Humanized monoclonal antibodies targeting monkeypox virus B6R protein and their applications

Abstract

This application relates to humanized monoclonal antibodies against monkeypox virus and their applications. By screening humanized mouse anti-B6R specific binding memory B cells with fully human antibodies, humanized high-neutralizing activity monkeypox virus antibodies B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1 were obtained. These antibodies have the characteristics of strong binding ability to monkeypox virus, good in vitro neutralization, high affinity, and in vivo protective effect.

Description

Technical Field

[0001] This application belongs to the field of antibody engineering, specifically relating to humanized monoclonal antibodies against monkeypox virus B6R protein and their applications. Background Technology

[0002] Monkeypox is caused by the monkeypox virus (MPXV) and is a zoonotic disease. It was first discovered in 1958 in a population of monkeys used in research and named "Monkeypox virus (MPXV)". In 1970, a nine-month-old boy in Zaire (now the Democratic Republic of Congo) was diagnosed with it, marking the first human case and demonstrating that monkeypox virus could infect humans. At that time, monkeypox outbreaks were mainly localized in countries in the tropical rainforest regions of Africa, with many cases reported in Central and West Africa. The mortality rates for the West African and Central African branches were 3.6% and 10.6%, respectively. Although monkeypox was primarily distributed in Central and West Africa for the past few decades, its spread has changed in recent years, with cases even appearing outside of Africa. The first case of monkeypox outside the African continent was reported in the United States in 2003, followed by reports in Israel, Nigeria, the United Kingdom, Singapore, and other countries. In particular, the global spread of monkeypox virus in 2022 has drawn significant public health attention. Amid the severe global monkeypox outbreak, my country faces a significant risk of imported cases.

[0003] Monkeypox can be transmitted 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 it is transmitted through sexual contact 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 B6R protein participates in the disruption of the glycosaminoglycan-mediated EEV outer membrane during EEV infection, thereby promoting the fusion of the inner membrane. Post-infection, EEV requires B6R to induce the formation of the actin tail, thereby accelerating the spread of uninfected cells; B6R plays a crucial role in viral infection. Antibodies targeting B6R primarily target VIG's EEV neutralizing ability, indicating that B6R has a prominent role as a target for the neutralizing activity of human antibodies against EEV.

[0006] Monoclonal antibodies recognizing different epitopes of B5 have been successfully prepared, including chimpanzee-human fusion MAb 8AH8AL, rat MAb19C2, and human MAb h101, which have been shown to provide protection in mouse models. Epitope localization studies have identified two major neutralizing sites on B5, located at SCR1 and SCR2, and at the stem of B5, respectively. However, precise information on these antibodies and other potential neutralizing epitopes on B5, as well as the structural characterization of B5, remains elusive. Several human anti-B5 antibodies have also been characterized for their binding properties, and the crystal structure of hMB668 bound to B5 has been resolved.

[0007] Developing new monkeypox virus antibodies with strong affinity for antigens and good in vitro neutralization properties is very important and necessary. Summary of the Invention

[0008] Based on this, one embodiment of this application provides a humanized monoclonal antibody with strong affinity and good in vitro neutralization properties and its application.

[0009] The technical solution includes:

[0010] One embodiment of this application provides a humanized monoclonal antibody against monkeypox virus, including a heavy chain variable region and a light chain variable region.

[0011] The CDR sequence of the heavy chain variable region and the CDR sequence of the light chain variable region are selected from any one of terms a to e;

[0012] a. HCDR1 with amino acid sequence as shown in SEQ ID NO:5, HCDR2 with amino acid sequence as shown in SEQ ID NO:6, HCDR3 with amino acid sequence as shown in SEQ ID NO:7; and LCDR1 with amino acid sequence as shown in SEQ ID NO:8, LCDR2 with amino acid sequence as shown in SEQ ID NO:9, and LCDR3 with amino acid sequence as shown in SEQ ID NO:10.

[0013] b. HCDR1 with amino acid sequence as shown in SEQ ID NO:21, HCDR2 with amino acid sequence as shown in SEQ ID NO:22, HCDR3 with amino acid sequence as shown in SEQ ID NO:23; and LCDR1 with amino acid sequence as shown in SEQ ID NO:24, LCDR2 with amino acid sequence as shown in SEQ ID NO:25, and LCDR3 with amino acid sequence as shown in SEQ ID NO:26.

[0014] c. HCDR1 with amino acid sequence as shown in SEQ ID NO:37, HCDR2 with amino acid sequence as shown in SEQ ID NO:38, HCDR3 with amino acid sequence as shown in SEQ ID NO:39; and LCDR1 with amino acid sequence as shown in SEQ ID NO:40, LCDR2 with amino acid sequence as shown in SEQ ID NO:41, and LCDR3 with amino acid sequence as shown in SEQ ID NO:42.

[0015] d. HCDR1 with amino acid sequence as shown in SEQ ID NO:53, HCDR2 with amino acid sequence as shown in SEQ ID NO:54, HCDR3 with amino acid sequence as shown in SEQ ID NO:55; and LCDR1 with amino acid sequence as shown in SEQ ID NO:56, LCDR2 with amino acid sequence as shown in SEQ ID NO:57, and LCDR3 with amino acid sequence as shown in SEQ ID NO:58.

[0016] e. HCDR1 with the amino acid sequence shown in SEQ ID NO:69, HCDR2 with the amino acid sequence shown in SEQ ID NO:70, HCDR3 with the amino acid sequence shown in SEQ ID NO:71; and LCDR1 with the amino acid sequence shown in SEQ ID NO:72, LCDR2 with the amino acid sequence shown in SEQ ID NO:73, and LCDR3 with the amino acid sequence shown in SEQ ID NO:74.

[0017] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.

[0018] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:18.

[0019] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:33, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:34.

[0020] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:50.

[0021] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:65, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:66.

[0022] In one embodiment, a heavy chain constant region and a light chain constant region are also included.

[0023] One embodiment of this application also provides a nucleic acid molecule including a nucleotide sequence encoding the humanized monoclonal antibody.

[0024] In one embodiment, the nucleotide sequence comprises a nucleotide sequence encoding the heavy chain variable region and a nucleotide sequence encoding the light chain variable region, the nucleotide sequences being selected from any one of the following groups:

[0025] (1) Heavy chain variable region as shown in SEQ ID NO:3 of nucleotide sequence, and light chain variable region as shown in SEQ ID NO:4 of nucleotide sequence;

[0026] (2) Heavy chain variable region as shown in SEQ ID NO:19 of nucleotide sequence, and light chain variable region as shown in SEQ ID NO:20 of nucleotide sequence;

[0027] (3) Heavy chain variable region as shown in SEQ ID NO:35 of nucleotide sequence, and light chain variable region as shown in SEQ ID NO:36 of nucleotide sequence;

[0028] (4) Heavy chain variable region as shown in SEQ ID NO:51 of nucleotide sequence, and light chain variable region as shown in SEQ ID NO:52 of nucleotide sequence;

[0029] (5) Heavy chain variable region as shown in SEQ ID NO:67 of nucleotide sequence, and light chain variable region as shown in SEQ ID NO:68 of nucleotide sequence.

[0030] An embodiment of this application also provides an expression vector comprising the nucleic acid molecule.

[0031] One embodiment of this application also provides a host cell comprising the nucleic acid molecule or the expression vector.

[0032] An embodiment of this application also provides a method for preparing the humanized monoclonal antibody, comprising the following steps:

[0033] The host cells are cultured, and the humanized monoclonal antibody is collected from the resulting cell culture.

[0034] One embodiment of this application also provides a detection reagent or detection kit, including the humanized monoclonal antibody.

[0035] One embodiment of this application also provides a drug comprising the humanized monoclonal antibody.

[0036] One embodiment of this application also provides the application of the humanized monoclonal antibody in the detection of monkeypox virus B6R protein for non-diagnostic purposes.

[0037] One embodiment of this application also provides the application of the humanized monoclonal antibody in the preparation of a detection product for monkeypox virus.

[0038] One embodiment of this application also provides the use of the humanized monoclonal antibody in the preparation of medicaments for the prevention or treatment of monkeypox virus and / or vaccinia virus.

[0039] Compared with traditional technologies, this application has the following advantages:

[0040] The applicant's bio-based monoclonal antibody exhibits strong binding ability to monkeypox virus, good in vitro neutralization, high affinity, and in vivo protective activity. Its sequence is completely different from previously reported monkeypox virus antibodies, providing a product for the detection and neutralization of monkeypox virus and making it possible to provide monkeypox virus products with in vivo protective activity. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 SDS-PAGE results of molecular sieve purification of monkeypox virus B6R protein;

[0043] Figure 2 The results show the kinetic curves of the B6R antibody and B6R.

[0044] Figure 3 The results show the neutralization curve of B6R antibody against VACV-WR.

[0045] Figure 4 The figures show a schematic diagram of the experiment and curves showing the survival and weight changes of mice after viral infection. The top figure is a schematic diagram of the experiment, and the bottom figure is a curve showing the survival and weight changes of mice after viral infection. Detailed Implementation

[0046] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided below. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0048] In this document, the term "and / or" includes any and all combinations of one or more of the related listed items.

[0049] This application obtained humanized MPXV antibodies B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1 with high neutralizing activity by screening humanized mouse memory B cells that specifically bind to B6R with fully human antibodies. Finally, new humanized monoclonal antibodies against monkeypox virus with specificity, strong binding ability to monkeypox virus, good in vitro neutralization, and high affinity were screened out.

[0050] The specific technical solutions include:

[0051] I. Preparation and purification of monkeypox virus B6R monoclonal antibody

[0052] Five monoclonal antibodies against monkeypox virus B6R were obtained through expression and purification of the B6R protein, sorting of specific memory B cells that bind to the B6R protein, and expression and purification of B6R antibodies against monkeypox virus. These antibodies are B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1. 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 monoclonal antibodies are as follows. The variable regions of the light and heavy chains are different from existing sequences and have specificity.

[0053] II. Performance Validation of Monoclonal Antibodies

[0054] 1. Antigen and antibody neutralization test

[0055] The neutralizing activity of the antibodies was detected by the plaque assay. The neutralization curves of B6R antibodies against VCV-WR showed that the PRNT50 values ​​of B6R-16A1, B6R-22D12, B6R-22F9, B6R-22H1, and B6-21G7 were 0.542 μg / mL, 0.291 μg / mL, 0.856 μg / mL, 0.245 μg / mL, and 8.07 μg / mL, respectively.

[0056] 2. Surface plasmon resonance technology for detecting antibody-antigen affinity

[0057] Five antibodies, B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1, have good affinity for the B6R antigen.

[0058] 4. ELISA detection of B6R antigen and antibody binding activity

[0059] Antibodies B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1 can all bind to antigens and have a strong ability to bind to antigens.

[0060] 5. Animal protection experiments

[0061] In cases of viral infection, administration of antibodies B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1, respectively, can provide protection against monkeypox virus infection in mice.

[0062] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0063] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0064] The main experimental materials involved in the following embodiments include, but are not limited to:

[0065] Key pharmaceuticals and reagents: fully humanized antibody mice, model RenMab, Biocytogen Pharmaceuticals, Inc.; 14K chip, model 750-00021, Berkeley Lights.

[0066] Main instrument: Beacon sorter, model Beacon, Berkeley Lights.

[0067] Example 1: Preparation and purification of monkeypox virus monoclonal antibody

[0068] 1. Expression and purification of monkeypox virus B6R protein

[0069] The B6R gene was constructed and transfected into the eukaryotic expression vector pCAGGS into 293F cells. The B6R protein was expressed as a secretory protein. The cell supernatant was collected and purified using affinity chromatography and molecular sieves, and then identified by SDS-PAGE.

[0070] Specifically, S19-H279 (SEQ ID NO:81) of B6R was extracted, and after gene sequence optimization, it was constructed into the vector pCAGGS.

[0071] STCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYS FGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYH

[0072] like Figure 1 As shown, the SDS-PAGE results of monkeypox virus B6R protein purification were obtained. The protein flowed through a Superdex 200GL, and a UV 280 nM absorption peak was detected at a position of ~13 mL. The molecular weight of the protein in the SDS-PAGE was approximately ~35 kDa, and the obtained protein had high purity.

[0073] 2. Sorting of specific memory B cells that bind to B6R protein

[0074] The B6R 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.

[0075] Five monoclonal antibodies against monkeypox virus B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1 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 five monoclonal antibodies are as follows. The variable regions of the light and heavy chains are different from existing sequences, demonstrating specificity.

[0076] B6R-16A1 monoclonal antibody:

[0077] Heavy chain variable region amino acid sequence, SEQ ID NO:1:

[0078] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKKQFSLKVSSVTAADTAVYYCARRDWNSRFDPWGQGTLVTVSS

[0079] Amino acid sequence of the light chain variable region, SEQ ID NO:2:

[0080] EIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLVWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK

[0081] Nucleic acid sequence of the heavy chain variable region, SEQ ID NO:3:

[0082] CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAAGCAGTTCTCCCTGAAGGTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAAGGGACTGGAACTCGCGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0083] Nucleic acid sequence of the light chain variable region, SEQ ID NO:4:

[0084] GAAATTGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTACTTAGTTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGG CATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGAAATCAAA

[0085] The amino acid sequence of the CDR region of the B6R-16A1 antibody is shown in Table 1; the nucleic acid sequence of the CDR region of the B6R-16A1 antibody is shown in Table 2.

[0086] Table 1

[0087]

[0088] Table 2

[0089]

[0090] B6R-21G7 monoclonal antibody:

[0091] Heavy chain variable region amino acid sequence, SEQ ID NO:17:

[0092] QVQLQQSGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISSVDTSKNQFSLKLISVTAADTAVYYCARHGSGRGYYYYVMDVWGQGTTVTVSS

[0093] The amino acid sequence of the light chain variable region, SEQ ID NO:18:

[0094] EIVMTQSPATLSLSPGERATLSCGASQSISSSYLAWYQQKPGLAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDSAVYYCQQYGSSPTFGGGTKVEIK

[0095] Heavy chain variable region nucleic acid sequence, SEQ ID NO:19:

[0096] CAGGTGCAGCTGCAGCAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAATGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCC TCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGATCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAGACATGGTTCGGGGAGGGGCTACTACTACGTTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0097] Light chain variable region nucleic acid sequence, SEQ ID NO:20:

[0098] GAAATTGTGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCGGGGCCAGTCAGAGTATTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCTGGCGCCCAGGCTCCTCATCTATGATGCATCCA GCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTCTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCCACTTTCGGCGGAGGGACCAAGGTGGAAATCAAA

[0099] The amino acid sequence of the CDR region of the B6R-21G7 antibody is shown in Table 3; the nucleic acid sequence of the CDR region of the B6R-21G7 antibody is shown in Table 4.

[0100] Table 3

[0101]

[0102] Table 4

[0103]

[0104] B6R-22D12 monoclonal antibody:

[0105] Heavy chain variable region amino acid sequence, SEQ ID NO:33:

[0106] The amino acid sequence of the light chain variable region, SEQ ID NO:34, is as follows:

[0107] EIVMTQSPDSLAVSLGERATINCKSSQHILYSSNNDNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPYTFGQGTKLEIK

[0108] Heavy chain variable region nucleic acid sequence, SEQ ID NO:35:

[0109] CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTAC AACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTTGACACGTCCAAGAAACAGTTCTCCCTGAAGCTGAACTCTGTGACCGCCGCGGACACGGCTGGTATAACTGTGCGAGAAAGGCGGGGGGCAGTGGCTGGTACGGCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0110] Light chain variable region nucleic acid sequence, SEQ ID NO:36:

[0111] GAAATTGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGCATATTTTATACAGCTCCAACAATGACAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGG CATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAACAATATTATACTACTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

[0112] The amino acid sequence of the CDR region of the B6R-22D12 antibody is shown in Table 5; the nucleic acid sequence of the CDR region of the B6R-22D12 antibody is shown in Table 6.

[0113] Table 5

[0114]

[0115] Table 6

[0116]

[0117] B6R-22F9 monoclonal antibody:

[0118] Heavy chain variable region amino acid sequence, SEQ ID NO:49:

[0119] The amino acid sequence of the light chain variable region, SEQ ID NO:50, is as follows:

[0120] EIVMTQSPDSLAVSLGERATINCKSSQTVLYNSNNENYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISTLQAEDVAVYYCQQYYTTPLTFGGGTKVEIK

[0121] Heavy chain variable region nucleic acid sequence, SEQ ID NO:51:

[0122] CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCTTTTAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTGCAACCCGTCCCTCAAGAGTCGAGTCGCCATGTCAGTAGACCCGCCCAGGAAACAGTTTTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAAAAGTGGCTGGTACTGGGTGGTTCTATACTTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCA

[0123] Nucleic acid sequence of the light chain variable region, SEQ ID NO: 52:

[0124] GAAATAGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGTCAGACTGTTTTATACAATTCCAACAATGAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCACCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAGTATTATACTACTCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAAATCAAA

[0125] Among them, the amino acid sequences of the CDR regions of the B6R-22F9 antibody are shown in Table 7; the nucleic acid sequences of the CDR regions of the B6R-22F9 antibody are shown in Table 8.

[0126] Table 7

[0127]

[0128] Table 8

[0129]

[0130] B6R-22H1 monoclonal antibody:

[0131] Heavy chain variable region amino acid sequence, SEQ ID NO:65:

[0132] The amino acid sequence of the light chain variable region, SEQ ID NO: 66, is as follows:

[0133] EIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLFIYWSSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYSIPWTFGQGTKVEIK

[0134] Heavy chain variable region nucleic acid sequence, SEQ ID NO:67:

[0135] CAGGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGTTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAACACCAACTAC AACCCGTCCCTCAAGAGTCGAGTCACCACATCAGTAGACACGTCCAAGAAGCAGTTCTCCCTGAACCTGAGCTCTGTGACCGCCGCGGAAACGGCTGTGTACTACTGTGCGAGACGCCCTGGTGGGAGCCCCTGGTTCGACCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0136] Light chain variable region nucleic acid sequence, SEQ ID NO:68:

[0137] GAAATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGTTATTCATTTACTGGT CATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTTCTGTCAACAATATTACAGTATTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0138] The amino acid sequence of the CDR region of the B6R-22H1 antibody is shown in Table 9; the nucleic acid sequence of the CDR region of the B6R-22H1 antibody is shown in Table 10.

[0139] Table 9

[0140]

[0141]

[0142] Table 10

[0143]

[0144] 3. Antibody expression and purification

[0145] 3.1 Antibody Cloning and Construction: The antibody heavy chain variable region gene (VH) and the IgG1 constant region gene (CH) were fused into the pCAGGS vector (named pCAGGS-Heavy chain-full length), and the antibody light chain variable region gene (VL) and the constant region gene (CL) were fused into the pCAGGS vector (named pCAGGS-Light chain-full length).

[0146] 3.2 Antibody Expression and Purification: When co-transfecting 100 mL of 293F cells with both light and heavy chain plasmids, 36 μg of heavy chain plasmid and 75 μg of light chain plasmid were dissolved in 1 mL of HBS 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 cultured at 37°C in a shaker. After 5 days, the supernatant was harvested after centrifugation at 8000 rpm for 90 min. The supernatant was passed through a Protein A affinity column and eluted with 0.1 M mg lycine at pH 3.0 using an AKTA Purifier to obtain the antibody. The antibody was then concentrated and the medium was changed to PBS. SD-PAGE was used to detect the purified antibody. The purity was high, with the heavy chain size after dissociation being approximately 50 kDa and the light chain size approximately 25 kDa.

[0147] Example 3: Detection of antibody-antigen affinity using surface plasmon resonance technology

[0148] 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 is 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 is 10 μg / mL (adjustable as needed), and the antigen titers are 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM (adjustable as needed). The characteristics of B6R antigen-antibody affinity detection are shown in Table 11. Figure 2 .

[0149] Table 11 Characteristics of B6R antigen-antibody affinity assay

[0150]

[0151] Example 4 Antibody Neutralization Test

[0152] The neutralizing activity of the antibody was detected using the plaque assay, as briefly described below: The initial antibody concentration was 200 μg / mL, serially diluted 4-fold to create 10 gradients. 400 μL of each gradient was mixed with an equal volume of vaccinia virus VACV-WR dilution buffer (containing 10% complement and 100 μg / mL targeting IMV antibody) and incubated at 37°C for 1 h. Vero cells were pre-seeded into 12-well plates; cells with ~100% confluence were suitable for the neutralization experiment. After washing the cells twice, 400 μL of the antibody-virus mixture was added to each well, with two replicate wells for each antibody gradient and a control well containing only virus and no antibody. The cells were incubated at 37°C for 1 h. The antibody-virus mixture was discarded, and the cells were washed once. 1 mL of fixative was added to each 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% tissue cell fixative, then stained with crystal violet, counted, and the neutralizing activity PRNT50 of the antibody was calculated.

[0153] like Figure 3 As shown, the neutralization curves of B6R antibodies against VACV-WR show that the PRNT50 values ​​of B6R-16A1, B6R-22D12, B6R-22F9, B6R-22H1, and B6R-21G7 are 0.542 μg / mL, 0.291 μg / mL, 0.856 μg / mL, 0.245 μg / mL, and 8.07 μg / mL, respectively.

[0154] Example 5: Animal Protection Experiment

[0155] To further evaluate antibody applicability, antibodies with high neutralizing activity—B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1—were selected and administered to mice to assess their protective efficacy. Because monkeypox virus infection experiments require a high-level biosafety level 3 laboratory, the Western Reserve (VACV-WR) strain of vaccinia virus (belonging to the Poxviridae family, the same family as monkeypox virus) was initially used for evaluation. Seven- to eight-week-old mice were intranasally infected with 5 LD50 and 10 LD50 VCV-WR, respectively. Four hours before and four hours after infection, 200 μg of antibody per mouse was administered intraperitoneally.

[0156] like Figure 4As shown, after infection with the 5LD50 virus, the body weight of all mice in the irrelevant antibody group decreased by more than 20% on day 6, while the body weight of mice injected with antibodies B6R-16A1, B6R-22D12, B6R-22F9, and B6R-22H1 showed no significant change. In the B6RR-21G7 antibody group, one mouse experienced a body weight decrease of more than 20% on day 7. After infection with the 10LD50 virus, the body weight of all mice in the irrelevant antibody group decreased by more than 20% on day 5, while the body weight of mice injected with antibodies B6R-16A1, B6R-22D12, and B6R-22F9 showed no significant change.

[0157] Table 12 shows the survival rates of mice after infection with different viruses and after antibody injection.

[0158] Table 12 Survival rates of mice after infection with different viruses and antibody injection.

[0159]

[0160] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0161] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A humanized monoclonal antibody targeting the monkeypox virus B6R protein, characterized in that, Includes variable regions of heavy chains and variable regions of light chains; The CDR sequences of the heavy chain variable region and the light chain variable region: The amino acid sequences are as shown in SEQ ID NO:5 for HCDR1, SEQ ID NO:6 for HCDR2, and SEQ ID NO:7 for HCDR3; and the amino acid sequences are as shown in SEQ ID NO:8 for LCDR1, SEQ ID NO:9 for LCDR2, and SEQ ID NO:10 for LCDR3.

2. The humanized monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:

2.

3. The humanized monoclonal antibody according to claim 1 or 2, characterized in that, It also includes the heavy chain constant region and the light chain constant region.

4. A nucleic acid molecule, characterized in that, Includes a nucleotide sequence encoding the humanized monoclonal antibody according to any one of claims 1-3.

5. An expression vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. The method for preparing the humanized monoclonal antibody according to any one of claims 1-3, characterized in that, Includes the following steps: The host cells of claim 6 are cultured, and the humanized monoclonal antibody is collected from the resulting cell culture.

8. A detection reagent or detection kit, characterized in that, Including the humanized monoclonal antibody as described in any one of claims 1-3.

9. A drug, characterized in that, Including the humanized monoclonal antibody as described in any one of claims 1-3.

10. The use of the humanized monoclonal antibody according to any one of claims 1-3 in any of the following: (1) Application in the detection of monkeypox virus B6R protein for non-diagnostic purposes. (2) Application in the preparation of detection products for monkeypox virus; (3) Use in the preparation of medicines for the prevention or treatment of monkeypox virus and / or vaccinia virus.

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