Monoclonal antibody 5e9 recognizing human r9ap protein and uses thereof

Abstract

The application belongs to the technical field of antibodies, and discloses a monoclonal antibody 5E9 for recognizing human R9AP protein and application thereof. The monoclonal antibody or antigen binding fragment thereof has high affinity to R9AP protein and can significantly inhibit EBV infection on epithelial cells and B cells. The antibody can be used for detecting the presence or level of R9AP protein in a sample, detecting diseases caused by high expression of R9AP, and preventing EB virus infection and / or treating and / or preventing diseases caused by EB virus infection.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to a monoclonal antibody 5E9 that recognizes human R9AP protein and its application background technology.

[0002] R9AP (Retinal G protein-coupled receptor interacting protein) is a protein located on the cell membrane that plays a crucial role in photoreceptor cells of the retina. Encoded by the R9AP gene, it participates in intracellular signal transduction processes related to rhodopsin.

[0003] The primary function of the R9AP protein is to regulate the function of rhodopsin in the retina. It interacts with the G protein-coupled receptor complex of rhodopsin, helping to maintain a stable connection between rhodopsin and G proteins, thereby promoting the transmission of visual signals. Particularly during photoreception, rhodopsin is activated by light, triggering a series of signal transduction responses, and the R9AP protein is an indispensable part of this process. At the molecular level, R9AP interacts with rhodopsin and its related molecules (such as G proteins) to ensure that visual signals can be rapidly and effectively transmitted from external light stimulation to the nervous system, thus generating visual perception. The absence or dysfunction of R9AP may lead to impaired visual function and even trigger some hereditary eye diseases. However, R9AP expression is also associated with the occurrence of various tumors, and it may play a potential role in the occurrence and development of certain tumors. Specifically, the relationship between high R9AP expression and the following tumor types has been preliminarily explored: 1. Breast cancer: Some studies have shown that R9AP may have high expression levels in breast cancer cells, especially in some advanced or invasive breast cancers. High expression of R9AP may be associated with tumor cell proliferation, migration, and invasiveness. 2. Ovarian Cancer: R9AP has been found to be highly expressed in ovarian cancer cells, and this high expression may be associated with the survival and drug resistance of ovarian cancer cells. This high expression may promote tumor cell growth by regulating signaling pathways related to cell survival and growth. 3. Colorectal Cancer: In colorectal cancer, some studies have found high levels of R9AP expression, which is associated with tumor malignancy and metastatic potential. R9AP may affect the growth and migration of colorectal cancer cells by participating in G protein-coupled receptor signaling. 4. Lung Cancer: R9AP is also highly expressed in certain types of lung cancer, particularly non-small cell lung cancer. This high expression may be associated with tumor drug resistance, proliferation, and metastatic ability. 5. Gastric Cancer: In gastric cancer studies, high expression of R9AP protein is thought to be associated with the invasive behavior and poor prognosis of gastric cancer. 6. Liver Cancer: High expression of R9AP has also been observed in some liver cancer studies, which may be related to the growth, division, and drug resistance characteristics of liver cancer cells.

[0004] Meanwhile, recent studies have found that R9AP is an important universal receptor for EB virus infection of cells and is involved in both B cell and epithelial cell infection processes. Therefore, antibodies against R9AP can inhibit EB virus from recognizing R9AP and further infecting cells.

[0005] Currently, there is no effective vaccine against EBV, and there are no specific treatments for diseases caused by EBV infection. Treatment for infectious mononucleosis mostly involves antiviral drugs such as acyclovir. While these drugs can alleviate symptoms to some extent, they cannot eliminate EBV from B lymphocytes and the pharyngeal epithelium. Therefore, developing an antibody against R9AP could serve as an important treatment method to block EBV infection. 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] The fourth aspect of this invention aims to provide a method for preparing the monoclonal antibody or its antigen-binding fragment of the first aspect of this invention or the recombinant protein of the second aspect.

[0010] The fifth aspect of this invention is to provide a coupling material.

[0011] The sixth 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 fifth aspect in the preparation of products.

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

[0013] An eighth aspect of the present invention is to provide a vaccine.

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

[0015] In a first aspect, the present invention provides a monoclonal antibody against EB virus R9AP or an antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region;

[0016] The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3;

[0017] The CDR-H1, CDR-H2, and CDR-H3 are amino acid sequences as shown in SEQ ID NO: 4, namely CDR1, CDR2, and CDR3.

[0018] The light chain variable region includes CDR-L1, CDR-L2 and CDR-L3;

[0019] The CDR-L1, CDR-L2, and CDR-L3 are amino acid sequences as shown in SEQ ID NO: 15, namely CDR1, CDR2, and CDR3.

[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: 10, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SAS, and SEQ ID NO: 18, respectively, and the CDR is defined using the IMGT definition scheme.

[0021] 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: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, and the CDR is defined using the Kabat 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: 8, SEQ ID NO: 9, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, 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: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, 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: 4; or

[0026] a2) An amino acid sequence of SEQ ID NO: 4 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: 4; or

[0027] a3) has 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: 4 and has the same amino acid sequence as the protein shown in SEQ ID NO: 4.

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

[0029] b1) SEQ ID NO: 15; or

[0030] b2) The amino acid sequence of SEQ ID NO: 15 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: 15; or

[0031] b3) has 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: 15 and has the same amino acid sequence as the protein shown in SEQ ID NO: 17.

[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 heavy chain further comprises a heavy chain signal peptide; and / or

[0036] The light chain also includes a light chain signal peptide.

[0037] Preferably, the amino acid sequence of the heavy chain signal peptide comprises:

[0038] e1) The amino acid sequence consisting of amino acids 1-19 in SEQ ID NO: 3; or

[0039] e2) An amino acid sequence having the same function as the amino acid sequence described in e1) by substitution and / or deletion and / or addition of one or more amino acids; or

[0040] 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).

[0041] Preferably, the amino acid sequence of the light chain signal peptide comprises:

[0042] f1) The amino acid sequence consisting of amino acids 1-19 in SEQ ID NO: 14; or

[0043] f2) An amino acid sequence of the amino acid sequence described in f1) 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

[0044] 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).

[0045] Preferably, the amino acid sequence of the human R9AP protein comprises:

[0046] g1) SEQ ID NO: 1 amino acid sequence; or

[0047] 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

[0048] The amino acid sequences described in g3) and g1) have 99%, 98%, 97%, 96%, 95%, 94% or 93% homology and have the same function as the proteins described in g1).

[0049] 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

[0050] Optional tag sequences to assist in expression and / or purification.

[0051] 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.

[0052] A third aspect of the invention provides biological materials relating to the monoclonal antibody or its antigen-binding fragment of the first aspect of the invention, or the recombinant protein of the second aspect of the invention, said biological material comprising at least one of h1) to h16):

[0053] h1) A nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention, or a recombinant protein of the second aspect of the present invention;

[0054] h2) contains an expression cassette containing the nucleic acid molecule described in h1);

[0055] h3) A carrier containing the nucleic acid molecule described in h1);

[0056] h4) A carrier containing the expression box described in h2);

[0057] h5) A transgenic cell line containing the nucleic acid molecules described in h1);

[0058] h6) Transgenic cell lines containing the expression cassette described in h2);

[0059] h7) A transgenic cell line containing the vector described in h3);

[0060] h8) A transgenic cell line containing the vector described in h4);

[0061] h9) Microorganisms containing the nucleic acid molecules described in h1);

[0062] h10) Microorganisms containing the expression cassette described in h2);

[0063] h11) contains microorganisms containing the carrier described in h3);

[0064] h12) contains microorganisms that carry the vector described in h4);

[0065] h13) contains viruses containing the nucleic acid molecules described in h1);

[0066] h14) A virus containing the expression cassette described in h2);

[0067] h15) contains a virus with the vector described in h3);

[0068] h16) contains the virus of the vector described in h4).

[0069] Preferably, the transgenic cell line does not contain propagation material.

[0070] 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.

[0071] A fourth aspect of the present invention provides a method for preparing 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, which is obtained by expression and purification using the biological material described in the third aspect of the present invention.

[0072] A fifth aspect of the present invention provides a conjugate comprising at least one of a monoclonal antibody or an antigen-binding fragment thereof from the first aspect of the present invention and a recombinant protein from the second aspect of the present invention;

[0073] 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.

[0074] Preferably, the detectable marker is selected from radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, or any combination thereof.

[0075] 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.

[0076] A sixth aspect of the present invention provides 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 fifth aspect in the preparation of a product;

[0077] The product includes at least one of the following: drug, reagent, test plate, reagent kit, and test chip.

[0078] Preferably, the drug has at least one of the functions i1) to i2):

[0079] i1) Prevention of EB virus infection;

[0080] i2) Treatment and / or prevention of diseases caused by EB virus infection.

[0081] Preferably, the reagent, detection plate, detection chip, or reagent kit has at least one function among j1) to j2):

[0082] j1) Detect the presence or level of R9AP protein in the sample;

[0083] j2) Detection of diseases associated with high R9AP expression.

[0084] Preferably, the disease includes at least one of breast cancer, ovarian cancer, colorectal cancer, lung cancer, stomach cancer, and liver cancer.

[0085] A seventh aspect of the present invention provides a product comprising at least one of k1) to k3):

[0086] k1) A monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention;

[0087] k2) The recombinant protein of the second aspect of the present invention;

[0088] k3) The coupling of the fifth aspect of the present invention;

[0089] The product includes at least one of reagents, test plates, reagent kits, and test chips.

[0090] Preferably, the reagent, detection plate, detection chip, or reagent kit has at least one function among j1) to j2):

[0091] j1) Detect the presence or level of R9AP protein in the sample;

[0092] j2) Detection of diseases associated with high R9AP expression.

[0093] Preferably, the disease includes at least one of breast cancer, ovarian cancer, colorectal cancer, lung cancer, stomach cancer, and liver cancer.

[0094] An eighth aspect of the present invention provides a medicament comprising at least one of 11) to 14):

[0095] l1) A monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention;

[0096] l2) The recombinant protein of the second aspect of the present invention;

[0097] l3) Benzene invented the third aspect of biomaterials;

[0098] l4) The coupling of the fifth aspect of the present invention.

[0099] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0100] Preferably, the drug has at least one of the functions i1) to i2):

[0101] i1) Prevention of EB virus infection;

[0102] i2) Treatment and / or prevention of diseases caused by EB virus infection.

[0103] 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.

[0104] Preferably, the drug comprises a vaccine.

[0105] Preferably, a vaccine comprises at least one of l1) to l4) and an adjuvant:

[0106] l1) A monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention;

[0107] l2) The recombinant protein of the second aspect of the present invention;

[0108] l3) Benzene invented the third aspect of biomaterials;

[0109] l4) The coupling of the fifth aspect of the present invention.

[0110] The beneficial effects of this invention are:

[0111] This invention provides a monoclonal antibody against human R9AP protein or its antigen-binding fragment thereof. This monoclonal antibody or its antigen-binding fragment has high affinity for human R9AP protein and can detect its expression level at multiple levels. Simultaneously, it can significantly inhibit EBV infection of epithelial cells and B cells, and therefore can be used to detect the presence or level of R9AP protein in samples, detect diseases caused by high R9AP expression, and prevent and / or treat and / or prevent diseases caused by EBV infection. Attached Figure Description

[0112] Figure 1 This is a graph showing the ELISA results of the binding of monoclonal antibody 5E9 to R9AP protein.

[0113] Figure 2 This is a graph showing the BLI binding detection results of monoclonal antibody 5E9 and R9AP protein.

[0114] Figure 3 This is a graph showing the results of WB-specific binding detection of monoclonal antibody 5E9 and R9AP protein.

[0115] Figure 4This is a diagram showing the neutralization results of monoclonal antibody 5E9 in EB virus epithelial cells.

[0116] Figure 5 This is a diagram showing the neutralization results of monoclonal antibody 5E9 in EB virus B cells. Detailed Implementation

[0117] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0118] 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.

[0119] Example 1: Preparation, animal immunization, and antibody screening of human R9AP protein

[0120] 1.1 Expression and purification of R9AP protein

[0121] 1.1.1 Experimental Materials

[0122] (1) Expression vector: eukaryotic expression vector: pGEX-6P-1(+) (Addgene).

[0123] (2) Expression system: prokaryotic expression system cells DE3.

[0124] (3) Reagents and consumables: TB culture medium (Haibo Biotechnology), GST tag protein purification agarose beads (Cytiva) and other conventional reagents and consumables were all purchased commercially.

[0125] (4) Gene: The human R9AP gene was optimized and synthesized by Nanjing Genscript Biotech Co., Ltd., and inserted into the pGEX-6P-1 vector, hereinafter referred to as GST-R9AP.

[0126] The R9AP extracellular recombinant protein consists of 210 amino acid residues in total, as follows:

[0127] MAREECKALLDGLNKTTACYHHLVLTVGGSADSQNLRQELQKTRQKAQELAVSTCARLTAVLRDRGLAADERAEFERLWVAFSGCLDLLEADMRRALELGAAFPLHAPRRPLVRTGVAGASSGVAARALSTRSLRLEAEGDFDVADLRELEREVLQVGEMIDNMEMKVNVPRWTVQARQAAGAELLSTVSAGPSSVVSLQERGGGCDPRK (SEQ ID NO: 1).

[0128] Its DNA expression sequence is 630 base pairs in total, specifically:

[0129] ATGGCGAGGGAGGAGTGCAAGGCGCTGCTGGACGGGCTCAACAAGACGACTGCGTGCTACCACCACCTGGTGCTGACCGTCGGTGGCTCGGCGGACTCGCAGAACCTGCGGCAGGAGCTGCAAAAGACGCGCCAGAAGGCGCAGGAGCTGGCGGTGTCCACCTGCGCCCGGCTGACTGCTGTGCTGCGCGACCGGGGCCTGGCCGCCGACGAGCGCGCCGAGTTCGAGCGGCTCTGGGTGGCCTTCTCGGGCTGCCTGGACCTGTTGGAAGCGGACATGCGACGCGCGCTGGAGCTGGGCGCCGCGTTCCCGCTGCACGCGCCGCGGCGGCCGCTGGTGCGCACAGGTGTGGCTGGCGCCTCCTCCGGCGTGGCGGCGCGCGCGCTGAGCACCCGCAGCCTGCGGCTCGAGGCGGAGGGCGACTTCGACGTCGCGGACCTGCGGGAGCTGGAGCGCGAGGTCCTTCAGGTGGGCGAGATGATCGACAACATGGAGATGAAGGTCAACGTGCCCCGCTGGACCGTGCAAGCCCGGCAGGCGGCGGGCGCCGAGCTCCTGTCCACGGTCAGCGCCGGCCCCTCCTCGGTCGTGTCCTTGCAGGAGCGCGGGGGGGGTTGCGACCCCAGGAAG (SEQ ID NO: 2).

[0130] 1.1.2 Protein expression

[0131] Step 1: Transformation and Cloning

[0132] (1) Add the synthesized plasmid to the freeze-thawed DH5a bacteria and let it stand on ice for 5 minutes.

[0133] (2) Place the bacteria with added plasmid in a shaking metal bath and heat shock at 42℃ and 120rpm for 60-90 seconds.

[0134] (3) Let the bacteria stand on ice to cool down for 5 minutes.

[0135] (4) Use a spreader to spread the bacteria on TB medium plates containing ampicillin resistance and incubate overnight.

[0136] (5) The next day, pick a single colony from the surface using a sterile pipette tip and add it to 500 mL of culture medium. Add 0.1 mg / mL ampicillin and incubate at 37°C and 220 rpm for 12-16 hours.

[0137] (6) After culturing the bacterial culture for about 8 hours, use Nanodrop to measure its OD value until the OD value is about 1.8-2.0.

[0138] (7) Adjust the incubator to 18°C ​​and 220 rpm to cool down the bacterial solution.

[0139] (8) Add 1 mM IPTG solution to the bacterial culture and continue culturing for 8-12 hours.

[0140] Step 3: Prokaryotic protein purification

[0141] (1) Centrifuge the cultured bacterial solution at 4000 rpm and 4℃ for 10 minutes to collect the bacterial precipitate and discard the supernatant.

[0142] (2) Use resuspension buffer (250mM NaCl, 50mM HEPES pH8.0, 2% Glycerol, 30mM Midazole, 1mM DTT) to resuspend the precipitate at a rate of 50mL of resuspension buffer per 1L of bacterial culture.

[0143] (3) Use a pressure crusher to crush, resuspend and precipitate three times at 4℃ and 800Pa pressure.

[0144] (4) Centrifuge the crushed precipitate at 15000g and 4℃ for 30 minutes to separate the supernatant.

[0145] (5) Filter the supernatant twice with a 0.22um filter membrane.

[0146] (6) Use the siphon method to transfer the supernatant to the GST beads purification column so that the supernatant completely passes through the purification column. If repeated recovery is required, the supernatant can be recovered in this step and purified again.

[0147] (7) Use resuspension buffer to balance the purification column to three column volumes.

[0148] (8) Use elution buffer (250mM NaCl, 50mM HEPES pH8.0, 2% Glycerol, 20mM GSH, 1mM DTT) to elute the purification column for two column volumes to obtain the eluent.

[0149] (9) Samples of bacterial lysate, centrifugation supernatant, centrifugation precipitate, flow-through, equilibration solution and elution solution were collected and subjected to SDS-PAGE to verify their protein expression and purification.

[0150] (10) Use an ultrafiltration tube of appropriate size to concentrate the eluent so that the volume after concentration is less than 1 mL. Centrifuge the concentrated eluent at 12000 g and 4 °C for 5 minutes to remove its precipitate.

[0151] (11) The concentrated eluent was purified by molecular sieve using an Akta purifier equipped with a Superdex200 Increase 10 / 300GL molecular sieve column, and the protein sample at the molecular sieve elution peak position was collected and verified by SDS-PAGE with the previous sample.

[0152] 2.2.5 Protein Preservation

[0153] (1) After confirming the purity by SDS-PAGE, the concentration of the concentrated protein was detected by BCA kit. The simplified method is as follows.

[0154] (2) Prepare BSA standard by diluting samples 10 times starting from 1 mg / mL into 7 gradients.

[0155] (3) In a 96-well plate, add 0, 1, 2, 4, 8, 12, 16, and 20 µl of standard to the wells of the 96-well plate, and add standard diluent to make up to 20 µl. This is equivalent to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml, respectively.

[0156] (4) Add an appropriate volume of sample to the wells of the 96-well plate. If the sample volume is less than 20 µl, add standard diluent to bring it up to 20 µl. Please record the sample volume.

[0157] (5) Add 200µl of BCA working solution to each well and place at 37ºC for 20-30 minutes.

[0158] (6) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at a wavelength of 562 nm.

[0159] (7) Use the absorbance and concentration of the standard to create a standard curve, and import the absorbance value of the sample well to calculate its concentration.

[0160] (8) After determining the concentration, dilute the protein to 1 mg / mL, store it in a 1.5 mL EP tube, freeze the protein with liquid nitrogen, and then store it in a -80℃ freezer for long-term storage. If it needs to be prepared and used immediately, it does not need to be frozen and can be used directly in the experiment.

[0161] 1.2 EBV R9AP Rabbit Immunization Experiment

[0162] 1.2.1 Vaccine Preparation

[0163] (1) Take out the GST-R9AP protein and freeze-thaw it. The dosage is 50 μg / animal. Use sterile PBS as the solvent for the antigen.

[0164] (2) Mix 500 μL of antigen solution with an equal volume of 500 μL of complete Freund's adjuvant to form a dose of 1 mL / rabbit, and place on a rotating shaker at 4°C overnight.

[0165] 1.2.2 Spleen collection for rabbit immunization

[0166] (1) The rabbits were fed with conventional feed and water, and were immunized at 12 weeks of age. They were of the New Zealand White rabbit breed.

[0167] (2) Fix the rabbit, and use a 2mL syringe to manually shake the vaccine again, then draw it up and inject it into the rabbit via intramuscular injection in the leg.

[0168] (3) Observe the rabbit for 3-5 minutes, and release it after it resumes normal activity.

[0169] (4) The vaccine immunization process is from week 0 to week 3 to week 6 to week 8, and the spleen collection time is 10 weeks.

[0170] (5) When collecting the spleen of a rabbit, the spinal dislocation method was used to kill it in accordance with animal welfare and standard animal experimental procedures. Its blood was collected and its spleen was dissected.

[0171] 1.3 EBV R9AP Rabbit B Cells: R9AP-Specific Cell Isolation and Cloning

[0172] 1.3.1 Steps for separating spleen lymphocytes:

[0173] (1) Ten weeks after immunization, rabbits were euthanized by cervical dislocation, and their peripheral lymph nodes and spleen were separated and placed in EP tubes.

[0174] (2) Add appropriate amounts of DNase and type IV collagenase to 1640 medium to prepare tissue digestion solution, and add appropriate amounts of digestion solution according to the size of lymph nodes and spleen.

[0175] (3) Using the pressure plug in the sterile syringe, place the tissue on a 70 μm cell sieve and crush it until the tissue is almost broken and eroded. Then rinse the cell sieve with an appropriate amount of fresh culture medium so that the remaining cells are rinsed into the 50 mL centrifuge tube below.

[0176] (4) Add red blood cell lysis buffer and incubate at room temperature for 10 minutes.

[0177] (5) Centrifuge at 2000 g, 18℃ for 5 min.

[0178] (6) Wash twice with fresh culture medium.

[0179] 1.3.2 B lymphocyte antigen staining and sorting

[0180] (1) Wash cells with PBS to a concentration of 1*10⁻⁶. ^8 Transfer the cells to a flow cytometry tube, centrifuge at 3000 g, 18°C ​​for 5 minutes, and discard the supernatant.

[0181] (2) Add 5 μL of Fc receptor blocking antibody solution to 100 μL PBS, resuspend the cells and incubate on ice for 30 minutes.

[0182] (3) Based on the cell grouping, prepare flow cytometry antibody dilution buffer using PBS. For the first use of the antibody, different dilution gradients can be set. Select the optimal gradient according to the antibody-to-PBS ratio of 1:20, 1:50, 1:100, 1:200, and 1:500. Add 100 μL of the diluted flow cytometry antibody and cell viability dye to resuspend the cells for staining, and incubate at 4°C in the dark for 30 minutes. Rabbit IgG and B220 antibodies were used as B cell separation reagents, and R9AP was used as the R9AP-specific B cell separation reagent.

[0183] (4) Wash the cells with PBS. Use 3000 g, 4℃, centrifuge for 5 minutes to wash the cells. Repeat 1 to 2 times and discard the supernatant.

[0184] (5) The cells were resuspended in a flow cytometer with PBS and the cells were sorted by flow cytometry using a BD Rhaspody single-cell sorter.

[0185] (6) Add IgG + B220 + The 0.2% of cells with the strongest R9AP positive signal were sorted into 96-well plates.

[0186] 1.3.3 B cell sequence cloning and amplification

[0187] (1) By using universal cloning primers for rabbit IgG heavy and light chains, PCR was performed on B cells in a 96-well plate to obtain the heavy and light chain sequences of a single B cell.

[0188] The forward (F) and reverse (R) primer sequences for the heavy chain VH are as follows:

[0189] F: ggtggttcctctagatcttcctcctctggtggcggtggctcgggcggtggtgggCAGTCGBTGGAGGAGT (SEQ ID NO: 22).

[0190] R: gctggccggcctggccactagt TGARGAGACGGTGACC (SEQ ID NO: 23).

[0191] The forward (F) and reverse (R) primer sequences for the light chain VL are as follows:

[0192] F: ggtggttcctctagatcttcctcctctggtggcggtggctcgggcggtggtgggCAGTCGBTGGAGGAGT (SEQ ID NO: 24)

[0193] R: gctggccggcctggccactagt TGARGAGACGGTGACC (SEQ ID NO: 25).

[0194] (2) By linking the upstream of the variable region of the antibody heavy chain to the CMV fragment and the downstream of the constant region of rabbit IgG1, the complete heavy chain fragment can be expressed; while by linking the upstream of the variable region of the antibody light chain to the CMV fragment and the downstream of the constant region of the light chain κ / λ, the complete light chain fragment can be expressed. Antibody expression can be achieved by co-transfecting plasmids containing the full-length sequences of the antibody heavy and light chains into 293F cells. Antibody purification can be achieved using protein A beads, and a total of 9 antibodies were expressed using this method.

[0195] (3) The obtained antibodies were subjected to R9AP affinity assay using ELISA, and the 5E9 antibody with the highest affinity was selected for further verification.

[0196] Example 2 Preparation of monoclonal antibody 5E9 (monoclonal antibody) against human R9AP protein

[0197] By linking the variable region of the antibody heavy chain upstream to the CMV fragment and downstream to the constant region of human IgG1, the complete heavy chain fragment can be expressed; similarly, by linking the variable region of the antibody light chain upstream to the CMV fragment and downstream to the constant region of the light chain κ / λ, the complete light chain fragment can be expressed. Co-transfection of plasmids containing the full-length sequences of the antibody heavy and light chains into 293F cells achieves antibody expression, and protein A beads can be used for antibody purification.

[0198] The 5E9 heavy chain contains 128 amino acid residues, specifically:

[0199] METGLRWLLLVAVLKGVQC QSLEESGGRLVTPGTPLTLSCKASGFDFS SYWMI WVRQAPGEGLEYIG I ITKSGNTYYANWAKG RFTISKTSTTVDLKITSPTTEDTATYFC TSNFKL WGQGTLVTVSS (SEQ ID NO: 3);

[0200] The underlined portion of the sequence represents the amino acid sequence of the heavy chain variable region (SEQ ID NO: 4). The italicized portions represent the three complementary regions CDR-H1 (GFDFSSYW) in the heavy chain variable region. ， SEQ ID NO: 10), CDR-H2 ( ITKSGNT, SEQ ID NO: 26) and CDR-H3 ( TSNFKL, The amino acid sequence of SEQ ID NO: 27 (IMGT definition scheme). The bolded portion is the signal peptide. * indicates a stop codon.

[0201] 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.

[0202] Table 11. CDR-H1, CDR-H2, and CDR-H3 in the variable region of the heavy chain, defined by other CDR schemes.

[0203]

[0204] The 5E9 variable region light chain consists of 132 amino acid residues, specifically:

[0205] MDTRAPTQLLGLLLLWLPGATFA QVLTQTASPVSAAVGGTVTINC QSSQSVYNNNN LAWFQQKPGQPP KVLIT SAS TLASGVPSRFSGSGSGTQFTLTISDLECDDAAAAYYC AGGYSGNIYT FGGGTDVVVK (SEQ ID NO: 14);

[0206] The underlined portion of the sequence represents the amino acid sequence of the light chain variable region (SEQ ID NO: 15). The underlined and bolded portions represent the three complementarity-determining regions CDR-L1 (QSVYNNNN) within the light chain variable region. ， SEQ ID NO: 28), CDR-L2 ( SAS ) and CDR-L3 ( AGGYSGNIYT, The amino acid sequence of SEQ ID NO: 18 (IMGT definition scheme). The bolded portion is the signal peptide. * indicates a stop codon.

[0207] 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.

[0208] Table 12 CDR-H1, CDR-H2, and CDR-H3 in the variable region of light chains with other defined CDR schemes

[0209]

[0210] Example 3 Affinity determination of 5E9

[0211] 3.1 Detection of 5E9 binding capacity using enzyme-linked immunosorbent assay (ELISA)

[0212] ELISA can be performed according to conventional methods in the art. In this embodiment, the specific operation is as follows: R9AP protein is prepared in PBS solution at a concentration of 10 μg / mL, and 100 μL is added to each well of an ELISA high-adhesion plate. Initial coating is performed at room temperature for 3 hours, and then the liquid is discarded. A 0.2 mg / mL BSA solution is prepared and added to the ELISA plate. The wells are blocked at room temperature for 6 hours, and then the liquid is discarded. Then, 5E9 antibody is serially diluted 1:10 and added to the ELISA plate. Initial binding is performed at room temperature for 2 hours, and then the liquid is discarded. Next, a 1:5000 dilution of goat anti-rabbit IgG-HRP secondary antibody is prepared and added to the ELISA plate. Secondary binding is performed at room temperature for 30 minutes, and then the liquid is discarded. Finally, TMB chromogenic reagent is added and incubated at room temperature for 15 minutes, and the incubation is stopped using dilute hydrochloric acid. The A280 reading in each well is detected using a microplate reader to reflect the R9AP antibody binding ability of each well; a higher reading indicates a stronger binding strength. The results are as follows. Figure 1 As shown, the A280 reading of the 5E9 antibody still showed a high level of binding even at a 1:512000 dilution, while the control unrelated IgG antibody showed no R9AP binding ability at all, reflecting its higher binding ability.

[0213] 3.2 The affinity of antibody 5E9 was determined using biomembrane interference (BLI) technique.

[0214] BLI can be performed according to conventional methods in the art. In this embodiment, the specific operation is as follows: The biosensor (Sartorius Octet® SA probe from Germany) is immersed in a buffer solution (a mixture of KB buffer, 0.1 wt% BSA, and 0.02 v / v% Tween 20) for equilibration. Then, it is removed and immersed in a solution containing 5 μg / mL R9AP-Biotin (biotin-labeled R9AP protein). The R9AP antigen in the solution binds to the surface of the SA (streptavidin) biosensor, increasing the thickness of its surface membrane. Then, the biosensor with a known concentration of cured antigen is immersed in the buffer solution as a baseline. By immersing the biosensor with a known concentration of cured antigen in a sample solution containing 31.3~500 nM 5E9 antibody for about 120 seconds, the specific binding between antigen and antibody leads to an increase in membrane thickness. The biosensor with 5E9 antibody is then immersed in the buffer solution for dissociation for about 180 seconds, causing the antibody to detach from the biosensor surface, resulting in a decrease in membrane thickness. By monitoring the thickness of the biofilm layer of the biosensor in real time during the experiment, the kinetic constants of the test sample (5E9 antibody) can be obtained. The results are as follows: Figure 2 As shown: KD(M) of the 5E9 antibody = 2.6 * 10 -8 M; This indicates that the 5E9 antibody has a high affinity for the R9AP antigen. 3.3 The ability of antibody 5E9 to specifically bind to R9AP was determined using Western blotting.

[0215] Western blotting (WB) was performed according to conventional methods in the art. In this embodiment, the specific procedure was as follows: R9AP full-length plasmid was overexpressed in 293 cells via PEI. Cell lysis buffer was then collected using WB lysis buffer. After thorough mixing, the fully lysed cell contents were collected and centrifuged to remove the precipitate. SDS-PAGE was then performed, and the samples were transferred to a 0.45 μm PVDF membrane using an electroporator. 5E9 antibody and R9AP positive antibody (Sigma, catalog number #HPA049791-100UL) were then added to the PVDF membrane, respectively, and incubated at 4°C for 8 hours. The antibodies were then discarded. Subsequently, 1:2000 dilution of goat anti-rabbit IgG-HRP secondary antibody and 1:4000 dilution of rabbit anti-mouse IgG-HRP secondary antibody were added, and the membranes were incubated at room temperature for 30 minutes. The antibodies were then discarded. Finally, horseradish peroxidase substrate was added for color development, and images were taken using a BioRad illuminator. The results are as follows: Figure 3 As shown, the 5E9 antibody and the well-validated Sigma R9AP antibody exhibit the same specific binding band position for R9AP, indicating that the 5E9 antibody has a specific binding to the R9AP antigen.

[0216] Example 4: Neutralization activity assay of 5E9 antibody

[0217] (1) Preparation of EBV virus:

[0218] 1) CNE2 cells infected with EBV-GFP were cultured in an incubator at 37 degrees Celsius (5v / v% CO2) using RPMI 1640 + 5v / v% FBS (the virus has been disclosed in the literature: An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus). When the cells reached 90% confluence (10 cm dish), TPA was added to a final concentration of 20 ng / mL and NaB (sodium butyrate) to induce induction. The medium was changed after 12 hours.

[0219] 2) Collect the culture supernatant 48–72 h after changing the medium to isolate and purify the virus. Directly aspirate the supernatant, centrifuge it, and filter it through a 0.45 μm filter. After concentration, resuspend it in serum-free RPMI 1640. Immediately use the resuspended virus for infection or store it at -80 degrees Celsius.

[0220] (2) Detection of neutralizing activity of 5E9 monoclonal antibody in epithelial cells

[0221] 1) Lay 1x10mm PVC in each well of a 96-well plate. 6 Add 100 μL of DMEM medium containing 10% FBS to each well for 293T epithelial cells.

[0222] 2) On the second day, take the 5E9 monoclonal antibody from the above examples and adjust the concentration to 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 5E9 antibody diluted with DMEM to the first well (the first well does not contain RPMI 1640 medium).

[0223] 3) After a 2-fold serial dilution (for serial dilution, 60 μL will be aspirated from the first well and added to the second well, and so on, with 60 μL aspirated from the last well and discarded, resulting in a final volume of 60 μL per well); add 60 μL of virus diluent to each well (the virus is diluted with DMEM medium to a titer of approximately 4*10). 6 The solution ( / mL) was incubated at 37°C for 2 hours and then added to the 293T cells plated the previous day. The cells were then incubated at 37°C for 48 hours before analysis.

[0224] 4) After digesting 293T cells with trypsin, a cell suspension was prepared. The infection rate was detected by flow cytometry. The inhibition rate (neutralization efficiency, %) of the antibody in the 293T epithelial cell infection model was calculated by detecting the reduction in the number of GFP-positive cells in the antibody-treated group compared with the infection control group (with an equal volume of DMEM added).

[0225] The results are as follows Figure 4 As shown: Monoclonal antibody 5E9 at a concentration of 400 μg / mL can significantly inhibit EBV infection of epithelial cells, where the vertical axis represents the EBV infection efficiency.

[0226] Example 5: Detection of neutralizing activity of 5E9 monoclonal antibody in B cells

[0227] 1) Take the 5E9 monoclonal antibody from the above examples and adjust its concentration to 2 mg / mL. Add 60 μL of RPMI 1640 medium to each well of a new 96-well plate. Add 90 μL of 100 μg / mL 5E9 antibody diluted with RPMI 1640 to the first well (the first well does not contain RPMI 1640 medium).

[0228] 2) After a 3-fold serial dilution (for serial dilution, 30 μL will be aspirated from the first well and added to the second well, and so on, with 30 μL aspirated from the last well and discarded, resulting in a final volume of 60 μL per well); add 60 μL of virus diluent to each well (the virus is diluted with DMEM medium to a titer of approximately 4*10). 6 After incubating at 37°C for 2 hours, add 1*10 mL to each well. 6 Raji cells were incubated at 37 degrees Celsius for 48 hours before being analyzed.

[0229] 3) Raji cells were aspirated to prepare cell suspensions, and the infection rate was detected by flow cytometry. The inhibition rate (neutralization efficiency, %) of the antibody in the Raji B cell infection model was calculated by detecting the reduction in the number of GFP-positive cells in the antibody-treated group compared with the infection control group (with an equal volume of RPMI1640 added).

[0230] The results are as follows Figure 5 As shown: Monoclonal antibody 5E9 can significantly inhibit EBV infection of B cells at a concentration of 400 μg / mL, where the vertical axis represents the EBV infection efficiency.

Claims

1. A monoclonal antibody against human R9AP protein or an antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3; The CDR-H1, CDR-H2, and CDR-H3 are the CDR1, CDR2, and CDR3 amino acid sequences in the variable region of the heavy chain as shown in SEQ ID NO: 4; The light chain variable region includes CDR-L1, CDR-L2 and CDR-L3; The CDR-L1, CDR-L2, and CDR-L3 are the amino acid sequences CDR1, CDR2, and CDR3 in the variable region of the light chain as shown in SEQ ID NO: 15; The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SAS, and SEQ ID NO: 18, respectively. The CDRs are defined using 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: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. The CDRs are defined using the Kabat 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: 8, SEQ ID NO: 9, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, 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: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. The CDR is defined using the Contact definition scheme.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that: The monoclonal antibody or its antigen-binding fragment comprises at least one of full-length antibody, Fab, Fab', F(ab')2, Fv, and scFv.

3. A recombinant protein comprising the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 2; and a tag sequence for assisting expression and / or purification.

4. Biological material relating to the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 2, or the recombinant protein as described in claim 3, wherein the biological material is at least one of h1) to h16): h1) A nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 2, or a recombinant protein of claim 3; h2) contains an expression cassette of 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 carry the vector described in h4); h13) contains viruses containing the nucleic acid molecules described in h1); h14) contains the expression cassette described in h2); h15) contains a virus with the vector described in h3); h16) contains the virus of the vector described in h4).

5. A method for preparing the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 2, or the recombinant protein as described in claim 3, comprising the following steps: It is obtained by expression and purification using the biological material described in claim 4.

6. A conjugate comprising the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 2, or the recombinant protein as described in claim 3; And the coupling part; The coupling portion is at least one of a detectable marker and a radionuclide.

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 2; (2) The recombinant protein according to claim 3; (3) The biomaterial as described in claim 4; (4) The coupling compound according to claim 6; The product includes at least one of the following: drug, reagent, test plate, reagent kit, and test chip; The drug has at least one of the functions i1) to i2): i1) Prevention of EB virus infection; i2) Prevention of diseases caused by EB virus infection; The reagent, detection plate, detection chip, or kit has at least one function among j1) to j2): j1) Detect the presence or level of R9AP protein in the sample; j2) Detection of diseases associated with high R9AP expression.

8. A drug comprising at least one of (l1) to (l4): (l1) The monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 2; (l2) The recombinant protein according to claim 3; (l3) The biomaterial as described in claim 4; (l4) The coupling as described in claim 6; The drug also contains a pharmaceutically acceptable carrier.

9. A kit for detecting EB virus, said kit comprising at least one of (l1) to (l4): (l1) The monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 2; (l2) The recombinant protein according to claim 3; (l3) The biomaterial as described in claim 4; (l4) The coupling compound according to claim 6.

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