucleic acid molecule encoding the light chain of the monoclonal antibody or its antigen-binding fragment according to the first aspect of the present invention comprises:

[0083] a221) The nucleotide sequence shown in SEQ ID NO: 36; or

[0084] a222) A nucleotide sequence of SEQ ID NO: 36 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 36; or

[0085] a223) has 80%, 85% or 90% or more homology with SEQ ID NO: 36 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 36.

[0086] A fourth aspect of the present invention provides a conjugate comprising at least one of the monoclonal antibody or antigen-binding fragment thereof described in the first aspect of the present invention and the recombinant protein described in the second aspect of the present invention;

[0087] 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.

[0088] Preferably, the detectable marker is selected from radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, or any combination thereof.

[0089] Preferably, 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 (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes, chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0090] A fifth aspect of the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof described in the first aspect of the invention, the recombinant protein described in the second aspect, the biomaterial described in the third aspect, and / or the conjugate described in the fourth aspect in the preparation of a product;

[0091] The product includes at least one of the following: drug, reagent, test plate, reagent kit, and test chip.

[0092] Preferably, the drug has at least one of the functions i1) to i2):

[0093] i1) Prevention of EB virus infection;

[0094] i2) Treatment and / or prevention of diseases caused by EB virus infection.

[0095] Preferably, the reagent, detection plate, detection chip, or kit has at least one function among j1) to j3):

[0096] j1) Detect the presence or level of BARF1 protein in the sample;

[0097] j2) Detection of EB virus;

[0098] j3) Diagnose diseases caused by EB virus infection.

[0099] Preferably, the disease includes at least one of the following: nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disorders, and infectious mononucleosis.

[0100] Preferably, the drug comprises a vaccine.

[0101] A sixth aspect of the present invention provides a product comprising at least one of k1) to k3):

[0102] k1) The monoclonal antibody or antigen-binding fragment thereof as described in the first aspect of the present invention;

[0103] k2) The recombinant protein described in the second aspect of the present invention;

[0104] k3) The coupling described in the fourth aspect of the present invention;

[0105] The product includes at least one of reagents, test plates, reagent kits, and test chips.

[0106] Preferably, the product has at least one function among j1) to j3):

[0107] j1) Detect the presence or level of BARF1 protein in the sample;

[0108] j2) Detection of EB virus;

[0109] j3) Diagnose diseases caused by EB virus infection.

[0110] Preferably, the disease includes at least one of the following: nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disorders, and infectious mononucleosis.

[0111] A seventh aspect of the present invention provides a medicament comprising at least one of 11) to 14):

[0112] l1) The monoclonal antibody or antigen-binding fragment thereof as described in the first aspect of the present invention;

[0113] l2) The recombinant protein described in the second aspect of the present invention;

[0114] l3) The biomaterials described in the third aspect of the invention;

[0115] l4) The coupling compound described in the fourth aspect of the present invention.

[0116] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0117] Preferably, the drug has at least one of the functions i1) to i2):

[0118] i1) Prevention of EB virus infection;

[0119] i2) Treatment and / or prevention of diseases caused by EB virus infection.

[0120] Preferably, the disease includes at least one of the following: nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disorders, and infectious mononucleosis.

[0121] Preferably, the drug comprises a vaccine.

[0122] Preferably, a vaccine comprises at least one of l1) to l4) and an adjuvant:

[0123] l1) The monoclonal antibody or antigen-binding fragment thereof as described in the first aspect of the present invention;

[0124] l2) The recombinant protein described in the second aspect of the present invention;

[0125] l3) The biomaterials described in the third aspect of the invention;

[0126] l4) The coupling compound described in the fourth aspect of the present invention.

[0127] The eighth aspect of the present invention is to provide a method for preparing a monoclonal 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.

[0128] The beneficial effects of this invention are:

[0129] This invention provides a monoclonal antibody against EBV BARF1 or its antigen-binding fragment thereof, which has a high affinity for BARF1 protein (KD(M) = 3.33E-12) and can significantly inhibit the growth of EBV-related tumors in mice; it can also be used to detect the presence or level of BARF1 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. Attached Figure Description

[0130] Figure 1 This is a graph showing the affinity test results between monoclonal antibody 1B6 and BARF1 protein.

[0131] Figure 2 This is a flow cytometry diagram of BARF1 expression on the cell surface detected by monoclonal antibody 1B6.

[0132] Figure 3 This is a diagram showing the results of monoclonal antibody 1B6 targeting EBV-positive tumors in mice.

[0133] Figure 4 This is a diagram showing the results of monoclonal antibody 1B6 inhibiting the growth of EBV-positive tumors in mice. Detailed Implementation

[0134] The present invention will be further described in detail below through specific embodiments.

[0135] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0136] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0137] Example 1: Preparation of monoclonal antibody (mAb) against EBV BARF1 protein (EBV BARF1)

[0138] 1.1 Preparation of EBV BARF1 recombinant protein

[0139] The BARF1 protein plays an important role in the process of EBV invading epithelial cells and B cells. In the embodiments of the present invention, the inventors selected BARF1 as a bait protein to screen for specific antibodies.

[0140] The selected original BARF1 sequence includes the KOZAK sequence, BARF1 signal peptide, BARF1 protein (21-221aa), (G4S) linker region, His tag, and stop codon; specifically as follows: 5'--3' (SEQ ID NO: 1); wherein, the sequence composed of nucleotides 1-6 in SEQ ID NO: 1 is the KOZAK sequence, and the sequence composed of nucleotides 7-66 in SEQ ID NO: 1 is the BARF1 signal peptide sequence;The sequence consisting of nucleotides 67-669 in SEQ ID NO: 1 is the BARF1 protein (21-221aa) sequence; the sequence consisting of nucleotides 670-714 in SEQ ID NO: 1 is the (G4S) linker region sequence; the sequence consisting of nucleotides 715-732 in SEQ ID NO: 1 is the His tag sequence; and the sequence consisting of nucleotides 733-735 in SEQ ID NO: 1 is the stop codon.

[0141] The original BARF1 sequence was ligated into the mammalian cell expression vector pcDNA3.1+ (Invitrogen) as follows:

[0142] (1) Amplification of the BARF1 protein gene:

[0143] 50 μL of NEB was selected. The PCR amplification reaction system (Table 1) was used to amplify the original BARF1 sequence described above. The upstream primer was 5'-CTAGTCCAGTGTGGTGGAATTCGCCACCATGGCCAGGTTCATCGC-3' (SEQ ID NO: 2); the downstream primer was 5'-CTCTAGACTCGAGCGGCCGCTTAGTGGTGGTGGTGGTGGTGGCTGCCGCCGCCGCCAGAGCCTCCTCCGCCGGAGCCTCCTCCTCCTTGCGACAAGTATCCAG-3' (SEQ ID NO: 3).

[0144] The amplified target fragment was analyzed by agarose gel electrophoresis. The band with the correct molecular weight was cut under UV light, and the PCR product was recovered according to the instructions of the commercially available kit.

[0145] Table 1. PCR amplification reaction system of BARF1 original sequence

[0146] Components content 5×Reaction Buffer 10μL dNTPs 1μL Upstream primer (10 μM) 2.5μL Downstream primer (10 μM) 2.5μL EBV genomic DNA template 1μL DNA polymerase 0.5μL High GC Enhancer (Q5 kit from NEB) 10μL <![CDATA[ddH2O]]> Add to 50μL

[0147] (2) Enzyme digestion and ligation of the target fragment and vector:

[0148] The vector used was the eukaryotic expression plasmid pcDNA3.1+. The vector was digested with EcoRI and NotI (using a 50 μL digestion reaction system (Table 2)).

[0149] Table 2 Enzyme digestion reaction system

[0150] Components content 10×CutSmart Buffer 5μL EcoRI-HF 1μL NotI-HF 1μL Target fragment or vector 5μg <![CDATA[ddH2O]]> Add to 50μL

[0151] Enzyme digestion was performed at 37°C for 2–3 hours. After running on an agarose gel, the digested vector was recovered using a gel extraction kit to obtain a linearized vector. The insert fragment was directly recovered using a DNA purification kit. Ligation was then performed using a 10 μL ligation reaction mixture (Table 3). The reaction was carried out at 37°C for 30 minutes to obtain the ligation product.

[0152] Table 3 Connection Reaction System

[0153]

[0154]

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

[0156] Add the ligation product to freshly thawed DH5α competent cell suspension, incubate on ice for 30 min, heat shock at 42°C for 90 s, and return to ice for 5 min. Add 200 μL of LB medium and incubate at 30°C with gentle shaking for 40 min. Spread the culture medium onto ampicillin-resistant LB agar plates and incubate overnight at 37°C.

[0157] Select a single cloned colony for sequencing verification. Once the sequencing results are correct, the target recombinant plasmid is obtained; extract a large number of plasmids.

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

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

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

[0161] Since the obtained BARF1 recombinant protein has a 6×His tag at its C-terminus, it can be purified using a nickel column affinity chromatography. The specific procedure is as follows: filter the supernatant containing the target protein obtained above through 0.65 μm filter paper, pass it through a nickel column bead binding chamber three times, wash the beads three times with 30 mM imidazole, and then elute the target protein with 500 mM imidazole. Further purification is then performed using gel filtration chromatography. Specifically, the protein eluted from the nickel column is concentrated using a 3 kDa concentrator to a volume less than 1 mL. Finally, purification is performed using a Superdex 200 Increase 10 / 300GL.

[0162] The final amino acid sequence of the target protein is: VTAFLGERVTLTSYWRRVSLGPEIEVSWFKLGPGEEQVLIGRMHHDVIFIEWPFRGFFDIHRSANTFFLVVTAANISHDGNYLCRMKLGETEVTKQEHLSVVKPLTLSVHSERSQFPDFSVLTVTCTVNAFPHPHVQWLMPEGVEPAPTAANGGVMKEKDGSLSVAVDLSLPKPWHLPVTCVGKNDKEEAHGVYVSGYLSQGGGGSGGGGSGGGSHHHHHHH (SEQ ID NO: 4); wherein, the sequence composed of amino acid residues 1 to 201 in SEQ ID NO: 4 is the BARF1 protein (21-221aa) sequence, the sequence composed of amino acid residues 202 to 216 is the (G4S) linker region sequence, and the sequence composed of amino acid residues 217 to 222 is the His tag sequence.

[0163] 1.2 Construction of phage antibody library

[0164] (1) Total RNA extraction:

[0165] Blood from nasopharyngeal carcinoma (NPC) patients was diluted 1:1 with PBS (10 mL blood + 10 mL PBS). 20 mL of the diluted solution was carefully spread over 15 mL of lymphocyte separation medium, maintaining the separation interface. The mixture was centrifuged at 2000 rpm for 20 min at room temperature. The centrifugation speed was then slowly reduced, and the intermediate mononuclear cell layer was transferred to a new 15 mL centrifuge tube. PBS was added to bring the volume to 15 mL, and the tube was centrifuged at 300 g for 20 min at room temperature. The supernatant was transferred to a new 50 mL centrifuge tube, centrifuged at 300 g for 20 min at room temperature, and the supernatant was discarded. The cell pellets in both the 15 mL and 50 mL centrifuge tubes were resuspended with 1 mL of Trizol (i.e., 2 mL Trizol for every 10 mL of blood).

[0166] Add 4 mL of chloroform to 20 mL of Trizol-resuspended cell pellet. Vortex for 15 seconds and incubate at room temperature for 5 minutes. Centrifuge at 4000 g for 30 minutes at 4°C. After centrifugation, the pellet will separate into layers. Carefully transfer the top clear layer to a new 50 mL centrifuge tube free of RNase and DNase using a pipette. Add isopropanol in a 1:1 ratio to each tube, mix thoroughly by inverting several times, and incubate 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 to a 1.5 mL centrifuge tube. Spin the pellet several times to ensure thorough contact with the liquid. Centrifuge at 7500 g for 5 minutes, remove the supernatant, and retain the pellet. Keep the centrifuge tube open and allow it to dry at room temperature for 10 minutes. Add 400 μL of enzyme-free water and incubate at 55°C for 10 minutes to ensure complete RNA dissolution, yielding total RNA.

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

[0168] (2) RNA reverse transcription:

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

[0170] The specific steps are as follows:

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

[0172] Aliquot the reaction solution from the above reaction into 8-tube PCR instruments and place them in a PCR instrument. Briefly centrifuge and incubate at 70°C for 5 min to denature the RNA. After the reaction, cool rapidly on ice. Then, prepare the remaining reaction solution into the 8-tube PCR instruments according to the proportions in Table 5. Mix the reaction solutions in Table 5, briefly centrifuge, and place the centrifuge tubes into a PCR instrument. Incubate at 45°C for 60 min, then at 75°C for 15 min. Cool on ice to obtain cDNA after reverse transcription of total RNA. Store at 4°C.

[0173] Table 4. 5μL reverse transcription reaction system

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

[0175] Table 5 Other reaction solution ratios

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

[0177] (3) PCR amplification:

[0178] Using the cDNA obtained in step (2) as a template, a single-chain antibody fragment (ScFv) was amplified by two rounds of PCR using NEB Q5 high-fidelity DNA polymerase. 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, and the reaction system is shown in Table 6. The PCR reaction used a three-temperature method: denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute per cycle, for a total of 35 cycles. 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] In each primer, the lowercase part represents the restriction enzyme site; the Vλ forward primer is used in pairs with the Vλ-linker reverse primer, the Vκ forward primer is used in pairs with the Vκ-linker reverse primer, and the linker-VH forward primer is used in pairs with the linker-VH reverse primer.

[0187] Table 6 First-round PCR reaction system

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

[0189] After the reaction, all PCR products were subjected to 1.5% agarose gel electrophoresis, and bands with target fragment sizes of approximately 320 bp (corresponding to VL) and 350 bp (corresponding to VH) were excised and recovered. DNA was recovered using the NEB DNA GEL purification kit according to the kit instructions. The collected DNA solution was the first-round PCR amplification product; after concentration determination, it was stored at 4°C.

[0190] The second round of PCR was performed using the amplified products from the first round of PCR as templates. The reaction system for the second round of PCR 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 adhesive recycling products 80ng VH + 80ng VL dNTP Mix 1μL 5×Reaction Buffer 10μL High GC Enhancer 10μL Forward primer OF (10 μM) 2.5μL Reverse primer OR (10 μM) 2.5μL polymerase 0.5μL <![CDATA[ddH2O]]> Add to 50μL

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

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

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

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

[0197] Table 8. Vector Enzyme Digestion System

[0198] Components content Vector (pComb3XSS) 20μg SpeI 10μL SacI 10μL 10×CutSmart Buffer 50μL Quick CIP 5μL <![CDATA[ddH2O]]> Add to 500μL

[0199] Table 9 Enzyme digestion system for the target fragment (ScFv fragment)

[0200] Components content scFv 5μg SpeI 5μL SacI 5μL CutSmart 10×buffer 25μL <![CDATA[ddH2O]]> Add to 250μL

[0201] Enzyme digestion conditions: incubation at 37℃ for 2 hours, followed by incubation at 80℃ for 3 minutes. After digestion, the vector digestion products were run on a gel and then excised using the NEB DNA purification and recovery kit (do not expose to UV light); the ScFv fragment digestion products did not require gel running and could be directly recovered.

[0202] After purification via enzymatic digestion, the concentration of the recovered product was measured. Then, the digested vector and ScFv digested product were ligated according to the ligation reaction system shown in Table 10. The ligation system was incubated overnight (16–24 h) at 37°C. After ligation, the ligation product was recovered using a NEBDNA purification and recovery kit. The concentration of the recovered ligation product was measured and recorded, and stored at 4°C.

[0203] Table 10 Connection Reaction System

[0204] Components content 10×T4 reaction buffer 2μL ScFv fragment (after enzyme digestion) 68ng Vector (after enzyme digestion) 100ng T4 ligase 1μL <![CDATA[ddH2O]]> Add to 20μL

[0205] 1.3 Construction of bacterial libraries:

[0206] (1) Preparation of TG1 Escherichia coli competent cells:

[0207] Strawberry strain TG1 was streaked onto 2×YT solid medium and incubated overnight at 37°C. A single colony was picked from the streak plate and transferred to 10 mL of 2×YT medium, incubated overnight at 37°C and 220 rpm. The bacteria were then inoculated into 100 mL of 2×YT medium at a 1:100 dilution and incubated at 37°C and 250 rpm for 40 min. The OD value was measured, and then measured every 20 min until OD600 = 0.3–0.35. The bacterial suspension was collected, centrifuged at 3200 g at 0–4°C for 10 min, the supernatant was discarded, and the suspension was placed on ice. 40 mL of pre-chilled ddH2O was added for resuspending, and the suspension was centrifuged again at 3200 g at 0–4°C for 10 min, the supernatant was discarded, and the suspension was placed on ice. 1 mL of pre-chilled ddH2O was added for resuspending, and the suspension was transferred to a 1.5 mL pre-chilled EP tube. The suspension was centrifuged at 10000 g at 4°C for 30 s. This process was repeated once. Discard the supernatant, place on ice, add 400 μL of pre-cooled ddH2O to resuspend, and obtain TG1 Escherichia coli competent cell suspension.

[0208] The Comb3XSS recombinant plasmid containing the ScFv fragment obtained in the above examples was used to construct an E. coli library using electroporation. Specifically, 100 ng of the Comb3XSS recombinant plasmid containing the ScFv fragment was added to 50 μL of the above TG1 E. coli competent cell suspension using a pre-chilled pipette tip. The mixture was gently blown to mix and transferred to a pre-chilled 1 mm electroporation cuvette. After confirming the mixture was at the bottom of the cuvette and free of air bubbles, electroporation was performed at 1800 V and 1 mm spacing. Immediately after completion, 1000 μL of 37°C SOC medium was added. The mixture was removed from the cuvette and incubated at 37°C with shaking at 180 rpm for 90 min. A 10-fold serial dilution was performed using 2×YT liquid medium, resulting in six dilutions (each diluted 10^6 times). 1 10 2 10 3 10 4 10 5 10 6 (Multiple times). For each gradient, 5 μL was evenly added dropwise to 2×YT-GA solid medium, air-dried, and then 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:

[0209] E(pfu / ug) = N × D × 10;

[0210] Where E is the competent cell efficiency (unit: pfu / ug), D is the dilution factor, and N is the number of single clones on the plate at the corresponding dilution factor.

[0211] Repeat the above method for 100 electroporation transformation reactions. Spread the revived bacterial culture evenly onto 100 2×YT-GA2 45mm square agar plates, air-dry, and incubate overnight at 37°C upside down. Take one of the overnight incubated square plates, add 6 mL of 2×YT liquid medium to the surface of each plate, and gently scrape off the colonies from all 100 plates with a spreader. Collect the bacterial culture into a 50 mL centrifuge tube, add glycerol to a final concentration of 20%, and the bacterial library is obtained.

[0212] Take 10 μL of bacterial culture into 990 μL of 2×YT liquid medium and measure OD600 using NanoDrop. Calculate and record the total OD600 of the bacterial library.

[0213] T OD600 =M OD600 ×100;

[0214] Among them, T OD600 Total bacterial OD600, M OD600 This is the measured OD600.

[0215] 1.4 Phage Library Construction:

[0216] Take an appropriate amount from the bacterial library above into a 1.5 mL EP tube. The formula for calculating the bacterial volume is as follows:

[0217]

[0218] Where V is the volume of the transferred bacterial culture (in μL), and OD600 is the total OD600 of the constructed bacterial library.

[0219] Transfer the culture medium to 100 mL of 2×YT-GA liquid medium to achieve an initial OD600 of 0.1. Incubate at 37°C and 250 rpm in a shaker until the OD600 reaches 0.5–0.55. Calculate and add helper phage M13K07 according to the following formula to achieve a bacteria:phage ratio of 1:20. The formula for calculating the amount of phage to add is:

[0220]

[0221] Where V is the volume of helper phage added (in mL), and T helper-phage The titer of the helper phage used is given, and OD600 is the OD600 value of the bacterial culture.

[0222] Incubate at 37°C and 220 rpm for 30 min in a constant temperature shaker. Centrifuge at 3200g for 5 min to collect TG1 bacteria, discard the supernatant, resuspend the precipitate and transfer it to 100 mL of 2×YT-AK liquid medium, and incubate overnight at 30°C and 250 rpm in a constant temperature shaker.

[0223] Transfer the overnight culture to a new 50 mL centrifuge tube and centrifuge at 4000 g for 30 min at 4 °C. Collect the supernatant and add 1 / 4 volume of pre-chilled 20% PEG / 2.5 M NaCl (4 °C). Mix thoroughly and incubate on ice for 30 min. Centrifuge at 4000 rpm for 30 min at 4 °C, discard the supernatant, and invert the tube on paper for 2 min. Resuspend the precipitate in 1 mL PBS and centrifuge at 12000 rpm for 20 min at 4 °C. Collect the supernatant and add 1 / 4 volume of pre-chilled 20% PEG / 2.5 M NaCl solution. Mix well and incubate on ice for 10 min. Centrifuge at 12000 rpm for 10 min at 4 °C, discard the supernatant, and resuspend the precipitate in 1 mL PBS. Centrifuge at 12000 rpm for 2 min at 4 °C, collect the supernatant (this is the phage library), add glycerol to a final concentration of 20%, and store at -80 °C.

[0224] The titer of the phage library was determined as follows: The phage library was added to 10 mL of 2×YT liquid medium and incubated at 37°C and 250 rpm for approximately 45–60 min, until the OD600 reached 0.5–0.55. 10 μL of the cultured phage library was then serially diluted 10-fold (a total of 13 dilutions). For each dilution, 90 μL of untreated TG1 bacterial culture was added, mixed, and incubated at 37°C for 20 min. Then, 5 μL from each dilution was added dropwise to 2×YT-GA solid medium, air-dried, and incubated overnight at 37°C. The phage cells were counted, and the number of phage particles per milliliter of phage solution was calculated according to the phage library titer formula.

[0225] T(pfu / ml) = N × D × 400;

[0226] Where 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.

[0227] 1.5 Screening for BARF1 protein-specific antibodies

[0228] Antibodies that specifically bind to the BARF1 protein were screened using an antigen solid-phase adsorption screening method.

[0229] The specific steps are as follows:

[0230] Dissolve 50 μg of the BARF1 protein obtained above in 2 mL of PBS and coat the immunotubes overnight at 4°C (positive tubes). Simultaneously coat the tubes with a negative control protein (50 μg BSA) as a negative control. Discard the coated protein, wash three times with 2 mL of PBS, and then block with 2 mL of 3% BSA (dissolved in PBS T) at room temperature for 2 h. Discard the blocking solution, take 100 μL of the obtained phage library, dilute with 2 mL of PBS, and incubate in an immunotube for 1 h. Discard the liquid, wash five times with 2 mL of PBST for 5 min each time. Then wash five times with 2 mL of PBS for 5 min each time. Simultaneously, take 1 mL of overnight TG1 saturated bacterial culture and add it to 100 mL of 2×YT liquid medium, incubate at 37°C and 150 rpm until OD600 = 0.5 (approximately 1.5 h). Discard the washing solution, and wash with 1 mL of 0.1 mg / mL Trypsin at room temperature for 30 min. Add 1 mL of the eluent to 10 mL of the cultured TG1 bacterial suspension (OD = 0.5), infect at 37°C and 150 rpm for 30 min; then centrifuge at 4°C and 3000 g for 10 min. Resuspend the pellet in 1 mL of 2×YT liquid medium. Spread 1 mL of the resuspended product from the positive immunotherapy tube evenly onto a 245 mm × 245 mm 2×YT solid culture dish and incubate overnight (16–20 h) at 37°C. Measure the phage titer of the positive and negative control tubes using the same method as described in the previous example.

[0231] Based on the phage titer comparison results, 5 mL of 2×YT liquid medium was added to the culture dish with high phage titer after overnight culture. All colonies were scraped off with a spreader, and a sub-phage library was prepared according to the method described in the above examples. The above operation was repeated until an antibody with the highest affinity for BARF1 (compared to the negative control) was screened.

[0232] Unless otherwise specified, the rotation speed for antigen coating, blocking, incubation, washing, and elution processes is 15 rpm.

[0233] Use ELISA to detect the antibody with the highest affinity.

[0234] Take 10 μL of the bacterial culture containing the antibody with the highest affinity for BARF1 and dilute it with 1 ml of 2×YT liquid medium. Streak the colonies onto a 2×YT-GA solid culture plate and incubate overnight at 37°C for 16–20 h. Pick 192 single colonies and transfer them to a 96-well plate (each well containing 200 μL of 2×YT-GA liquid) and incubate at 37°C until saturation. Transfer 2 μL of the saturated bacterial culture to a new 96-well plate (each well containing 200 μL of 2×YT-A liquid) to make the initial OD approximately 0.03, and incubate at 37°C for 2.5–3 h until the OD is approximately 0.5. Add 0.1 μL of the above helper phage to each well and infect at 37°C for 30 min. Add 0.2 μL of kanamycin to each well and incubate overnight at 30°C. Coat the protein (- / +) plate overnight. Centrifuge the overnight cultured 96-well plate at 4°C and 3400g for 5 min. Discard the liquid in the ELISA plate and wash once with 350 μL PBS. Block with 350 μL 3% BSA (PBST) at 37°C for 1 h. Discard the blocking solution, wash once with 350 μL PBST, and tap the plate to remove the liquid. Add 140 μL 3% BSA (PBST) to each well, then add 60 μL of the above phage library expression antibody (the antibody with the highest affinity, as the primary antibody), and incubate at 37°C for 1 h. Discard the liquid (primary antibody), wash 5 times with 350 μL PBST, and tap the plate. Add 100 μL M13 Antibody (HRP) as the secondary antibody (1:8000 ratio relative to the blocking solution), incubate at 37°C for 1 h, discard the liquid (secondary antibody), wash 5 times with 350 μL PBST, and tap the plate. Add 100 μL of TMB to the light-protected solution for 2-3 minutes, then terminate the reaction with 100 μL of dilute hydrochloric acid (concentrated hydrochloric acid: water = 1:12). Read the OD450 and OD630 values ​​of the ELISA plate. In this embodiment, the inventor named the antibody with the best performance 1B6.

[0235] 1.6 Expression and purification of monoclonal antibodies

[0236] By linking the variable region of the antibody heavy chain upstream to the CMV fragment, downstream to the constant region of human IgG1, and the polyA fragment, the complete heavy chain fragment can be expressed. Similarly, by linking the variable region of the antibody light chain upstream to the CMV fragment, downstream to the constant region of the light chain κ / λ, and the polyA fragment, the complete light chain fragment can be expressed. Co-transfection of plasmids containing the full-length sequences of both the antibody heavy and light chains into 293T cells achieves antibody expression, and protein A beads can be used for antibody purification.

[0237] The 1B6 full-length heavy chain contains 477 amino acid residues (excluding *), specifically:

[0238]

[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 CDR-H1 (SEQ ID NO: 15), CDR-H2 (SEQ ID NO: 16), and CDR-H3 (SEQ ID NO: 17) within the heavy chain variable region (IMGT definition scheme). The italicized portion represents the signal peptide. Amino acids 142–471 in SEQ ID NO: 13 constitute the heavy chain constant region. * indicates a stop codon.

[0241] Table 11 shows CDR-H1, CDR-H2, and CDR-H3 in the variable region of the heavy chain, which are defined by other CDR schemes.

[0242] Table 11 shows CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region, defined by other CDR schemes.

[0243]

[0244] The gene encoding the full-length heavy chain 1B6 consists of 1434 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 CDR-H1, CDR-H2, and CDR-H3 within the heavy chain variable region (IMGT definition scheme). 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 1B6 full-length light chain consists of 232 amino acid residues (excluding *), specifically:

[0248]

[0249] In this sequence, the underlined portion represents the amino acid sequence of the light chain variable region (SEQ ID NO: 28). The underlined and bolded portions represent the amino acid sequences (IMGT definition scheme) of the three complementarity-determining regions CDR-L1 (SEQ ID NO: 29), CDR-L2 (GNS), and CDR-L3 (SEQ ID NO: 30) within the light chain variable region. The italicized portion represents the signal peptide. Amino acids 127–232 in SEQ ID NO: 27 constitute the light chain constant region. * indicates a stop codon.

[0250] Table 12 shows CDR-H1, CDR-H2, and CDR-H3 in the variable region of this light chain, which are defined by other CDR schemes.

[0251] Table 12 CDR-H1, CDR-H2, and CDR-H3 in the variable region of light chains, defined by other CDR schemes.

[0252]

[0253] The gene encoding the full-length light chain 1B6 consists of 699 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 CDR-L1, CDR-L2, and CDR-L3 within 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.

[0256] Effect Example

[0257] 1. The affinity of antibody 1B6 was determined using biomembrane interference (BLI) technique.

[0258] BLI can be performed according to conventional methods in the art. In this embodiment, the specific operation is as follows: the biosensor (Sartorius, Germany) is used... The SA probe was equilibrated in a buffer solution (a mixture of KB buffer, 0.1% BSA, and 0.02% Tween 20). Then, it was removed and immersed in a solution containing 10 μg / mL BARF1-Biotin (biotin-labeled BARF1 protein). The BARF1 antigen in the solution bound to the SA (streptavidin) bioprobe surface, increasing its surface membrane thickness. A baseline was then established by immersing a biosensor with a known concentration of cured antigen in the buffer solution. By immersing the biosensor with the cured known concentration of antigen in a sample solution containing 7.8–500 nM 1B6 antibody for approximately 120 seconds, the specific binding between antigen and antibody led to an increase in membrane thickness. The biosensor with the bound 1B6 antibody was then immersed in the buffer solution for approximately 180 seconds for dissociation, causing the test antibody (1B6 antibody) to detach from the biosensor surface, resulting in a decrease in membrane thickness. The kinetic constants of the test sample (1B6 antibody) were obtained by real-time monitoring of the biosensor biomembrane thickness during the experiment. Results are as follows: Figure 1 As shown: the KD(M) of the 1B6 antibody is 3.33E-12, indicating that the 1B6 antibody has a very high affinity for the BARF1 antigen.

[0259] 2.1 Binding ability of B6 antibody to BARF1 on cell surface

[0260] (1) Establish a stable B16 cell line expressing BARF1: The specific procedure is as follows:

[0261] Construction of stable BARF1 lentivirus strain: 2.5 μg of psPAX2 plasmid, 2.5 μg of pMD2.G plasmid, and 5 μg of pLVX-BARF1 plasmid were added to Opti-MEM medium and mixed thoroughly. 30 μl of PEI was added, and the mixture was shaken vigorously and allowed to stand for 15 min. The mixture was then gently added dropwise to 10 cm culture dishes of 293T cells (cell density 60%). After incubation in a cell culture incubator for 4 hours, the cell culture medium was changed. After 48 hours, 40 ml of the cell culture supernatant (i.e., lentivirus solution) was transferred to a 50 ml centrifuge tube, centrifuged at 4500 g at room temperature for 5 minutes, filtered through a 0.45 μm filter, and 10 ml of 5×PEG was added. The mixture was incubated overnight at 4°C. The next day, the lentivirus solution was centrifuged at 4500 g at room temperature for 1 hour, the supernatant was discarded, and the pellet was resuspended in 2 ml of serum-free medium. Add an appropriate amount of resuspended lentivirus solution to B16-LUC cell culture medium, incubate for 4 hours, then change the medium. Add puro antibiotic at a 1:1000 ratio for stable cell selection; the surviving cells are the successfully constructed stable cell lines. Further validation of the stable cell lines was performed using Western blotting.

[0262] (2) Flow cytometry: The specific steps are as follows:

[0263] B16-LUC tumor cells and B16-LUC BARF1 overexpressing stable cell lines were digested separately from culture dishes with 0.25% trypsin solution, transferred to 15ml centrifuge tubes, centrifuged, and then repeatedly resuspended in PBS and washed twice by centrifugation. B16-LUC and B16-LUC-BARF1 cells were then divided into 200μl portions per tube at a cell density of 5 x 10⁻⁶ cells / mL. 6 Cells / ml, 100 μL FSV700 live / dead dye (BD Horizon) per tube TM Incubate Fixable Viability Stain 700 (1:1000 dilution) at room temperature for 30 minutes, wash twice with PBS, and then take 100 μl of Fixation / Perm solution (BD Cytofix / Cytoperm). TM The cells were fixed at room temperature for 30 minutes using the Fixation / Permeabilization Kit, washed twice with PBS, and 100 μL of fluorescent 1B6 monoclonal antibody (AbFluor 647, 0.005 mg / ml, see 3-(1)) was added to each cell type. The cells were incubated at room temperature for 30 minutes, washed twice with PBS, and then resuspended in 200 μl of PBS before being loaded onto the flow cytometer. Cell surface markers were detected using the APC-A fluorescent channel on a Beckman CytoFLE X flow cytometer. Cell populations were divided into gating zones, and the results are as follows: Figure 2 As shown, the 1B6 monoclonal antibody can specifically bind to BARF1 on the surface of stable B16-LUC BARF1 overexpressing cell lines.

[0264] 3.1 Mouse EBV-positive tumor-targeting assay using B6 monoclonal antibody

[0265] (1) Antibody-conjugated fluorescence: Using an antibody-conjugated fluorescence kit (LinKine TMAntibody fluorescence conjugation was performed using the AbFluor 647 Labeling Kit Booklet. First, prepare 100 μl of the 1B6 antibody solution to be labeled, with a concentration of 10 mg / ml (ensuring an initial concentration of at least 2 mg / ml). The buffer solution was PBS solution with pH 7.4 (the solution must not contain amino components or glycine, and the pH should be controlled between 6.5 and 8.0). Add 50 μl of AbFluor 647 labeling solution to the 1B6 antibody solution to be labeled, and gently mix with a pipette. Then, transfer 25 μl of pre-activated AbFluor 647 solution to the mixture from the previous step (the mixture of 1B6 antibody and AbFluor 647 labeling solution). Add deionized water to a volume of 500 μl, mix gently, and incubate in a 37°C incubator for 1 hour in the dark. Add 450 μl of PBS to the incubated mixture, mix gently, transfer to a 50 kDa purification column, and centrifuge at 12000 × g at 4 °C for 10 minutes. Discard the filtrate, add another 450 μl of PBS to the purification column, and centrifuge again under the same conditions. Invert the centrifuged filter column into a clean test tube, centrifuge at 4000 × g at 4 °C for 2 minutes to collect the target fluorescently conjugated antibody. The labeling method for the control antibody is the same as in 1B6.

[0266] (2) Construction of mouse EBV-positive tumor model: The specific operation is as follows:

[0267] Culture of EBV-positive tumor cells C-666: After reviving C-666 cells from liquid nitrogen, they were cultured in 1640 medium (containing 10% fetal bovine serum) in 10cm cell culture dishes until the density reached 80%-90%.

[0268] Digestion and Counting of C-666 Tumor Cells: The tumor cell supernatant culture medium was discarded. After washing once with 2 ml of PBS, 2 ml of 0.25% trypsin solution was added and the cells were incubated at 37°C for digestion. Under a microscope, cell cytoplasm retraction was observed, and the cells no longer adhered to each other in sheets. 4 ml of fresh culture medium (containing 10% fetal bovine serum) was added to terminate the digestion. The adherent tumor cells were gently pipetted off and transferred to clean 15 ml centrifuge tubes. The tubes were centrifuged at 200×g at room temperature for 5 min, and the supernatant was discarded to obtain the target tumor cells. The cells were resuspended in PBS, centrifuged twice, and then resuspended again in PBS. The tumor cells were counted using a hemocytometer and aliquoted into 1.5 ml EP tubes (300,000 cells / 100 μl). Preheated matrix gel was added and mixed thoroughly before subcutaneous implantation in mice.

[0269] Nude mouse preparation: Purchase 20 four-month-old female nude mice (Balb / c Nude). The EBV-positive C-666 tumor cells containing matrix gel prepared in the above steps were implanted into the right lower abdomen of the mice using a 1ml syringe. The cell density was 500,000 cells / 100μl. When withdrawing the syringe, pressure was applied to the injection site to prevent cell leakage. Tumor growth was continuously monitored to ensure successful establishment of the mouse EBV-positive tumor model.

[0270] (3) In vivo imaging experiment in mice: After the EBV-positive tumor model in mice was successfully established, 200 μg / 100 μl of the above-mentioned fluorescently conjugated antibody was injected intraperitoneally. Fluorescent in vivo imaging was performed on mice at 12 h, 24 h, 48 h, 72 h, and 96 h after intraperitoneal injection. The results at 48 h are as follows: Figure 3 As shown, the 1B6 antibody with red fluorescence is specifically enriched at the tumor site, indicating that the 1B6 monoclonal antibody has good targeting properties for EBV-positive tumors.

[0271] 4.1 Mouse EBV-positive tumor suppression experiment using B6 monoclonal antibody

[0272] (1) Construction of mouse EBV positive tumor model: The steps are the same as those in point 3 for tumor model construction;

[0273] (2) Tumor suppression experiment:

[0274] The established mouse tumor models were randomly divided into two groups of six mice each: a 1B6 monoclonal antibody treatment group and an IgG treatment group. Each mouse in each group was marked by ear clipping. Before administration, the weight and tumor size of each mouse were measured. Each mouse received 2 mg / 200 μl of the drug every three days. Before each administration, the weight and tumor size of each mouse were measured to monitor changes. When the tumor became too large, the mouse's weight decreased significantly, or obvious signs of endangerment (significant cyanosis of the lips and significant respiratory depression) appeared, the animals were euthanized as the humane endpoint of this experiment. A total of eight administrations were administered. The results are as follows: Figure 4 As shown, the tumors in the 1B6 monoclonal antibody-treated mice grew slower and were smaller than those in the control group, indicating that the 1B6 monoclonal antibody has a significant inhibitory effect on mouse tumors.

[0275] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody against EBV BARF1 or an antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment comprising a heavy chain and a light chain: The heavy chain 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: 14; The light chain comprises: 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:

28.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that: The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 30, respectively. The CDRs are defined using the Kabat scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 are shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 29, and SEQ ID NO: 30, respectively. The amino acid sequence of CDR-L2 is GNS. The CDR is defined according to the IMGT scheme. 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: 20, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 30, respectively. The CDRs are defined according to the Chothia 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: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively. The CDR is defined using the Contact definition scheme. The amino acid sequence of the heavy chain variable region includes: a1) SEQ ID NO.14; or a2) An amino acid sequence of SEQ ID NO. 14 with one or more amino acid substitutions and / or deletions and / or additions that have the same function as the protein shown in SEQ ID NO. 14; or a3) has 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% homology with 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 includes: b1) SEQ ID NO.28; or b2) An amino acid sequence of SEQ ID NO. 28 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO. 28; or b3) has 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% homology with SEQ ID NO. 28 and has the same function as the protein shown in SEQ ID NO.

28.

3. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that: The monoclonal antibody or its antigen-binding fragment comprises at least one of Fab, Fab', F(ab')2, Fv, scFv or bispecific antibody; The heavy chain further includes a heavy chain constant region; and / or The light chain also includes a light chain constant region; The amino acid sequence of the heavy chain constant region includes: c1) The amino acid sequence consisting of amino acids 142–471 in SEQ ID NO: 13; or c2) An amino acid sequence having the same function as the amino acid sequence described in c1) by substitution and / or deletion and / or addition of one or more amino acids; or c3) 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 with the amino acid sequence described in c1). The amino acid sequence of the light chain constant region includes: d1) The amino acid sequence consisting of amino acids 127 to 232 in SEQ ID NO: 27; or d2) An amino acid sequence having the same function as the amino acid sequence described in d1) by substitution and / or deletion and / or addition of one or more amino acids; or The amino acid sequence described in d3) 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 described in d1).

4. A recombinant protein comprising: the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3; and Tag sequences that assist in expression and / or purification.

5. A biological material relating to the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3, or the recombinant protein as described in claim 4, wherein the biological material comprises at least one of h1) to h12): h1) A nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 3 or the recombinant protein of claim 4; h2) contains an expression cassette containing the nucleic acid molecule described in h1); h3) A carrier containing the nucleic acid molecule described in h1); h4) A carrier containing the expression box described in h2); h5) A transgenic cell line containing the nucleic acid molecules described in h1); h6) Transgenic cell lines containing the expression cassette described in h2); h7) A transgenic cell line containing the vector described in h3); h8) A transgenic cell line containing the vector described in h4); h9) Microorganisms containing the nucleic acid molecules described in h1); h10) contains microorganisms containing the expression cassette described in h2); h11) contains microorganisms containing the carrier described in h3); h12) contains microorganisms that contain the carrier described in h4).

6. A conjugate comprising: at least one of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3 and the recombinant protein as described in claim 4; And a coupling portion, which includes at least one of a detectable marker, a drug, or a toxin.

7. The application of at least one of (1) to (4) in the preparation of the product; (1) The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3; (2) The recombinant protein according to claim 4; (3) The biomaterial as described in claim 5; (4) The coupling compound according to claim 6; The product contains at least one of a drug, a reagent, or a test strip; The drug has at least one of the functions i1) to i2): i1) Prevention of EB virus infection; i2) Treatment and / or prevention of diseases caused by EB virus infection; The reagent or detection plate has at least one function among j1) to j3): j1) Detect the presence or level of BARF1 protein in the sample; j2) Detection of EB virus; j3) Diagnose diseases caused by EB virus infection.

8. A product comprising at least one of k1) to k3): k1) The monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3; k2) The recombinant protein according to claim 4; k3) The coupling as described in claim 6; The product contains at least one of a reagent or a test strip.

9. A drug comprising at least one of (l1) to (l4): (l1) The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3; (l2) The recombinant protein according to claim 4; (l3) The biomaterial as described in claim 5; (l4) The coupling as described in claim 6; The drug also contains a pharmaceutically acceptable carrier.

10. A vaccine comprising at least one of (l1) to (l4) and an adjuvant: (l1) The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3; (l2) The recombinant protein according to claim 4; (l3) The biomaterial as described in claim 5; (l4) The coupling compound according to claim 6.

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