A monoclonal antibody against monkeypox virus and application thereof

Abstract

The present application relates to monkeypox virus human monoclonal antibody and its application, through screening human antibody humanization mouse anti A29L specific binding memory B cell, the human source high neutralization activity monkeypox virus antibody A29L-1B3, A29L-1B2, A29L-1A5 and A29L-1A2 obtained, have the characteristics such as strong monkeypox virus binding ability, good in-vitro neutralization, affinity, have in-vivo protective effect, and are completely different from the monkeypox virus antibody sequence reported, provide the product for the detection and neutralization of monkeypox virus, and provide the possibility for providing the monkeypox virus product with in-vivo protective effect.

Description

Technical Field

[0001] This invention relates to the field of antibody engineering, and more particularly to human monoclonal antibodies against monkeypox virus and their applications. Background Technology

[0002] Monkeypox virus (MPXV) belongs to the family Poxviridae, subfamily Chordopoxvirinae, and genus Orthopoxvirus (OPXV). The genus Orthopoxvirus contains more than 10 members, including smallpox virus (VARV), cowpox vaccine virus (VACV), rinderpest virus (CPXV), camelpox virus (CMLV), and several new species isolated from infected humans or primates since 2010. Based on comparisons between VACSV and MPXV, the surface proteins of poxviruses show approximately 93–98% sequence similarity, suggesting they play similar roles in the process of entering the body.

[0003] Monkeypox is a zoonotic disease whose natural host is not yet clearly identified. It primarily spreads among rodents and non-human primates. Transmission can occur through various means, including contact with damaged skin tissue. Furthermore, recent transmission of monkeypox virus in non-endemic countries has shown a link to sexual contact; the virus has been detected in bodily fluids such as semen, although whether sexual transmission is possible remains unclear. Symptoms after infection include swollen lymph nodes, muscle aches, and permanent scarring after the pustules rupture.

[0004] The eradication of smallpox in 1980 and the subsequent cessation of smallpox vaccination have resulted in lower immunity to ornithovirus among younger populations, which may be one of the reasons for the current MPXV outbreak. With the spread of various monkeypox virus strains in non-endemic countries, not only are transmission routes diverse and insidious, and infection symptoms atypical, but monkeypox virus also mutates rapidly during human-to-human transmission, with a mutation rate far exceeding expectations, posing an increasing threat to global public health. Although monkeypox cases are currently low in China, with the increasing movement of people both domestically and internationally, how to prevent and control monkeypox infection has become a critical issue that urgently needs to be addressed. Furthermore, there is currently no specific antiviral therapy approved for the treatment of human MPXV infection. We urgently need more research on this virus to better prevent and treat monkeypox.

[0005] To date, neutralizing antibodies have proven to be an effective treatment for viral diseases, including human immunodeficiency virus (HIV), influenza virus, and other flaviviruses. MPXV exists in two forms: extracellular enveloped virus (EEV) and intracellular mature virus (IMV) particles. The A29L and E8L proteins are envelope proteins on the surface of IMV and play a crucial role in viral infection.

[0006] Three monoclonal antibodies, 9F8, 3A1, and 2D1, recognizing different epitopes of MPXV A29L, have been successfully prepared. All three have been shown to possess strong, broad-spectrum binding and neutralizing activity against orthopoxvirus, with 9F8 exhibiting the best neutralizing activity and providing synergistic protection against orthopoxvirus. The binding properties of several human anti-D8 antibodies have also been characterized, and the crystal structures of three VACV-mAb variants—VACV-66, VACV-138, and VACV304—that bind to D8 have been resolved.

[0007] It is very important and necessary to develop new monkeypox virus neutralizing antibodies that are specific, have strong antigen-binding ability, good in vitro neutralization, high affinity, and in vivo protective effects. Summary of the Invention

[0008] The problem that the invention aims to solve:

[0009] To address the problems existing in the prior art, this invention screens humanized mouse memory B cells that specifically bind to A29L using fully human antibodies, obtaining humanized MPXV antibodies A29L-1B3, A29L-1B2, A29L-1A5, and A29L-1A2 with high neutralizing activity. Ultimately, it screens out new humanized monoclonal antibodies against monkeypox virus that are specific, have strong binding ability to monkeypox virus, good in vitro neutralization, and high affinity.

[0010] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0011] I. Preparation and purification of monoclonal antibody against monkeypox virus A29L

[0012] Four monoclonal antibodies against monkeypox virus A29L were obtained through expression and purification of monkeypox virus A29L protein, sorting of specific memory B cells that bind to A29L protein, and expression and purification of monkeypox virus A29L antibodies: A29L-1B3, A29L-1B2, A29L-1A5, and A29L-1A2. The amino acid sequences of the light and heavy chains, and the CDR1-3 regions of the light and heavy chains of these four monoclonal antibodies are as follows. The variable regions of the light and heavy chains are different from existing sequences and have specificity.

[0013] II. Performance Validation of Monoclonal Antibodies

[0014] (I) ELISA detection of A29L antigen and antibody binding activity

[0015] The four antibodies, A29L-1B3, A29L-1B2, A29L-1A5, and A29L-1A2, can all bind to antigens. As the antibody concentration increases, the binding activity increases, and the OD450 value increases.

[0016] (ii) Antigen and antibody neutralization test

[0017] The neutralizing activity of the A29L antibody against VCV-WR was determined by the plaque assay. The IC50 values ​​of A29L-1A2, A29L-1A5, A29L-1B2, and A29L-1B3 were 0.026, 0.085, 0.029, and 0.0411, respectively.

[0018] (III) Detection of antibody-antigen affinity using surface plasmon resonance technology

[0019] The four antibodies, A29L-1B3, A29L-1B2, A29L-1A5, and A29L-1A2, have good affinity for the antigen.

[0020] (iv) Animal protection experiments

[0021] In cases of low-dose viral infection, repeated administration of A29-1A2 antibody can provide protection against monkeypox virus infection in mice.

[0022] Compared with the prior art, the beneficial effects of this application are as follows:

[0023] The humanized monoclonal antibody of this invention has strong binding ability to monkeypox virus, good in vitro neutralization, high affinity, and in vivo protective effect. It is completely different from the reported monkeypox virus antibody sequence, providing a product for the detection and neutralization of monkeypox virus, and making it possible to provide monkeypox virus products with in vivo protective effect. Attached Figure Description

[0024] Figure 1 Results of SDS-PAGE purification of monkeypox virus A29L protein using molecular sieve;

[0025] Figure 2 SDS-PAGE results of Protein A purified antibody;

[0026] Figure 3 ELISA detection results of antibody-antigen binding activity;

[0027] Figure 4Neutralization curve results of A29L antibody against VACV-WR;

[0028] Figure 5 The kinetic curve results of A29L antibody and A29L;

[0029] Figure 6 The experiment diagram and the survival and weight change curves of mice after low-dose infection are shown.

[0030] A is a schematic diagram of the experiment.

[0031] B represents the survival and weight change curves of mice after low-dose infection. Detailed Implementation

[0032] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] Experimental materials

[0036] 1. Key medicines and reagents

[0037] name model Manufacturers Fully humanized antibody mice RenMab Biocytok Pharmaceutical Technology Co., Ltd. 14K chip 750-00021 Berkeley lights

[0038] 2. Main Instruments

[0039] Instrument Name model Manufacturers Beacon sorting machine Beacon Berkeley Lights

[0040] Example 1: Preparation and purification of monkeypox virus monoclonal antibody

[0041] 1. Expression and purification of monkeypox virus A29L protein

[0042] The A29L gene was constructed and transfected into the eukaryotic expression vector pCAGGS into 293F cells. A29L was expressed intracellularly, and after sonication, it was crudely separated by Strep affinity chromatography, further purified by molecular sieves, and identified by SDS-PAGE.

[0043] like Figure 1 As shown, the SDS-PAGE results of monkeypox virus A29L protein purification were obtained. The protein was passed through a Superdex 200PG, and a UV 280 nM absorption peak was detected at ~75 mL. The molecular weight of the protein in the SDS-PAGE was approximately ~20 kDa, and the obtained protein had high purity.

[0044] 2. Sorting of specific memory B cells that bind to A29L protein

[0045] The A29L gene was constructed into mRNA vectors, transcribed in vitro, and capped. Concentration and quality were then measured. Humanized mice (RenMab) received three immunizations at a dose of 2.5 μg / mouse. Serum was collected before immunization and 12 days after each immunization to detect antibody titers. Once the antibody titer reached an appropriate level, a booster immunization was administered. Lymph nodes from the humanized mice were then collected, ground into single cells, incubated with antigen molecules, and specifically sorted using a Beacon sorter. The variable region gene sequence of the antibody was obtained after reverse transcription and PCR amplification.

[0046] Four monoclonal antibodies against monkeypox virus A29L-1B3, A29L-1B2, A29L-1A5, and A29L-1A2 were obtained. The amino acid sequences of the light and heavy chains, and the CDR1-3 variable regions of the light and heavy chains of these four monoclonal antibodies are as follows. The variable regions of the light and heavy chains are different from existing sequences, demonstrating specificity.

[0047] The amino acid sequence information of the three CDR regions of the A29L-1B3 light chain is as follows:

[0048] A29L-1B3 Light Chain Serial number amino acid sequence CDR1 SEQ ID NO:1 QSLLYSDGSTY CDR2 SEQ ID NO:2 KVS CDR3 SEQ ID NO:3 MQGTHWPFT

[0049] The amino acid sequence information of the three CDR regions of the A29L-1B3 heavy chain is as follows:

[0050] A29L-1B3 Heavy Chain Serial number amino acid sequence CDR1 SEQ ID NO:4 GFTFSNYW CDR2 SEQ ID NO:5 INSVGSST CDR3 SEQ ID NO:6 GYSGY

[0051] The amino acid sequence of the A29L-1B3 light chain (SEQ ID NO:7)

[0052] DIVMTQSPLSLPVTLGQPASISCRSSQSLLYSDGSTYLNWFQQRPGQSPRRLIYKVSTRDSGVPDRFSGSGSGTDFTLKISRVEADDVGVYYCMQGTHWPFTFGPGTKVEIK

[0053] The amino acid sequence of the A29L-1B3 heavy chain (SEQ ID NO:8)

[0054] QVQLEQSGGGLVQPGGSLRLSCAASGFTFSNYWMYWVRQAPGKGLVWVSRINSVGSSTSYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCGYSGYLGQGTLVTVSS

[0055] This invention provides nucleotide sequences for editing the three CDR regions of the A29L-1B3 light chain, as follows:

[0056] A29L-1B3 Light Chain Serial number nucleotide sequence CDR1 SEQ ID NO:9 CAAAGCCTCCTATACAGTGATGGAAGCACCTAC CDR2 SEQ ID NO:10 AAGGTTTCT CDR3 SEQ ID NO:11 ATGCAAGGTACACACTGGCCATTCACT

[0057] This invention provides nucleotide sequences for editing the three CDR regions of the A29L-1B3 heavy chain, as follows:

[0058] A29L-1B3 Heavy Chain Serial number nucleotide sequence CDR1 SEQ ID NO:12 GGATTCACCTTCAGTAACTACTGG CDR2 SEQ ID NO:13 ATAAATAGTGTTGGGAGTAGCACA CDR3 SEQ ID NO:14 GGATATAGTGGCTAC

[0059] This invention provides a nucleotide sequence (SEQ ID NO:15) for editing the A29L-1B3 light chain.

[0060] GTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCCTATACAGTGATGGAAGCACCTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTAATTTATAAGGTTTCT ACCCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTTGGAGGCTGATGATGTTGGGGTTTATTACTGCATGCAAGGTACACACTGGCCATTCACTTTCGGCCCTGGGACCAAAGTGGAAATCAAA

[0061] This invention provides a nucleotide sequence (SEQ ID NO:16) for editing the A29L-1B3 heavy chain.

[0062] CAGGTGCAGCTGCAGCAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACA ACCCTCCCTCCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGGCCGTGTATTACTGTGCGAGACATAATAATAGCAGCAGTCCTCACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0063] The amino acid sequence information of the three CDR regions of the A29L-1B2 light chain is as follows:

[0064] A29L-1B2 Light Chain Serial number amino acid sequence CDR1 SEQ ID NO:17 QSVLYSSNNKNY CDR2 SEQ ID NO:18 WAS CDR3 SEQ ID NO:19 QQYYRTPPT

[0065] The amino acid sequence information of the three CDR regions of the A29L-1B2 heavy chain is as follows:

[0066] A29L-1B2 Heavy Chain Serial number amino acid sequence CDR1 SEQ ID NO:20 GGSISSYY CDR2 SEQ ID NO:21 IYYSGST CDR3 SEQ ID NO:22 ARHNNSSSPHFDY

[0067] The amino acid sequence of the A29L-1B2 light chain (SEQ ID NO:23)

[0068] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLFYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYRTPPTFGQGTKVEIK

[0069] The amino acid sequence of the A29L-1B2 heavy chain (SEQ ID NO:24)

[0070] QVQLQQSGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARHNNSSSPHFDYWGQGTLVTVSS

[0071] This invention provides nucleotide sequences for editing the three CDR regions of the A29L-1B2 light chain, as follows:

[0072]

[0073]

[0074] This invention provides nucleotide sequences for editing the three CDR regions of the A29L-1B2 heavy chain, as follows:

[0075] This invention provides a nucleotide sequence (SEQ ID NO:31) for editing the A29L-1B2 light chain.

[0076] GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGTTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCCCCTAAGCTGCTCTTTTACTGGG CATCTACGCGGGAATCCGGGGTCCCCGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGAACTCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0077] This invention provides a nucleotide sequence (SEQ ID NO:32) for editing the A29L-1B2 heavy chain.

[0078] CAGGTGCAGCTGCAGCAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAGACATAATAATAGCAGCAGTCCTCACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0079] The information of the CDR regions of the A29L-1A5 light chain is as follows:

[0080]

[0081]

[0082] The information of the CDR regions of the A29L-1A5 heavy chain is as follows:

[0083] A29L-1A5 Heavy Chain Serial number amino acid sequence CDR1 SEQ ID NO:36 GFTFSTYW CDR2 SEQ ID NO:37 IDTDGNIT CDR3 SEQ ID NO:38 ARETNYAPFDY

[0084] The amino acid sequence of the A29L-1A5 light chain (SEQ ID NO:39)

[0085] EIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLIISSLQAEDVAVYYCQQYYRTPPTFGQGTKVEIK

[0086] The amino acid sequence of the A29L-1A5 heavy chain (SEQ ID NO:40)

[0087] QVQLEQSGGGLVQPGGSLRLSCAASGFTFSTYWMHWVRQAPGKGLVWVSRIDTDGNITRYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCARETNYAPFDYWGQGALVTVSS

[0088] This invention provides nucleotide sequences for editing the three CDR regions of the A29L-1A5 light chain, as follows:

[0089] A29L-1A5 Light Chain Serial number nucleotide sequence CDR1 SEQ ID NO:41 CAGAGTGTTTATACAGGTCCAACAATAAGAACTAC CDR2 SEQ ID NO:42 TGGGCATCT CDR3 SEQ ID NO:43 CAACAATATTATAGAACTCCTCCGACG

[0090] This invention provides nucleotide sequences for editing the three CDR regions of the A29L-1A5 heavy chain, as follows:

[0091] A29L-1A5 Heavy Chain Serial number nucleotide sequence CDR1 SEQ ID NO:44 GGATTCACCTTCAGTACCTACTGG CDR2 SEQ ID NO:45 ATTGATACTGATGGGAATATCACA CDR3 SEQ ID NO:46 GCAAGAGAAACTAACTACGCCCCCTTTGACTAC

[0092] This invention provides a nucleotide sequence (SEQ ID NO:47) for editing the A29L-1A5 light chain.

[0093] GAAATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGGTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGG CATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTGTCAACAATATTATAGAACTCCTCCGACGTTCGGCCAAGGGACAAGGTGGAAATCAAA

[0094] This invention provides a nucleotide sequence (SEQ ID NO:48) for editing the A29L-1A5 heavy chain.

[0095] CAGGTGCAGCTGCAGCAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACA ACCCTCCCTCCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGGCCGTGTATTACTGTGCGAGACATAATAATAGCAGCAGTCCTCACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0096] The CDR information for the A29L-1A2 light chain is as follows:

[0097] A29L-1A2 Light Chain Serial number amino acid sequence CDR1 SEQ ID NO:49 QSVLYRSNNKNY CDR2 SEQ ID NO:50 WAS CDR3 SEQ ID NO:51 QQYYSIPYT

[0098] The CDR region information for the A29L-1A2 heavy chain is as follows:

[0099] A29L-1A2 Heavy Chain Serial number amino acid sequence CDR1 SEQ ID NO:52 GDSVSSNSTG CDR2 SEQ ID NO:53 TYYRSKWYK CDR3 SEQ ID NO:54 ASGAFDF

[0100] The amino acid sequence of the A29L-1A2 light chain (SEQ ID NO:55)

[0101] EIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNKNYLAWYQQKPGQPPELLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPYTFGQGTKLEIK

[0102] The amino acid sequence of the A29L-1A2 heavy chain (SEQ ID NO:56)

[0103] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSTGWNWIRQSPSRGLEWLGRTYYRSKWYKDYAVSVKSRIIINPDTSKNQFSLHLNSVAPEDTAVYYCASGAFDFWGQGTMVTVSS

[0104] This invention provides nucleotide sequences for editing the three CDR regions of the A29L-1A2 light chain, as follows:

[0105] A29L-1A2 Light Chain Serial number nucleotide sequence CDR1 SEQ ID NO:57 CAGAGTGTTTATACAGGTCCAACAATAAGAATTAC CDR2 SEQ ID NO:58 TGGGCATCT CDR3 SEQ ID NO:59 CAGCAATATTATAGTATTCCGTACACT

[0106] This invention provides nucleotide sequences for editing the three CDR regions of the A29L-1A2 heavy chain, as follows:

[0107] A29L-1A2 Heavy Chain Serial number nucleotide sequence CDR1 SEQ ID NO:60 GGGGACAGTGTCTCTAGCAACAGTACTGGT CDR2 SEQ ID NO:61 ACATACTACAGGTCCAAGTGGTATAAG CDR3 SEQ ID NO:62 GCAAGCGGTGCCTTTGATTTC

[0108] This invention provides a nucleotide sequence (SEQ ID NO:63) for editing the A29L-1A2 light chain.

[0109] GAAATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGGTCCAACAATAAGAATTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTGAGCTGCTCATTTACTGGG CATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTATTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA

[0110] This invention provides a nucleotide sequence (SEQ ID NO:64) for editing the A29L-1A2 heavy chain.

[0111] CAGGTGCAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTACTGGTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGT GGTATAAGGATTATGCAGTGTCTGTGAAAAGTCGAATAATCATAAATCCAGACACATCCAAAAACCAGTTCTCCCTGCACCTGAACTCTGTGGCTCCCGAGGACACGGGCTGTGTATTACTGTGCAAGCGGTGCCTTTGATTTCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA

[0112] 3. Antibody expression and purification

[0113] The heavy chain variable region gene (V) of the antibody H ) and IgG1 constant region gene (C H The antibody's light chain variable region gene (V) was fused into the pCAGGS vector (named pCAGGS-Heavy chain-full length) and constructed. L ) and constant region genes (C L The plasmid was fused into the pCAGGS vector (named pCAGGS-Light chain-full length). For transfection of 293F cells, 36 μg of heavy chain plasmid and 75 μg of light chain plasmid were dissolved in 1 mL of HBS for every 100 mL of cells and allowed to stand for 5 min. 300 μg of PEI was dissolved in 1 mL of HBS and allowed to stand for 5 min. The plasmid and PEI were mixed, allowed to stand for 20 min, and then added to the cells. The cells were then incubated at 37°C in a shaker.

[0114] Five days later, centrifuge at 8000 rpm for 90 min to obtain the supernatant. Pass water through the Protein A column using a peristaltic pump at a rate not exceeding 2 mL / min, for at least 15 mL. Then pass binding buffer A (20 mM Na3PO4, pH 7.0) for at least 15 mL. Finally, resuspend the column in the supernatant.

[0115] Protein A column was attached to an AKTA Purifier / Explorer / FPLC column. The column was washed with binding buffer A until the absorbance at 280 nm no longer changed. Elution was then performed with elution buffer B (0.1 M Glycine, pH 3.0). 1 / 10 volume of buffer C (1 M Tris, pH 9.0) was added to the collection tube beforehand. The target protein was collected and concentrated, then the solution was changed to PBS for later use. After SDS-PAGE analysis, the concentrated protein with acceptable purity was used for the next stage of experiments.

[0116] like Figure 2 As shown, the SDS-PAGE results of the purified Protein A antibody indicate that the antibody protein can be expressed. In non-reduced SDS-PAGE, the molecular weight is ~150kDa, while in reduced SDS-PAGE, the heavy chain is ~50kDa and the light chain is ~25kDa.

[0117] Example 2: ELISA detection of A29L antigen and antibody binding activity

[0118] The purified antibody was further tested for its binding activity with the antigen using ELISA. A29L was diluted to 4 μg / mL with ELISA coating buffer and added to 3690 plates at a rate of 50 μL / well to coat the microplates. The plates were incubated overnight at 4°C. Blocking was performed for 1 hour at room temperature with PBST solution containing 5% milk powder. The microplates were washed once with PBST solution.

[0119] Dilute A29L with PBST at a ratio of antibody molar to coating antigen molar of 3:1 (100 μL), then perform serial dilutions fourfold. Add 100 μL of the serially diluted antibody to each well and incubate at room temperature for 1 hour. Wash three times with PBST. Dilute HRP-labeled anti-human secondary antibody 4000-fold with PBST, add 100 μL to each well, and incubate at 37°C for 40 minutes. Wash five times with PBST, add 100 μL of TMB for color development, and react at room temperature for 10 minutes. Finally, add 100 μL of 2M HCl to each well to terminate the enzyme reaction. Read the absorbance (at 450 nm) using a microplate reader.

[0120] like Figure 3 As shown in the figure, the ELISA results of antibody-antigen binding activity were obtained. All four antibodies could bind to the antigen. As the antibody concentration increased, the binding activity increased and the OD450 value increased. However, different antibodies showed different binding activities, and there were significant differences in binding activities among the antibodies. Among them, 1A2 had the strongest binding activity at low concentrations.

[0121] Example 3: Antigen and Antibody Neutralization Test

[0122] The neutralizing activity of the antibodies was detected using the plaque assay, as briefly described below: The initial concentration of the antibodies (A29L-1A2, A29L-1A5, A29L-1B2, A29L-1B3, and the control) was 400 μg / mL, serially diluted 3-fold to create 10 gradients. 400 μL of each gradient was mixed with 400 μL of vaccinia virus diluent (containing 10% complement) and incubated at 37°C for 1 hour. A 12-well plate pre-inoculated with Vero cells (at approximately 100% confluence) was removed, and the cells were washed twice. 400 μL of the antibody-virus mixture was added to each well, with two replicates for each gradient. A control well containing only virus and no antibody was also included. The plates were incubated at 37°C for 1 hour. The antibody-virus mixture was discarded, and the cells were washed once. 1 mL of fixative was added per well, prepared by mixing methylcellulose and high-glucose medium (2×) at a 1:1 volume ratio. The cell culture plates were then placed in a cell culture incubator. After 48 hours, the cells were fixed with 8% fixative, then stained with crystal violet, counted, and the half-maximal inhibitory concentration (IC50) was calculated.

[0123] like Figure 4As shown, the neutralization curves of A29L antibodies against VACV-WR show that the IC50 values ​​of A29L-1A2, A29L-1A5, A29L-1B2, and A29L-1B3 are 0.026, 0.085, 0.029, and 0.0411, respectively.

[0124] Example 4: Detection of antibody-antigen affinity using surface plasmon resonance technology

[0125] Surface plasmon response (SPR) assays can be used to detect interactions between proteins in vitro. The kinetic parameters of protein binding are determined by detecting changes in the response value as the mobile phase flows over the chip surface. This was performed on a BIACORE 8K instrument. In SPR assays, one of the two proteins to be analyzed is typically immobilized on a metal chip surface, while the other is used as the mobile phase flowing over the chip surface, and the resulting change in response value is detected. Before the experiment, PBST should be filtered through a 0.22 μM filter and then autoclaved. The antibody concentration was 10 μg / mL (adjustable as needed), and the antigen titers were 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM (adjustable as needed). The characteristics of the A29L antigen-antibody affinity assay are shown in Table 1 and [Table data missing]. Figure 5 .

[0126] Table 1. Characteristics of A29L antigen-antibody affinity assay

[0127]

[0128] Example 5: Animal Protection Experiment

[0129] To further evaluate the suitability of the antibody, the antibody with the highest neutralizing activity, A29-1A2, was selected, and its protective effect was assessed in mice through multiple administrations. Since animal experiments involving monkeypox virus infection require a high-level biosafety level 3 laboratory, the vaccinia virus strain VAV-WR (belonging to the same family as monkeypoxvirus) was initially used for evaluation. 7-8 week old mice were intranasally infected with 5LD50 VAV-WR, and 200 μg / mouse was intraperitoneally injected with the antibody at 4 hours before infection and at 4, 24, 48, 72, and 96 hours after infection.

[0130] like Figure 6 As shown, after viral infection, the body weight of mice in both the irrelevant antibody group and the A29-1A2 group decreased by more than 20% on day 7.

[0131] In summary, this invention, through screening humanized mouse memory B cells specifically binding to A29L with fully human antibodies, obtained four novel humanized MPXV antibodies (A29L-1B3, 1B2, 1A5, and 1A2) with high neutralizing activity. These antibodies have sequences completely different from previously reported monkeypox virus antibodies, exhibiting strong antigen-binding ability and potent neutralizing VCV activity. Furthermore, they can protect mice from lethal doses of VCV-WR. These results suggest that these four humanized antibodies have clinical value in the treatment and prevention of monkeypox virus.

[0132] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A human monoclonal antibody A29L-1B3 that neutralizes monkeypox virus, characterized in that, The amino acid sequence of the light chain CDR1 of the human monoclonal antibody A29L-1B3 that neutralizes monkeypox virus is shown in SEQ ID NO:1, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:

3. The amino acid sequence of the heavy chain CDR1 of the human monoclonal antibody A29L-1B3 that neutralizes monkeypox virus is shown in SEQ ID NO:4, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO:5, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO:

6.

2. The human monoclonal antibody A29L-1B3 for neutralizing monkeypox virus as described in claim 1, characterized in that, The light chain amino acid sequence of the human monoclonal antibody A29L-1B3 that neutralizes monkeypox virus is shown in SEQ ID NO:7, and the heavy chain amino acid sequence of the human monoclonal antibody A29L-1B3 that neutralizes monkeypox virus is shown in SEQ ID NO:

8.

3. The use of the human monoclonal antibody A29L-1B3 for neutralizing monkeypox virus as described in any one of claims 1-2 in the preparation of products for detecting monkeypox virus.

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