A monoclonal neutralizing 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 E8L specific binding memory B cell, the human source high neutralization activity monkeypox virus antibody E8L-7H4, E8L-7H2, E8L-7E7, E8L-8D6 and E8L-7C9 obtained, with the characteristics of strong monkeypox virus binding capacity, good in vitro neutralization, affinity, with in vivo protection, completely different from the reported monkeypox virus antibody sequence, provide the product for the detection and neutralization of monkeypox virus, provide the possibility for the monkeypox virus product with in vivo protection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody engineering, in particular to a human monoclonal antibody against monkeypox virus and its application. BACKGROUND

[0002] Monkeypox virus (MPXV) belongs to Poxviridae, Chordopoxvirinae, Orthopoxvirus (OPXV). There are more than 10 members in Orthopoxvirus, including smallpox virus (VARV), cowpox (smallpox vaccine) virus (VACV), rinderpest virus (CPXV), camel pox virus (CMLV), and several new species isolated from infected humans or primates since 2010. According to the comparison between VACV and MPXV, the surface proteins between poxviruses show about 93-98% sequence similarity, suggesting that they play similar roles in the process of entering the body.

[0003] In 1958, monkeypox virus was first discovered in a group of monkeys used for research, and was named "Monkeypoxvirus (MPXV)". In 1970, a 9-month-old male infant was diagnosed as the first human case, indicating that monkeypox virus can infect humans.

[0004] Monkeypox is a zoonosis, and its natural host is not clear, mainly in rodents and non-human primates. It can be transmitted through various forms such as contact with damaged skin tissue. In addition, the recent spread of monkeypox virus in non-endemic countries shows a phenomenon related to sexual contact, and monkeypox virus has been found in body fluids such as semen, but whether it is transmitted through sexual contact is not clear. After infection, symptoms such as lymph node enlargement, muscle soreness, and permanent scarring after pustule rupture may occur.

[0005] Since the eradication of smallpox in 1980 and the subsequent cessation of smallpox vaccination in humans, the immune ability of the younger population to orthopoxviruses is low, which may be one of the reasons for the current MPOX outbreak. With the spread of various monkeypox virus strains in non-endemic countries, not only are the transmission routes diverse and hidden, the symptoms of infection are atypical, but the monkeypox virus also accelerates variation in interpersonal transmission, with a mutation rate far exceeding expectations, posing an increasingly greater threat to global public health security. Although there are not many cases of monkeypox in China at present, with population flow at home and abroad, how to prevent and treat monkeypox infection has become a key problem that needs to be solved urgently. And there is 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.

[0006] To date, neutralizing antibodies have proven to be an effective method for treating viral diseases, including human immunodeficiency virus (HIV), influenza virus, and other flaviviruses. MPXV exists in two forms of extracellular enveloped virus (EEV) and intracellular mature virus (IMV) particles. Among them, A29L and E8L proteins are the envelope proteins on the surface of IMV, which play a key role in the process of viral infection.

[0007] Three monoclonal antibodies recognizing different epitopes of MPXV A29L, 9F8, 3A1 and 2D1, have been successfully prepared, which all prove to have strong and broad binding and neutralizing activity to orthopoxviruses, and the neutralizing activity of 9F8 is the best, and it also has a synergistic protective effect on orthopoxviruses. Several human anti-D8 antibodies have been characterized for their binding properties, and the crystal structures of three VACV-mAb variants VACV-66, VACV-138 and VACV304 that bind to D8, respectively, have been resolved.

[0008] It is very important and necessary to develop new MPXV neutralizing antibodies that are specific, have strong antigen binding ability, good in vitro neutralizing activity, high affinity, and have in vivo protective effect. SUMMARY

[0009] Problems to be solved by the invention:

[0010] In view of the problems existing in the prior art, the humanized monoclonal antibodies E8L-7H2 and E8L-8D6 that are specific, have strong MPXV binding ability, good in vitro neutralizing activity, high affinity, and have in vivo protective effect, are finally screened out by screening humanized mouse anti-E8L and A29L specific binding memory B cells, obtaining human high neutralizing activity MPXV antibodies E8L-7H4, E8L-7H2, E8L-7E7, E8L-8D6 and E8L-7C9, A29L-1B3, A29L-1B2, A29L-1A5 and A29L-1A2.

[0011] To achieve the above-mentioned purpose, the specific technical solutions of the present application are as follows:

[0012] I. Preparation and purification of monkeypox virus E8L monoclonal antibody

[0013] Through expression and purification of monkeypox virus E8L, sorting of specific memory B cells combined with E8L protein, expression and purification of monkeypox virus E8L antibody, five kinds of monkeypox virus E8L monoclonal antibodies are obtained, which are E8L-7H2, E8L-7H4, E8L-7E7, E8L-8D6 and E8L-7C9; the amino acid sequences of the light chain and the heavy chain of the monkeypox virus monoclonal antibody, the light chain variable region CDR1-3 and the heavy chain variable region CDR1-3 are as follows, the light chain variable region and the heavy chain variable region are different from the existing sequences, and have specificity.

[0014] II. Performance verification of monoclonal antibody

[0015] (I) ELISA detects the binding activity of E8L antigen and antibody E8L-7H2

[0016] The five antibodies E8L-7H2, E8L-7H4, E8L-7E7, E8L-8D6 and E8L-7C9 can all bind to the antigen, and the binding activity is enhanced with the increase of the antibody concentration, and the OD450 value increases.

[0017] (II) Antigen and antibody neutralization test

[0018] The neutralization activity of the antibody is detected by the plaque method, and the neutralization curve result of the E8L antibody to VACV-WR is obtained, and the IC50 of E8L-7C9, E8L-7E7, E8L-8D6, E8L-7H2 and E8L-7H4 is 0.078, 0.060, 0.085, 0.075 and 0.102 μg / mL respectively.

[0019] (III) Surface plasmon resonance technology detects the affinity of antibody and antigen

[0020] The five antibodies E8L-7H2, E8L-7H4, E8L-7E7, E8L-8D6 and E8L-7C9 have good affinity to the antigen.

[0021] (IV) Animal protection test

[0022] E8L-7H2 and E8L-8D6 have a protection rate of 100% and 80% for monkeypox virus infection, and multiple administrations of E8L-7H2 or E8L-8D6 antibody can provide protection for monkeypox virus infected mice under low-dose virus infection.

[0023] Compared with the prior art, the beneficial effects of the present application are:

[0024] The humanized monoclonal antibody of the application has strong binding ability with monkeypox virus, good in-vitro neutralization, large affinity, and in-vivo protection effect, is completely different from the reported monkeypox virus antibody sequence, and provides a product for detection and neutralization of monkeypox virus, and provides a possibility for providing a monkeypox virus product with in-vivo protection effect. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 , monkeypox virus E8L protein purification molecular sieve SDS-PAGE results;

[0026] Figure 2 , antibody SDS-PAGE results after Protein A purification;

[0027] Figure 3 , ELISA detection antibody and antigen binding activity results;

[0028] Figure 4 , E8L antibody neutralization curve results of VACV-WR;

[0029] Figure 5 , E8L antibody and E8L kinetic curve results;

[0030] Figure 6 , experimental schematic diagram and mouse survival and body weight change curve after low-dose infection, wherein,

[0031] A is an experimental implementation schematic diagram,

[0032] B is a mouse survival and body weight change curve after low-dose infection. DETAILED DESCRIPTION

[0033] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art.

[0034] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0035] All materials, reagents, etc. in the following examples can be obtained from commercial channels unless otherwise specified.

[0036] Experimental materials

[0037] 1. Key drugs and reagents

[0038] Name Model Manufacturer Fully human antibody humanized mice RenMab BioScape Biotechnologies Co., Ltd. 14K chip 750-00021 Berkeley lights

[0039] 2. Main instruments

[0040] Instrument name Model Manufacturer Beacon sorter Beacon Berkeley Lights

[0041] Example 1: Preparation and purification of monkeypox virus monoclonal antibody

[0042] 1. Expression and purification of monkeypox virus E8L protein

[0043] The E8L gene was constructed and transfected into the eukaryotic expression vector pCAGGS into 293F cells. The E8L fusion His tag was expressed in the cell supernatant, and the cells were initially purified by nickel affinity chromatography, followed by further purification by molecular sieve and identification by SDS-PAGE.

[0044] like Figure 1 As shown, the SDS-PAGE results of monkeypox virus E8L protein purification were obtained. When the protein was passed through a Superdex 200PG, a UV 280 nM absorption peak was detected at a position of ~82 mL. The molecular weight of the protein in the SDS-PAGE was approximately ~40 kDa, and the obtained protein had high purity.

[0045] 2. Sorting of specific memory B cells that bind to E8L protein

[0046] The E8L gene was constructed into an mRNA vector, transcribed in vitro, and capped. Concentration and quality were then determined. 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.

[0047] Five monkeypox viruses, E8L-7H2, E8L-7H4, E8L-7E7, E8L-8D6, and E8L-7C9, were obtained as monoclonal antibodies. The amino acid sequences of the light and heavy chains, and the CDR1-3 variable regions of the light and heavy chains of these five monkeypox virus monoclonal antibodies are as follows. The variable regions of the light and heavy chains are different from existing sequences, demonstrating specificity.

[0048] The amino acid sequence information of the three CDR regions of the E8L-7H2 light chain is as follows:

[0049] E8L-7H2 light chain Sequence ID Amino acid sequence CDR1 SEQ ID NO: 1 QDISSY CDR2 SEQ ID NO: 2 DAS CDR3 SEQ ID NO: 3 QQFDNLPLT

[0050] The amino acid sequence information of the three CDR regions of the E8L-7H2 heavy chain is as follows:

[0051]

[0052]

[0053] Amino acid sequence of E8L-7H2 light chain (SEQ ID NO: 7)

[0054] EIVMTQSPSSLSASVGDRVTITCRASQDISSYLNWFQQKPGKAPNLLIYDASNLKTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQFDNLPLTFGGGTKVEIK

[0055] Amino acid sequence of E8L-7H2 heavy chain (SEQ ID NO: 8)

[0056] QVQLVQSGGGVVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSLISGDGGNTYYADSVRGRFTISRDNSKNSLYLKMNSLRTEDTALYYCAKAELFLTGWGQGTLVTVSS

[0057] The present application provides edited nucleotide sequences of the three CDR regions of the E8L-7H2 light chain, as follows:

[0058] E8L-7H2 light chain Sequence ID Nucleotide sequence CDR1 SEQ ID NO: 9 CAGGACATTAGCAGCTAT CDR2 SEQ ID NO: 10 GATGCATCC CDR3 SEQ ID NO: 11 CAACAGTTTGATAATCTCCCGCTCACT

[0059] The present application provides edited nucleotide sequences of the three CDR regions of the E8L-7H2 heavy chain, as follows:

[0060] E8L-7H2 heavy chain Sequence ID Nucleotide sequence CDR1 SEQ ID NO: 12 GGATTCACCTTTGATGATTATGCC CDR2 SEQ ID NO: 13 ATTAGTGGGGATGGTGGTAACACA CDR3 SEQ ID NO: 14 GCAAAAGCGGAACTTTTTTTGACAGGC

[0061] The present application provides edited nucleotide sequences of the E8L-7H2 light chain (SEQ ID NO: 15)

[0062] GAAATTGTGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGACATTAGCAGCTATTTAAATTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTACGATGCATCCAATTTGAAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTTTGATAATCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAAATCAAA

[0063] The present application provides a nucleotide sequence of E8L-7H2 heavy chain (SEQ ID NO: 16)

[0064] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTACAACCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGTCAAGCTCCAGGGAAGGGTCTGGAGTGGGTCTCTCTTATTAGTGGGGATGGTGGTAACACATACTATGCAGACTCTGTGAGGGGCCGATTCACCATCTCCAGAGACAACAGCAAAAACTCCCTGTATCTGAAAATGAACAGTCTGAGAACTGAGGACACCGCCTTGTATTACTGTGCAAAAGCGGAACTTTTTTTGACAGGCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0065] The amino acid sequence information of the three CDR regions of the E8L-7H4 light chain is as follows:

[0066] E8L-7H4 light chain Sequence ID Amino acid sequence CDR1 SEQ ID NO: 17 QGIRND CDR2 SEQ ID NO: 18 AAS CDR3 SEQ ID NO: 19 LQDFSYPWT

[0067] The amino acid sequence information of the three CDR regions of the E8L-7H4 heavy chain is as follows:

[0068] E8L-7H4 heavy chain Sequence ID Amino acid sequence CDR1 SEQ ID NO: 20 GYSFTYYW CDR2 SEQ ID NO: 21 IYPGDSDT CDR3 SEQ ID NO: 22 ARHEYSGSSGVYYHGLDV

[0069] The amino acid sequence of the E8L-7H4 light chain (SEQ ID NO: 23)

[0070] EIVMTQSPSSLSASIGDRVTITCRASQGIRNDLGWYQQKPGKTPKLLIYAASSLQSGVPSRFSGSGSGTDFILAISSLQPEDFATYYCLQDFSYPWTFGQGTKVEIK

[0071] The amino acid sequence of the E8L-7H4 heavy chain (SEQ ID NO: 24)

[0072] QVQLVQSGAEVKKPGESLKISCKGSGYSFTYYWIGWVRQMPGKGLEWMGSIYPGDSDTRYSPSFQGQVTISADKSVSTAYLQWSSLEASDTAMYYCARHEYSGSSGVYYHGLDVWGQGTTVTVSS

[0073] The present application provides editing the nucleotide sequence of the three CDR regions of the E8L-7H4 light chain as follows:

[0074] E8L-7H4 light chain Sequence ID Nucleotide sequence CDR1 SEQ ID NO: 25 CAGGGCATTAGAAATGAT CDR2 SEQ ID NO: 26 GCTGCATCC CDR3 SEQ ID NO: 27 CTACAAGATTTCAGTTACCCGTGGACG

[0075] The present application provides editing the nucleotide sequence of the three CDR regions of the E8L-7H4 heavy chain as follows:

[0076]

[0077] The present application provides editing the nucleotide sequence of the E8L-7H4 light chain (SEQ ID NO: 31)

[0078] GAAATTGTGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTATAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAACCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTACAAAGTGGGGTCCCTTCAAGGTTCAGCGGCAGTGGATCTGGCACAGATTTCATTCTCGCCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTATTGTCTACAAGATTTCAGTTACCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0079] The present application provides editing the nucleotide sequence of the E8L-7H4 heavy chain (SEQ ID NO: 32)

[0080] CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCTACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGAAGCATCTATCCGGGTGACTCTGATACCAGATACAGTCCGTCCTTCCAAGGCCAGGTCACCATTTCAGCCGACAAGTCCGTCTCCACCGCCTACCTGCAGTGGAGCAGCCTGGAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACATGAATATAGTGGTTCGTCCGGGGTCTACTACCACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0081] The amino acid sequence information of the three CDR regions of the light chain of E8L-7E7 is as follows:

[0082] E8L-7E7 light chain Sequence ID Amino acid sequence CDR1 SEQ ID NO: 33 QSIRSW CDR2 SEQ ID NO: 34 KAS CDR3 SEQ ID NO: 35 QQYNSYSPWT

[0083] The amino acid sequence information of the three CDR regions of the heavy chain of E8L-7E7 is as follows:

[0084] E8L-7E7 heavy chain Sequence ID Amino acid sequence CDR1 SEQ ID NO: 36 GFTFSSYD CDR2 SEQ ID NO: 37 ISGSGGST CDR3 SEQ ID NO: 38 AKEGNWNYFDY

[0085] The amino acid sequence of the light chain of E8L-7E7 (SEQ ID NO: 39)

[0086] EIVMTQSPSTLSASVGDRVTITCRASQSIRSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSPWTFGQGTKVEIK

[0087] The amino acid sequence of the heavy chain of E8L-7E7 (SEQ ID NO: 40)

[0088] QVQVVQSGGGLVQPGESLRLSCAASGFTFSSYDMTWVRQAPGKGLEWVSVISGSGGSTYYADSVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCAKEGNWNYFDYWGQGTLVTVSS

[0089] The present application provides editing the nucleotide sequence of the three CDR regions of the E8L-7E7 light chain, as follows:

[0090] E8L-7E7 light chain Sequence ID Nucleotide sequence CDR1 SEQ ID NO: 41 CAGAGTATTCGTAGCTGG CDR2 SEQ ID NO: 42 GCTGCATCC CDR3 SEQ ID NO: 43 CTACAAGATTTCAGTTACCCGTGGACG SEQ ID NO:42 AAGGCGTCT CDR3 SEQ ID NO:43 CAACAATATAATAGTTATTCTCCGTGGACG

[0091] The present application provides editing the nucleotide sequence of the three CDR regions of the E8L-7E7 heavy chain, as follows:

[0092] E8L-7E7 heavy chain SEQ ID Nucleotide sequence CDR1 SEQ ID NO:44 GGATTCACCTTTAGCAGCTATGAC CDR2 SEQ ID NO:45 ATTAGTGGTAGTGGTGGTAGCACA CDR3 SEQ ID NO:46 GCGAAAGAGGGGAACTGGAACTACTTTGACTAC

[0093] The present application provides editing the nucleotide sequence of the E8L-7E7 light chain (SEQ ID NO: 47)

[0094] GAAATCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTCGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAATATAATAGTTATTCTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0095] The present application provides editing the nucleotide sequence of the E8L-7E7 heavy chain (SEQ ID NO: 48)

[0096] CAGGTCCAGGTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGGAGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGACATGACCTGGGTCCGCCAGGCTCCGGGGAAGGGGCTGGAGTGGGTCTCAGTTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGGGAACTGGAACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0097] The amino acid sequence information of the three CDR regions of the light chain of E8L-8D6 is as follows:

[0098] E8L-8D6 light chain SEQ ID Amino acid sequence CDR1 SEQ ID NO:49 QSLLHSNGYNY CDR2 SEQ ID NO:50 LVS CDR3 SEQ ID NO:51 MQALHTPLT

[0099] The amino acid sequence information of the three CDR regions of the heavy chain of E8L-8D6 is as follows:

[0100] E8L-8D6 heavy chain SEQ ID Amino acid sequence CDR1 SEQ ID NO:52 GGSISSYY CDR2 SEQ ID NO:53 IFYSGRT CDR3 SEQ ID NO:54 ARQDVDEVATI

[0101] The amino acid sequence of the light chain of E8L-8D6 (SEQ ID NO: 55)

[0102] EIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLVSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALHTPLTFGGGTKVEIK

[0103] The amino acid sequence of the heavy chain of E8L-8D6 (SEQ ID NO: 56)

[0104] QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIFYSGRTKYNPSLKSRVTISVDTSKKQFSLRLNSVTAADTAVYYCARQDVDEVATIWGQGTLVTVSS

[0105] The present application provides the nucleotide sequence of editing the three CDR regions of the light chain of E8L-8D6, as follows:

[0106] E8L-8D6 light chain SEQ ID Nucleotide sequence CDR1 SEQ ID NO:57 CAGAGCCTCCTCCATAGTAATGGATACAACTAT CDR2 SEQ ID NO:58 TTGGTTTCT CDR3 SEQ ID NO:59 ATGCAAGCTCTACATACTCCGCTCACT

[0107] The present application provides editing the nucleotide sequence of the CDR regions of the heavy chain of E8L-8D6, as follows:

[0108] E8L-8D6 heavy chain SEQ ID Nucleotide sequence CDR1 SEQ ID NO:60 GGTGGCTCCATCAGTAGTTACTAC CDR2 SEQ ID NO:61 ATATTTTACAGTGGGAGAACC CDR3 SEQ ID NO:62 GCGAGACAGGACGTGGATGAAGTGGCTACGATC

[0109] The present application provides editing the nucleotide sequence of the light chain of E8L-8D6 (SEQ ID NO: 63)

[0110] GAAATCGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTCCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCTGGGCAGTCTCCACAGCTCCTGATCTATTTGGTTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTTAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACATACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAAATCAAA

[0111] The present application provides editing the nucleotide sequence of the heavy chain of E8L-8D6 (SEQ ID NO: 64)

[0112] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATATTTTACAGTGGGAGAACCAAGTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCGGTAGACACGTCCAAGAAGCAGTTCTCCCTGAGGCTGAACTCTGTGACCGCCGCAGACACGGCCGTCTATTACTGTGCGAGACAGGACGTGGATGAAGTGGCTACGATCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0113] The amino acid sequence information of the three CDR regions of the light chain of E8L-7C9 is as follows:

[0114] E8L-7C9 light chain SEQ ID Amino acid sequence CDR1 SEQ ID NO:65 QSVISN CDR2 SEQ ID NO:66 GAS CDR3 SEQ ID NO:67 QQYNRWPRT

[0115] The amino acid sequence information of the three CDR regions of the heavy chain of E8L-7C9 is as follows:

[0116] E8L-7C9 heavy chain SEQ ID Amino acid sequence CDR1 SEQ ID NO:68 GGSFRDYY CDR2 SEQ ID NO:69 INHSGRT CDR3 SEQ ID NO:70 ARGGFLEWLFYFDY

[0117] The amino acid sequence of the light chain of E8L-7C9 (SEQ ID NO: 71)

[0118] EIVMTQSPATLSVSPGERATLSCRASQSVISNLAWYQQKPGQTPRLLIYGASTRATGIPARFSGSGSLTEFTLNISSLQSEDFAVYYCQQYNRWPRTFGPGTKVEIK

[0119] The amino acid sequence of the heavy chain of E8L-7C9 (SEQ ID NO: 72)

[0120] QVQLQQWGAGLLKPSETLSLTCAVYGGSFRDYYWNWIRQSPGKGLEWIGEINHSGRTNYNPSLKSRVIISVDTSKNQFSLKLSSVTAADTAMYYCARGGFLEWLFYFDYWGQGTLVTVSS

[0121] The present application provides the nucleotide sequence of the three CDR regions of the light chain of E8L-7C9, as follows:

[0122]

[0123]

[0124] The present application provides the nucleotide sequence of the three CDR regions of the heavy chain of E8L-7C9, as follows:

[0125] The present application provides the nucleotide sequence of the light chain of E8L-7C9 (SEQ ID NO: 79)

[0126] GAAATTGTGATGACCCAGTCTCCAGCCACCCTGTCTGTATCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTATCAGCAACTTAGCCTGGTATCAACAGAAACCTGGCCAGACTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTCTGACAGAATTCACTCTCAACATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAGGTGGCCTCGGACGTTCGGCCCAGGGACCAAGGTGGAAATCAAA

[0127] The present disclosure provides a nucleotide sequence of an edited E8L-7C9 heavy chain (SEQ ID NO: 80)

[0128] CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCCGTGATTACTACTGGAACTGGATCCGCCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGAACCAACTACAACCCGTCCCTCAAGAGTCGAGTCATCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTATGTATTACTGTGCGAGAGGGGGATTTTTGGAATGGTTATTCTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0129] 3. Expression and purification of the antibody

[0130] The variable region gene (V H ) of the heavy chain of the antibody was fused with the IgG1 constant region gene (C H ) to construct into the pCAGGS vector (named as pCAGGS-Heavy chain-full length), and the variable region gene (V L ) of the light chain of the antibody was fused with the constant region gene (C L) Fusion construct into pCAGGS vector (named pCAGGS-Light chain-full length). When transfect 293F cells, take 36 μg heavy chain plasmid and 75 μg light chain plasmid, dissolve in 1 mL HBS, stand for 5 min. Take 300 μg PEI, dissolve in 1 mL HBS, stand for 5 min. Mix plasmid and PEI, stand for 20 min, add to cells. Incubate at 37°C on a shaker.

[0131] After 5 days, centrifuge at 8000 r for 90 min to obtain supernatant. Connect Protein A column to AKTA Purifier / Explorer / FPLC, use binding buffer A (20 mM Na3PO4, pH 7.0) to wash column until the absorbance at 280 nm is no longer changed. Then use eluent B (0.1 M Glycine, pH 3.0) to elute, and add 1 / 10 volume of buffer C (1 M Tris, pH 9.0) in the collection tube in advance, collect the target protein and concentrate and change to PBS for use. After identification by SDS-PAGE, the qualified protein is concentrated for use in the next step.

[0132] After Protein A purification, the SDS-PAGE results of the antibody show that the antibody protein can be expressed, with a molecular weight of ~ 150 kDa in non-reducing SDS-PAGE, and a heavy chain of ~ 50 kDa and a light chain of ~ 25 kDa in reducing SDS-PAGE.

[0133] As shown in Figure 2 After Protein A purification, the SDS-PAGE results of the antibody show that the antibody protein can be expressed, with a molecular weight of ~ 150 kDa in non-reducing SDS-PAGE, and a heavy chain of ~ 50 kDa and a light chain of ~ 25 kDa in reducing SDS-PAGE.

[0134] Example 2, ELISA detection of E8L and A29L antigen and antibody E8L-7H2 binding activity

[0135] After purification, the antibody is further detected for its binding activity with antigen by ELISA. E8L and A29L are respectively diluted to 4 μg / mL with ELISA coating solution, and added to 3690 plates at an amount of 50 μL / well to coat the enzyme-labeled plate, and combined at 4°C overnight. Use PBST solution containing 5% milk powder to block at room temperature for 1 h. Wash the enzyme-labeled plate with PBST solution once.

[0136] Dilute A29L and E8L with PBST at a ratio of antibody molar to coating antigen of 3:1 per 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.

[0137] like Figure 3 As shown in the figure, the results of ELISA detection of antibody-antigen binding activity show that all nine antibodies can bind to the antigen. As the antibody concentration increases, the binding activity increases and the OD450 value increases. However, different antibodies show different binding activities, especially among the A29L antibodies, where there are significant differences in binding activity. Among them, 1A2 has the strongest binding activity at low concentrations.

[0138] Example 3: Antigen and Antibody Neutralization Test

[0139] The neutralizing activity of antibodies was detected using the plaque assay, as briefly described below: Antibodies (E8L-7C9, E8L-7E7, E8L-8D6, E8L-7H2, E8L-7H4, and control) were initially concentrated at 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 per gradient. A control well containing only virus and no antibody was 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.

[0140] like Figure 4 As shown, the neutralization curves of E8L antibodies against VACV-WR show that the IC50 values ​​of E8L-7C9, E8L-7E7, E8L-8D6, E8L-7H2, and E8L-7H4 are 0.078, 0.060, 0.085, 0.075, and 0.102 μg / mL, respectively.

[0141] Example 4: Detection of antibody-antigen affinity using surface plasmon resonance technology

[0142] Surface plasmon response (SPR) experiments can be used to detect the interaction between proteins in vitro, and the kinetic parameters of the binding of two proteins can be known by detecting the change of the response value of the mobile phase flowing through the chip surface. The experiment was performed on a BIACORE 8K instrument. In the surface plasmon resonance experiment, one of the two proteins to be analyzed is usually immobilized on the surface of the metal chip, and the other is used as the mobile phase flowing through the surface of the chip to detect the change of the response value caused thereby. Before the experiment, PBST should be filtered with a 0.22 μm filter and then autoclaved. The antibody concentration is 10 μg / mL (which can be adjusted appropriately), and the antigen concentration titers are 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM (which can be adjusted appropriately). The characteristics of the E8L antigen antibody affinity detection are shown in Table 1 and Figure 5 .

[0143] Table 1, Characteristics of E8L antigen antibody affinity detection

[0144]

[0145] Example 5, Animal protection test

[0146] In order to further evaluate the suitability of the antibodies, the antibodies with the highest neutralization activity, E8L-7H2, E8L-7H4, E8L-8D6 and A29-1A2, were selected for multiple dosing to evaluate the protective effect in mice. Since the monkeypox virus infection animal experiment needs to be carried out in a high-level biosafety level 3 laboratory, a preliminary evaluation was carried out using vaccinia virus (which belongs to the poxviridae family as monkeypox virus) VACV-WR strain. 7-8 week old mice were infected with 5 LD50 virus VACV-WR by intranasal instillation, and 200 ug of antibody was injected intraperitoneally at 4h before infection and 4h, 24h, 48h, 72h and 96h after infection, respectively.

[0147] As shown in Figure 6 , the body weight of the mice in the irrelevant antibody and A29-1A2 groups all decreased by more than 20% on the 7th day after virus infection. The body weight of the E8L antibody group decreased more slowly, and the body weight began to recover on the 9th day, with the protection rates of E8L-7H2 and E8L-8D6 being 100% and 80%, respectively. The above results show that multiple dosing of E8L-7H2 or E8L-8D6 antibodies can provide protection to mice under low-dose virus infection.

[0148] In summary, this invention, through screening humanized mouse memory B cells specifically binding to E8L with fully human antibodies, obtained four newly discovered humanized, highly neutralizing MPXV antibodies (E8L-7H4, 7H2, 7E7, 8D6, and 7C9). 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 five humanized antibodies have clinical value in the treatment and prevention of monkeypox virus.

[0149] 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 fall within the scope of protection claimed by the present invention.

Claims

1. A human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen, characterized in that, The amino acid sequence of the light chain CDR1 of the human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen is shown as SEQ ID NO: 1, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO: 2, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO: 3; The amino acid sequence of the heavy chain CDR1 of the human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen is shown as SEQ ID NO: 4, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO: 5, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO:

6.

2. The human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen of claim 1, characterized in that, The amino acid sequence of the light chain of the human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen is shown as SEQ ID NO: 7, and the amino acid sequence of the heavy chain of the human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen is shown as SEQ ID NO:

8.

3. The human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen of claim 1, characterized in that, The nucleotide sequence encoding the light chain CDR1 of the human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen is shown as SEQ ID NO: 9, the nucleotide sequence encoding the light chain CDR2 is shown as SEQ ID NO: 10, the nucleotide sequence encoding the light chain CDR3 is shown as SEQ ID NO: 11, the nucleotide sequence encoding the heavy chain CDR1 is shown as SEQ ID NO: 12, the nucleotide sequence encoding the heavy chain CDR2 is shown as SEQ ID NO: 13, and the nucleotide sequence encoding the heavy chain CDR3 is shown as SEQ ID NO:

14.

4. The human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen of claim 1, characterized in that, The nucleotide sequence of the edited light chain of the human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen is shown as SEQ ID NO: 15, and the nucleotide sequence of the edited heavy chain of the human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen is shown as SEQ ID NO:

16.

5. The use of the human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen according to any one of claims 1-4 in the preparation of a medicament for treating monkeypox virus.

6. The use of the human monoclonal antibody E8L-7H2 neutralizing monkeypox virus E8L antigen according to any one of claims 1-4 in the preparation of a product for detecting monkeypox virus.

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