Humanized monoclonal antibody aiming at monkey pox virus B6R protein and application thereof

Abstract

The invention relates to a humanized monoclonal antibody aiming at monkey pox virus and application of the humanized monoclonal antibody, human monkey pox virus antibodies B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9 and B6R-22H1 with high neutralization activity are obtained by screening memory B cells specifically combined with anti-B6R of a human antibody humanized mouse, and the human monkey pox virus antibodies have high neutralization activity. The monkey pox virus vaccine has the characteristics of strong combining capacity with monkey pox virus, good in-vitro neutrality, large affinity and in-vivo protection effect.

Description

Technical Field

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

[0002] Monkeypox is caused by the monkeypox virus (Mpox virus, MPXV) and is a zoonosis. In 1958, the monkeypox virus was first discovered in a group of monkeys used for research and was named "Monkeypox virus (MPXV)". In 1970, a 9-month-old baby boy in Zaire (now the Democratic Republic of the Congo) was diagnosed, which was the first human case, indicating that the monkeypox virus can infect humans. At this time, the monkeypox epidemic was mainly endemic in countries in the tropical rainforest regions of Africa, and many cases were reported in Central African and West African countries. The mortality rates caused by the West African and Central African branches were 3.6% and 10.6% respectively. Although monkeypox has been mainly distributed in Central Africa and West Africa in the past few decades, in recent years, the scope of monkeypox transmission has changed, and cases have even emerged in regions outside Africa. In 2003, monkeypox cases occurred in the United States, which was the first case outside the African continent. Subsequently, monkeypox was also reported in many countries such as Israel, Nigeria, the United Kingdom, and Singapore. Especially in 2022, the global spread of the monkeypox virus has attracted great attention in the public health field. Under the severe situation of the global monkeypox epidemic, China faces a great risk of importation.

[0003] Monkeypox can be transmitted in various forms such as through contact with damaged skin tissues. In addition, recently, the transmission of the monkeypox virus in non-endemic countries has shown a phenomenon related to sexual contact, and the presence of the monkeypox virus has been detected in body fluids such as semen, but it is not clear whether it is transmitted through sexual contact. After infection, symptoms such as swollen lymph nodes, muscle soreness, and permanent scars left after pustules rupture will appear.

[0004] Since the eradication of smallpox in 1980 and the subsequent cessation of smallpox vaccination in humans, the current young population has a relatively low immunity to orthopoxviruses, which may be one of the reasons for the current outbreak of MPOX. With the spread of various monkeypox virus strains in non-endemic countries, not only are the transmission routes diverse and hidden, the infection symptoms are atypical, but the monkeypox virus also accelerates mutation during human-to-human transmission, and its mutation rate far exceeds expectations, posing an increasing threat to global public health security. Although there are not many monkeypox cases in China at present, with the flow of people inside and outside the country, how to prevent and control monkeypox infection has become a key issue that needs to be solved urgently. And currently, no specific antiviral therapy has been 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 method for treating 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. Among them, the B6R protein is involved in disrupting the glycosaminoglycan-mediated outer membrane of EEV during EEV infection, thereby promoting the fusion of the inner membrane. After infection, EEV requires B6R to induce the formation of actin tails, thereby promoting the accelerated spread of uninfected cells. B6R plays a key role in the viral infection process. Antibodies against B6R are mainly responsible for the EEV neutralization ability of human VIG, indicating that B6R plays a prominent role as a target for the EEV neutralizing activity of human antibodies.

[0006] Monoclonal antibodies that recognize different epitopes of B5 have been successfully prepared. Chimpanzee-human fusion MAb 8AH8AL, rat MAb 19C2, and human MAb h101 have been shown to provide protection in a mouse model. Epitope mapping studies have identified two major neutralizing sites on B5, located in SCR1SCR2 and the stem of B5, respectively. However, the precise information on these antibodies and other potential neutralizing epitopes on B5, as well as the structural characteristics of B5, remain elusive. Some people have also characterized the binding properties of several human anti-B5 antibodies and solved the crystal structure of hMB668 bound to B5.

[0007] It is very important and necessary to develop new monkeypox virus antibodies with strong antigen affinity and good in vitro neutralization. Summary of the Invention

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

[0009] The technical solution includes:

[0010] An embodiment of the present application provides a humanized monoclonal antibody against monkeypox virus, including a heavy chain variable region and a light chain variable region;

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

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

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

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

[0015] d. HCDR1 with the amino acid sequence shown in SEQ ID NO:53, HCDR2 with the amino acid sequence shown in SEQ ID NO:54, and HCDR3 with the amino acid sequence shown in SEQ ID NO:55; and, LCDR1 with the amino acid sequence shown in SEQ ID NO:56, LCDR2 with the amino acid sequence shown in SEQ ID NO:57, and LCDR3 with the amino acid sequence 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, and 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 as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:2.

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

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

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

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

[0022] In one embodiment, it further includes a heavy chain constant region and a light chain constant region.

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

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

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

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

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

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

[0029] (5) The heavy chain variable region as shown in nucleotide sequence SEQ ID NO: 67 and the light chain variable region as shown in nucleotide sequence SEQ ID NO: 68.

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

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

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

[0033] Culturing the host cell and collecting the humanized monoclonal antibody from the obtained cell culture.

[0034] One embodiment of the present application also provides a detection reagent or detection kit comprising the humanized monoclonal antibody.

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

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

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

[0038] One embodiment of the present application also provides the application of the humanized monoclonal antibody in preparing a drug for preventing or treating monkeypox virus and / or vaccinia virus.

[0039] Compared with the traditional technology, the present application has the following beneficial effects:

[0040] The humanized monoclonal antibody of the present application has strong binding ability to monkeypox virus, good in vitro neutralization, large affinity, and has an in vivo protective effect. It is completely different from the reported monkeypox virus antibody sequences, providing products for the detection and neutralization of monkeypox virus, and providing the possibility for providing monkeypox virus products with in vivo protective effects. Description of the Drawings

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

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

[0043] Figure 2 Results of kinetic curve of B6R antibody and B6R

[0044] Figure 3 Results of neutralization curve of B6R antibody against VACV-WR

[0045] Figure 4 Schematic diagram of the experiment and curves of the survival and body weight changes of mice after virus infection. Among them, the upper figure is a schematic diagram of the experiment implementation, and the lower figure is a curve diagram of the survival and body weight changes of mice after virus infection Specific implementation manners

[0046] To make the above objects, features, and advantages of the present application more obvious and understandable, the following provides a detailed description of the specific implementation manners of the present application. Many specific details are set forth in the following description to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present application. Therefore, the present application is not limited by the specific embodiments disclosed below

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application

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

[0049] The present application screens memory B cells that specifically bind to humanized mouse anti-B6R of a fully human antibody, and obtains human-derived high-neutralizing activity MPXV antibodies B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1, and finally 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

[0050] Specific technical solutions include

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

[0052] Through the expression and purification of the monkeypox virus B6R protein, sorting of specific memory B cells bound to the B6R protein, and expression and purification of monkeypox virus B6R antibodies, five monkeypox virus B6R monoclonal antibodies were obtained, namely B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1. The amino acid sequences of the light and heavy chains, CDR1-3 of the light chain variable region, and CDR1-3 of the heavy chain variable region of these five monkeypox virus monoclonal antibodies are as follows. The light chain variable region and the heavy chain variable region are different from the existing sequences and are specific.

[0053] II. Performance Verification of Monoclonal Antibodies

[0054] 1. Antigen and Antibody Neutralization Test

[0055] The neutralization curve results of the B6R antibody against VACV-WR were detected by the plaque method. 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. Detection of Antibody-Antigen Affinity by Surface Plasmon Resonance Technology

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

[0058] 4. Detection of B6R Antigen-Antibody Binding Activity by ELISA

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

[0060] 5. Animal Protection Test

[0061] In the case of virus infection, administration of the antibodies B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1 can all provide protection to mice infected with monkeypox virus.

[0062] The implementation scheme of the present application will be described in detail below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions in the following embodiments, the guidance given in the present application shall be preferentially referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or by referring to the experimental methods known in the art.

[0063] In the following specific embodiments, regarding the measurement parameters of raw material components, if there is no special instruction, there may be slight deviations within the weighing accuracy range. Regarding temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

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

[0065] Key drugs and reagents: Humanized mouse with full human antibodies, model RenMab, Beijing Biocytogen Co., Ltd.; 14K chip, model 750-00021, Berkeley lights.

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

[0067] Example 1 Preparation and purification of monoclonal antibodies against monkeypox virus

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

[0069] The gene of B6R was constructed into the eukaryotic expression vector pCAGGS and transfected into 293F cells. The B6R protein was secreted and expressed by the cells. The cell supernatant was collected and purified by affinity chromatography and molecular sieve, and identified by SDS-PAGE.

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

[0071] STCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYH

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

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

[0074] The gene of B6R was constructed into the mRNA vector respectively. After in vitro transcription and capping, the concentration was measured and the quality was identified. Humanized mice with fully human antibodies (RenMab) were immunized 3 times at a dose of 2.5 μg per mouse. Serum was collected before immunization and 12 days after each immunization to detect antibody titers. When the antibody titer reached an appropriate level, after a booster immunization, the lymph nodes of humanized mice were taken, ground into single cells, incubated with antigen molecules, and then specific single B cells were sorted using a Beacon cell sorter. The variable region gene sequences of the antibodies were 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, CDR1-3 of the light chain variable region, and CDR1-3 of the heavy chain variable region of these five monoclonal antibodies against monkeypox virus are as follows. The light chain variable region and the heavy chain variable region are different from the existing sequences and are specific.

[0076] Monoclonal antibody B6R-16A1:

[0077] Amino acid sequence of the heavy chain variable region, 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] GAAATTGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTACTTAGTTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGAAATCAAA

[0085] Among them, 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] Amino acid sequence of the heavy chain variable region, SEQ ID NO:17:

[0092] QVQLQQSGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLISVTAADTAVYYCARHGSGRGYYYYVMDVWGQGTTVTVSS

[0093] 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] CAGGTGCAGCTGCAGCAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAATGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGATCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAGACATGGTTCGGGGAGGGGCTACTACTACTACGTTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

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

[0098] GAAATTGTGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCGGGGCCAGTCAGAGTATTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCTGGCGCCCAGGCTCCTCATCTATGATGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTCTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCCACTTTCGGCGGAGGGACCAAGGTGGAAATCAAA

[0099] Among them, the amino acid sequences of the CDR regions of the B6R-21G7 antibody are shown in Table 3; the nucleic acid sequences of the CDR regions of the B6R-21G7 antibody are shown in Table 4.

[0100] Table 3

[0101]

[0102] Table 4

[0103]

[0104] B6R-22D12 monoclonal antibody:

[0105] Amino acid sequence of the heavy chain variable region, SEQ ID NO:33:

[0106] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKKQFSLKLNSVTAADTAVYNCARKAGGSGWYGYWGQGTLVTVSS Amino acid sequence of the light chain variable region, SEQ ID NO:34:

[0107] EIVMTQSPDSLAVSLGERATINCKSSQHILYSSNNDNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPYTFGQGTKLEIK

[0108] Nucleic acid sequence of the heavy chain variable region, SEQ ID NO:35:

[0109] CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTTGACACGTCCAAGAAACAGTTCTCCCTGAAGCTGAACTCTGTGACCGCCGCGGACACGGCTGTGTATAACTGTGCGAGAAAGGCGGGGGGCAGTGGCTGGTACGGCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0110] Nucleic acid sequence of the light chain variable region, SEQ ID NO:36:

[0111] GAAATTGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGCATATTTTATACAGCTCCAACAATGACAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAACAATATTATACTACTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

[0112] Among them, 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] Amino acid sequence of the heavy chain variable region, SEQ ID NO:49:

[0119] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNCNPSLKSRVAMSVDPPRKQFSLKLSSVTAADTAVYYCARKVAGTGWFYTWGQGTPVTVSS Amino acid sequence of the light chain variable region, SEQ IDNO:50:

[0120] EIVMTQSPDSLAVSLGERATINCKSSQTVLYNSNNENYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISTLQAEDVAVYYCQQYYTTPLTFGGGTKVEIK

[0121] Nucleic acid sequence of the heavy chain variable region, SEQ ID NO:51:

[0122] CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCTTTTAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTGCAACCCGTCCCTCAAGAGTCGAGTCGCCATGTCAGTAGACCCGCCCAGGAAACAGTTTTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAAAAGTGGCTGGTACTGGGTGGTTCTATACTTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCA

[0123] Light chain variable region nucleic acid sequence, SEQ ID NO: 52:

[0124] GAAATAGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGTCAGACTGTTTTATACAATTCCAACAATGAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCACCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAGTATTATACTACTCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAAATCAAA

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

[0126] Table 7

[0127]

[0128] Table 8

[0129]

[0130] Monoclonal antibody B6R-22H1:

[0131] Amino acid sequence of the heavy chain variable region, SEQ ID NO:65:

[0132] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGNTNYNPSLKSRVTTSVDTSKKQFSLNLSSVTAAETAVYYCARRPGGSPWFDPWGQGTLVTVSS Amino acid sequence of the light chain variable region, SEQ ID NO:66:

[0133] EIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLFIYWSSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYSIPWTFGQGTKVEIK

[0134] Nucleic acid sequence of the heavy chain variable region, SEQ ID NO:67:

[0135] CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGTTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAACACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCACATCAGTAGACACGTCCAAGAAGCAGTTCTCCCTGAACCTGAGCTCTGTGACCGCCGCGGAAACGGCTGTGTACTACTGTGCGAGACGCCCTGGTGGGAGCCCCTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0136] Nucleic acid sequence of the light chain variable region, SEQ ID NO:68:

[0137] GAAATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGTTATTCATTTACTGGTCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTTCTGTCAACAATATTACAGTATTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0138] Among them, 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. Expression and purification of the antibody

[0145] 3.1 Antibody cloning and construction: The variable heavy chain gene (VH) of the antibody was fused with the IgG1 constant region gene (CH) and constructed into the pCAGGS vector (named pCAGGS-Heavy chain-full length), and the variable light chain gene (VL) of the antibody was fused with the constant region gene (CL) and constructed into the pCAGGS vector (named pCAGGS-Light chain-fulllength).

[0146] 3.2 Antibody Expression and Purification: When co-transfecting 100 mL of 293F cells with light chain and heavy chain plasmids, take 36 μg of heavy chain plasmid and 75 μg of light chain plasmid, dissolve them in 1 mL of HBS, and let it stand for 5 min. Take 300 μg of PEI and dissolve it in 1 mL of HBS, and also let it stand for 5 min. Mix the plasmid and PEI, let it stand for 20 min, and then add it to the cells. Culture in a constant temperature shaker at 37 °C. After 5 days, centrifuge at 8000 r for 90 min and harvest the supernatant. The supernatant flows through an affinity column of Protein A, and through an AKTA Purifier machine, the antibody is obtained after elution with 0.1 M Glycine, pH 3.0, and concentrated and exchanged to PBS. The purified antibody is detected by SD-PAGE, with high purity. The size of the heavy chain after antibody denaturation is approximately 50 kDa, and the size of the light chain is approximately 25 kDa.

[0147] Example 3 Detection of Antibody-Antigen Affinity by Surface Plasmon Resonance Technology

[0148] Surface plasmon resonance (SPR) experiments can be used to detect the interaction between proteins in vitro. By detecting the change in the response value of the mobile phase flowing through the surface of the chip, the kinetic parameters of the binding of two proteins can be known. It is carried out on the instrument BIACORE 8K. In surface plasmon resonance experiments, usually one of the two proteins to be analyzed is immobilized on the surface of a metal chip, and the other is used as the mobile phase to flow through the surface of the chip to detect the change in the response value it causes. Before the experiment, PBST should be filtered through a 0.22 μM filter membrane 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, 25 nM (which can be adjusted appropriately). The characteristics of the B6R antigen-antibody affinity detection are shown in Table 11 and Figure 2 .

[0149] Table 11 Characteristics of B6R Antigen-Antibody Affinity Detection

[0150]

[0151] Example 4 Antibody Neutralization Test

[0152] The neutralizing activity of the antibody was detected by the plaque assay, which is briefly described as follows: The initial concentration of the antibody was 200 μg / mL, and it was serially diluted 4-fold for 10 gradients. 400 μL of each gradient was mixed with an equal volume of the dilution of vaccinia virus VACV-WR (containing 10% complement and 100 μg / mL of the targeted IMV antibody) and incubated at 37 °C for 1 h. Vero cells were seeded into 12-well plates in advance and could be used for the neutralization experiment when the cell confluence reached ~100%. After the cells were rinsed twice, the mixed solution of the antibody and the virus was added, 400 μL per well. Two replicate wells were set for each antibody gradient, and a control well with only the virus but no antibody was set. The cells were incubated at 37 °C for 1 h. The mixed liquid of the antibody and the virus was discarded, the cells were rinsed once, and 1 mL per well of the fixing solution was added. The fixing solution was prepared by mixing methylcellulose and high-glucose culture medium (2×) at a volume ratio of 1:1. The cell plates were placed in an incubator for cell culture. After 48 h, 8% tissue cell fixing solution was added for fixation, and then crystal violet was added for staining and counting, and the neutralizing activity PRNT50 of the antibody was calculated.

[0153] As Figure 3 shown, for the neutralization curve results of the B6R antibody against VACV-WR, the PRNT50 values of B6R-16A1, B6R-22D12, B6R-22F9, B6R-22H1, and B6R-21G7 were 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 test

[0155] To further evaluate the antibody applicability, antibodies with relatively high neutralizing activities were selected: B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1, and their protective effects were evaluated at the mouse level by administration. Since animal experiments on monkeypox virus infection need to be carried out in a high-level biosafety level 3 laboratory, initially, the Western Reserve (VACV-WR) strain of vaccinia virus (which belongs to the same family of poxviruses as monkeypox virus) was used for evaluation. 7- to 8-week-old mice were infected intranasally with 5 LD50 and 10 LD50 of the virus VACV-WR, and the antibody was intraperitoneally injected at 200 μg per mouse 4 h before and 4 h after infection.

[0156] As Figure 4As shown, after being infected with 5 LD50 of the virus, the body weights of the mice in the irrelevant antibody group all decreased by more than 20% on the 6th day, while the body weights of the mice injected with antibodies B6R-16A1, B6R-22D12, B6R-22F9, and B6R-22H1 did not change significantly. In the group injected with antibody B6RR-21G7, the body weight of one mouse decreased by more than 20% on the 7th day. After being infected with 10 LD50 of the virus, the body weights of the mice in the irrelevant antibody group all decreased by more than 20% on the 5th day, while the body weights of the mice injected with antibodies B6R-16A1, B6R-22D12, and B6R-22F9 did not change significantly.

[0157] Table 12 shows the survival rates of mice after different virus infections and antibody injections.

[0158] Table 12 Survival Rates of Mice after Different Virus Infections and Antibody Injections

[0159]

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

[0161] The above-described embodiments merely represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the specification can be used to explain the content of the claims.

Claims

1. A humanized monoclonal antibody against monkeypox virus B6R protein, characterized in that: including a heavy chain variable region and a light chain variable region; 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 items a to e; a. HCDR1 with an amino acid sequence as shown in SEQ ID NO:5, HCDR2 with an amino acid sequence as shown in SEQ ID NO:6, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:7; and LCDR1 with an amino acid sequence as shown in SEQ ID NO:8, LCDR2 with an amino acid sequence as shown in SEQ ID NO:9, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:10; b. HCDR1 with an amino acid sequence as shown in SEQ ID NO:21, HCDR2 with an amino acid sequence as shown in SEQ ID NO:22, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:23; and LCDR1 with an amino acid sequence as shown in SEQ ID NO:24, LCDR2 with an amino acid sequence as shown in SEQ ID NO:25, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:26; c. HCDR1 with an amino acid sequence as shown in SEQ ID NO:37, HCDR2 with an amino acid sequence as shown in SEQ ID NO:38, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:39; and LCDR1 with an amino acid sequence as shown in SEQ ID NO:40, LCDR2 with an amino acid sequence as shown in SEQ ID NO:41, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:42; d. HCDR1 having an amino acid sequence as shown in SEQ ID NO:53, HCDR2 having an amino acid sequence as shown in SEQ ID NO:54, and HCDR3 having an amino acid sequence as shown in SEQ ID NO:55; and LCDR1 having an amino acid sequence as shown in SEQ ID NO:56, LCDR2 having an amino acid sequence as shown in SEQ ID NO:57, and LCDR3 having an amino acid sequence as shown in SEQ ID NO:58; e. HCDR1 with an amino acid sequence as shown in SEQ ID NO:69, HCDR2 with an amino acid sequence as shown in SEQ ID NO:70, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:71; and LCDR1 with an amino acid sequence as shown in SEQ ID NO:72, LCDR2 with an amino acid sequence as shown in SEQ ID NO:73, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:

74.

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; and / or, 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; and / or, 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; and / or, 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; and / or, 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.

3. The humanized monoclonal antibody according to claim 1 or 2, characterized in that: Also included are the heavy chain constant region and the light chain constant region.

4. A nucleic acid molecule, characterized in that It comprises a nucleotide sequence encoding the humanized monoclonal antibody according to any one of claims 1 to 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 a humanized monoclonal antibody according to any one of claims 1 to 3, characterized in that: The steps include: Cultivating the host cell according to claim 6, and collecting the humanized monoclonal antibody in the obtained cell culture.

8. A detection reagent or a detection kit, characterized in that: The invention comprises the humanized monoclonal antibody according to any one of claims 1 to 3.

9. A drug, characterized in that The invention comprises the humanized monoclonal antibody according to any one of claims 1 to 3.

10. Use of the humanized monoclonal antibody according to any one of claims 1 to 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 monkeypox virus detection products; (3) Use in the preparation of drugs for preventing or treating monkeypox virus and / or vaccinia virus.

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