Monoclonal antibody specifically targeting monkey pox virus and application thereof

Abstract

The invention relates to the field of antibody engineering, in particular to a monoclonal antibody specifically targeting a monkey pox virus and application of the monoclonal antibody. According to the invention, by screening mouse memory B cells specifically combined with monkey pox virus envelope protein A35R, monkey pox virus antibodies 1H4, 3H3, 3H6, 13H1 and 17H1 with high neutralization activity are obtained. Wherein 17H1 has excellent affinity and neutralizing efficacy on monkey pox virus and has a strong in-vivo protection effect, and 100% of survival rate of challenged mice is achieved in prevention and treatment experiments. The sequence of the antibody is completely different from that of an existing monkey pox virus antibody, and a new candidate product is provided for detection, diagnosis, prevention and treatment of the monkey pox virus.

Description

Technical Field

[0001] This invention belongs to the field of antibody engineering, and in particular relates to monoclonal antibodies that specifically target monkeypox virus and their applications. Background Technology

[0002] From 2022 to 2025, the global monkeypox epidemic will continue. The risk of imported cases in my country will place dual pressure on both port control and community prevention.

[0003] At the prevention and treatment levels, existing methods have significant limitations. The traditional smallpox vaccine ACAM2000 has a high incidence of myocarditis (approximately 1 / 175), limiting its applicability to certain populations. The currently approved monkeypox vaccine JYNNEOS has a neutralizing antibody seroconversion rate of only 55%–65%, and its protective efficacy against the new monkeypox virus IIb branch circulating since 2022 has decreased by up to 30%, resulting in less than ideal immunization efficacy. More seriously, approximately 45% of monkeypox-infected individuals are also infected with HIV-1, and when their CD4+ T cell count is below 200 cells / μL, their specific anti-monkeypox antibody levels can decrease by up to 10-fold, highlighting the serious inadequacy of current immunization strategies in this high-risk population. Therefore, developing a highly effective and broad-spectrum anti-monkeypox virus strategy is urgently needed.

[0004] Monoclonal antibodies have shown significant potential as a treatment for infectious diseases; however, no specific monoclonal antibody against monkeypox virus has yet entered clinical development. Similar to other orthopoxviruses, monkeypox virus exists in both extracellular enveloped virus (EEV) and intracellular mature virus (IMV) forms during infection. The A35R protein, a key antigen on the monkeypox virus EEV membrane, is highly homologous to the vaccine virus A33R, forming a homodimer and significantly promoting viral transmission between cells. Further immunological studies suggest that high levels of A35R-specific binding antibodies can still be detected in individuals vaccinated against smallpox more than 40 years later, indicating that this protein is a potential target for inducing durable immune memory.

[0005] Despite the promising prospects of the A35R target, the neutralization mechanism and in vivo protective effect of its specific antibodies remain unclear. Furthermore, due to biosafety restrictions and the low frequency of antigen-specific B cells in the peripheral blood of recovered patients, directly isolating and screening antibodies from natural sources of infection faces challenges such as resource scarcity, complex procedures, and the risk of missing rare and highly efficient clones, making high-throughput development difficult.

[0006] Against this backdrop, the development of monkeypox virus monoclonal antibodies that can specifically target the A35R antigen, possess high binding activity and strong virus neutralization capacity, and provide effective protection in vivo is not only of great scientific significance, but also an urgent need to address the current monkeypox epidemic and potential future threats. Summary of the Invention

[0007] The problem that this invention aims to solve is: To address the problems existing in the prior art, this invention provides a monkeypox virus monoclonal antibody that can specifically target the A35R antigen, has high binding activity and strong virus neutralization ability, and can provide effective protection in vivo, as well as its application.

[0008] To achieve the above objectives, the specific technical solution of the present invention is as follows: A monoclonal antibody 17H1 that neutralizes monkeypox virus, wherein the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:1, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO:3. The amino acid sequence of the light chain CDR1 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:4, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO:5, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:6.

[0009] In some embodiments, the heavy chain amino acid sequence of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:7, and the light chain amino acid sequence of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:8.

[0010] In some embodiments, the nucleotide sequence encoding the heavy chain CDR1 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:9, the nucleotide sequence encoding the heavy chain CDR2 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:10, the nucleotide sequence encoding the heavy chain CDR3 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:11, the nucleotide sequence encoding the light chain CDR1 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:12, the nucleotide sequence encoding the light chain CDR2 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:13, and the nucleotide sequence encoding the light chain CDR3 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:14.

[0011] In some embodiments, the nucleotide sequence of the monoclonal antibody heavy chain encoding the monkeypox virus neutralizing antibody is shown in SEQ ID NO:15, and the nucleotide sequence of the monoclonal antibody light chain encoding the monkeypox virus neutralizing antibody is shown in SEQ ID NO:16.

[0012] Secondly, this application relates to the use of the aforementioned monoclonal antibody 17H1, which neutralizes monkeypox virus, in the preparation of products for detecting monkeypox virus.

[0013] Thirdly, this application relates to the use of the aforementioned monoclonal antibody 17H1, which neutralizes monkeypox virus, in the preparation of a medicament for the prevention and treatment of monkeypox virus.

[0014] Compared with the prior art, the beneficial effects of this invention application are as follows: This invention screens mouse anti-A35R specific high-affinity plasma B cells and uses them to create a chimeric antibody against both humans and mice, obtaining a newly discovered monkeypox virus antibody, 17H1, with high neutralizing and protective activity. This antibody differs from previously reported monkeypox virus antibodies in sequence; it exhibits strong antigen-binding ability, with a binding affinity as high as 90.3 pM; it possesses excellent neutralizing potency, with an IC50 of only 1.73 nM (259.3 ng / mL) in the presence of complement; and it demonstrates strong in vivo protective activity, achieving 100% survival in both preventative and therapeutic mouse models challenged with lethal doses of monkeypox virus. Furthermore, 17H1 can protect mice from lethal doses of monkeypox virus. Therefore, 17H1 has great potential as a candidate drug for the detection, prevention, and treatment of monkeypox virus. Attached Figure Description

[0015] Figure 1 ELISA was used to detect the binding of five isolated monoclonal antibodies to the monkeypox virus A35R protein.

[0016] Figure 2 SPR was used to detect the affinity activity of five isolated monoclonal antibodies for monkeypox virus A35R protein.

[0017] Figure 3 FRNT was used to detect the neutralizing effect of five isolated monoclonal antibodies against monkeypox virus.

[0018] Figure 4 The preventive and therapeutic effects of 17H1 antibody on fatal monkeypox virus infection in mice were investigated. In the figure, A and B are the body weight change curves and survival curves of BALB / c mice in the preventive administration group after monkeypox virus challenge, respectively, and C and D are the body weight change curves and survival curves of BALB / c mice in the treatment administration group after monkeypox virus challenge, respectively. Detailed Implementation

[0019] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the techniques and experimental methods used in the following embodiments are conventional techniques and experimental methods well known to those skilled in the art.

[0020] Unless otherwise specified, all experimental materials used in the following examples are commercially available.

[0021] Experimental materials 1. Key reagents and consumables mRNA in vitro transcription kit (T7 High Yield RNA Transcription kit), catalog number E131-01A, supplier: Suzhou Nearshore Protein Technology Co., Ltd.; N1-methyl-pseudouridine triphosphate (N1-Me-pUTP), catalog number NMPUTP001, supplier: Jiangsu Shenji Biotechnology Co., Ltd.; Cap analog (CAP GAG(3'OMe)), catalog number CAP3111, supplier: Jiangsu Shenji Biotechnology Co., Ltd.; Plasma cell discovery cDNA synthesis kit (Opto Plasma B Discovery cDNA Synthesis Kit), catalog number 750-02030, supplier: Berkeley Lights; Sanger sequencing preparation kit for mouse plasma cell discovery (Opto Plasma B Discovery Sanger Prep Kit, Mouse), catalog number 750-01004, supplier: Berkeley Lights; OptoSelect ® 20K chip, part number 750-00019, supplier Berkeley Lights.

[0022] 2. Main instruments and equipment Single-cell light guide system, model Beacon, supplier Berkeley Lights.

[0023] Example 1: Preparation and Immunization of A35R-FC mRNA-LNP mRNA Vaccine The A35R segment of MPXV (GenBank MT350282.1; Arg58 to Thr181) was extracted and fused with the Fc segment of the human IgG1 heavy chain to obtain A35R-Fc. After optimizing its gene sequence, it was constructed into an mRNA vector in our laboratory. After in vitro transcription, its encapsulation efficiency and concentration were measured.

[0024] After in vitro transcription and capping, the concentration was determined and the quality was identified.

[0025] The amino acid sequence of A35R is as follows: RALNQCMSANKAAITDSAVAVAAASSTHRKVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT BALB / c mice were then immunized with A35R-Fc mRNA-LNP twice via intramuscular injection, 3 weeks apart, at a dose of 5 μg per mouse. Serum was collected 10 days after the two immunizations to detect antibody titers. Antibody titers were measured when the A35R antibody titer reached 10. 6 At that time, a booster immunization was administered. Five to seven days later, the spleens of the candidate mice were harvested, ground into single cells, incubated with antigen molecules, and then processed using a Beacon system based on photofluidization. ® A high-throughput single-cell photoconductive system was used to specifically sort individual B cells. The variable region gene sequence of the antibody was obtained by reverse transcription and PCR amplification using a cDNA synthesis kit and then sequenced.

[0026] Five monkeypox virus monoclonal antibodies, 1H4, 3H3, 3H6, 13H1 and 17H1, were obtained. Among them, the amino acid sequences of the heavy and light chains, the variable regions CDR1-3 of the heavy chain and CDR1-3 of the light chain of monkeypox virus monoclonal antibody 17H1 are as follows. The variable regions of the heavy chain and the variable regions of the light chain are different from the existing sequences and have specificity.

[0027] The amino acid sequence information of the three CDR regions of the 17H1 heavy chain is as follows:

[0028] The amino acid sequence information of the three CDR regions of the 17H1 light chain is as follows:

[0029] The amino acid sequence of the 17H1 heavy chain (SEQ ID NO:7): GVHSQVQLKQSGPELVKPGASVKVSCKASGYAFTNYNMYWVKQSHGKSLEWIGYIDPYNGGTSYNQKFKGKVTLTVDKSSSTAYMHLNSLTSEDSAVYYCARRASMDYWGQGTTLTVSS The amino acid sequence of the 17H1 light chain (SEQ ID NO:8): GVHSDIQMTQSPSSMYASLGESVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK This invention also provides nucleotide sequences encoding the three CDR regions of the 17H1 heavy chain, as follows:

[0030] This invention also provides nucleotide sequences encoding the three CDR regions of the 17H1 light chain, as follows:

[0031] This invention provides a nucleotide sequence encoding the 17H1 heavy chain (SEQ ID NO:15): GGCGTGCACAGCCAGGTGCAGCTGAAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACGCTTCACCAACTACAACATGTACTGGGTGAAGCAGAGCCACGGCAAGAGCCTGGAGTGGATCGGCTACATCGACCCCTACAACG GCGGCACCAGCTACAACCAGAAGTTCAAGGGCAAGGTGACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGCACCTGAACAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCCGCGCCAGCATGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC This invention provides a nucleotide sequence encoding the 17H1 light chain (SEQ ID NO:16): GGCGTGCACAGCGACATCCAGATGACCCAGAGCCCCAGCAGCATGTACGCCAGCCTGGGCGAGAGCGTGACCATCACCTGCAAGGCCAGCCAGGACATCAACAGCTACCTGAGCTGGTTCCAGCAGAAGCCCGGCAAGAGCCCCAAGACCCTGATCTACCGCGCCA ACCGCCTGGTGGACGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGCCAGGACTACAGCCTGACCATCAGCAGCCTGGAGTACGAGGACATGGGCATCTACTACTGCCTGCAGTACGACGAGTTCCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG Example 2: Antibody Expression and Purification Antibody clone construction: The variable region gene (VH) of the heavy chain and the variable region gene (VL) of the light chain of the antibody were analyzed by online software IgBLAST and IMGT, and then fused into human IgG1 heavy and light chain constant region expression vectors (AbVec2.0-IGHG1 and AbVec1.1-IGKC, respectively).

[0032] Antibody expression and purification: Light chain and heavy chain plasmids were co-transfected into 100 mL of 293F cells at a mass ratio of 1:1.5 and cultured in a shaker at 37°C. After 5 days of culture, the supernatant was harvested by centrifugation at 12000 rpm for 10 min. The supernatant was filtered through a 0.45 μm filter and purified by Protein A affinity column. The antibody was obtained by elution with 0.1 M glycine (pH 3.0). Simultaneously, 1 M NaOH (pH 9.0) was added at a volume ratio of 10:1 to neutralize the antibody immediately, and the solution was concentrated and transferred to PBS for storage.

[0033] Example 3: Antibody Binding Activity Detection Antibody activity was detected by ELISA: commercially available A35R protein (Antibody system, Franch, Cat) was used. #EVV13101) was coated at a concentration of 1 μg / ml, 100 ng / well, and 100 μL / well, and incubated overnight at 4°C. The plate was washed with PBST, residual liquid was removed, and 200 μL / well of 5% skim milk powder was added for blocking, and the plate was incubated at room temperature for 2 hours. After washing and removing residual liquid, the five antibodies obtained above (1H4, 3H3, 3H6, 13H1, and 17H1) were diluted to a concentration of 0.5 μg / mL, and serially diluted 12 times (2-fold) at 100 μL / well, incubated at room temperature for 2 hours. The plate was washed 5 times with PBST, and residual liquid was removed. Human IgG-HRP-labeled secondary antibody was added at 100 μL / well, and the plate was incubated at room temperature for 1 hour. After washing 5 times with PBST, residual liquid was removed, and chromogenic reagent was added at 100 μL / well, and chromogenic reaction was performed for 5 minutes. Stop solution was added at 50 μL / well. The OD 450 values ​​were read and analyzed.

[0034] Experimental results are as follows Figure 1 As shown, all five monoclonal antibodies could bind well to the A35R antigen. The EC50 values ​​of the antibodies were 2.82 ng / mL for 1H4, 4.2 ng / mL for 3H3, 8.26 ng / mL for 3H6, 4.34 ng / mL for 13H1, and 8.26 ng / mL for 17H1. The binding activity increased with increasing antibody concentration, and the OD450 value increased.

[0035] Example 4: Antibody Affinity Assay Surface plasmon response (SPR) was used to detect in vitro protein-protein interactions. Analysis was performed using a Biacore 8K instrument. Prior to the experiment, PBST was filtered through a 0.22 μM filter and then autoclaved. For binding kinetics assays, the antibody concentration was initially set at 5 μg / mL (adjustable as needed), and serially diluted antibodies were injected onto the surface of a conjugated A35R antigen chip containing approximately 200 RU; data were fitted using a 1:1 binding model.

[0036] The affinity and binding properties of the 5 antibodies to the A35R antigen are as follows: Figure 2 As shown, 17H1 has a strong binding affinity of 90.3 pM.

[0037] Example 5: Antibody Neutralization The neutralizing activity of the five antibodies was assessed using a focus reduction neutralization test (FRNT), as briefly described below: The initial antibody concentration was 10 μg / mL, serially diluted 3-fold to create eight gradients. The mixture was then incubated at 37°C for 1 h with an equal volume of MPXV (diluent containing 10% rabbit complement) at a titer of 200 FFUs. The mixture was added to 96-well plates pre-inoculated with Vero cells, 50 μL per well, with two replicates for each antibody gradient. A control well containing only virus and no antibody was also included. The plates were incubated at 37°C for 18 h before collection. The antibody-virus mixture was discarded, and the cells were washed once with PBS. Cells were fixed with 4% paraformaldehyde for 30 min, 200 μL per well. Cells were then permeabilized with 0.2% Triton X-100 for 10 min and incubated at room temperature for 2 h with horseradish peroxidase-labeled vaccinia virus polyclonal antibody (Invitrogen, PA1-73192). Colorimetric analysis was performed using KPL TrueBlue peroxidase substrate (Seracare Life Sciences, 5510-0030). The number of monkeypox virus spots was quantified using an EliSpot microplate reader, and the neutralizing antibody titer was calculated and expressed as a 50% inhibitor dose (ID50), which represents a 50% reduction in monkeypox virus lesions at serum dilution compared to the viral control.

[0038] Experimental results are as follows Figure 3 The results of the neutralization curve of A35R antibody against MPXV are shown. The neutralization IC50 of 17H1 is 1.73 nM, or 259.3 ng / mL, which indicates strong neutralizing activity.

[0039] Example 6: Antibody Protective Effect To further evaluate the in vivo prophylactic and therapeutic effects of the 17H1 antibody, its protective efficacy was assessed in mice. (The data were analyzed using a 1×10⁻⁶ mouse assay.) 6 Eight-week-old BALB / c mice were infected nasally with a lethal dose of MPXV (50 μL / mouse) via FFU. 20 μg (1 mg / kg) of 17H1 antibody was injected intraperitoneally 24 hours before infection and 100 μg (5 mg / kg) after infection. An equivalent dose of irrelevant IgG antibody served as a control.

[0040] The results are as follows Figure 4 As shown, after infection with a lethal dose of MPXV virus, in both prevention and treatment experiments, the body weight of mice in the unrelated antibody group decreased by more than 25% on day 6, while the body weight of mice in the prophylactic injection group with 17H1 antibody ( Figure 4 A) or the post-infection treatment group ( Figure 4In mice (C), body weight gradually increased from day 5, and within 2 weeks it basically returned to its initial weight before challenge, preventing ( Figure 4 B) or treatment ( Figure 4 D) All mice in the experimental antibody administration group survived completely, meaning that the 17H1 monoclonal antibody had a 100% protection rate against monkeypox virus infection in both prevention and treatment.

[0041] In summary, this application screened mouse anti-A35R specific high-affinity plasma cells and constructed a human-mouse chimeric antibody, obtaining the monkeypox virus antibody 17H1 with high neutralizing and protective activity. This newly discovered antibody differs from previously reported monkeypox virus antibodies in sequence and exhibits strong antigen-binding ability. Furthermore, it can protect mice from lethal doses of monkeypox virus. These results suggest that 17H1 has the potential to be a candidate drug for the prevention and treatment of monkeypox virus.

[0042] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A monoclonal antibody 17H1 that neutralizes monkeypox virus, characterized in that, The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:1, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO:

3. The amino acid sequence of the light chain CDR1 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:4, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO:5, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:

6.

2. The monoclonal antibody 17H1 for neutralizing monkeypox virus as described in claim 1, characterized in that, The heavy chain amino acid sequence of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:7, and the light chain amino acid sequence of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:

8.

3. The monoclonal antibody 17H1 for neutralizing monkeypox virus as described in claim 1, characterized in that, The nucleotide sequence encoding the heavy chain CDR1 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:9; the nucleotide sequence encoding the heavy chain CDR2 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:10; the nucleotide sequence encoding the heavy chain CDR3 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:11; the nucleotide sequence encoding the light chain CDR1 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:12; the nucleotide sequence encoding the light chain CDR2 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:13; and the nucleotide sequence encoding the light chain CDR3 of the monoclonal antibody 17H1 that neutralizes monkeypox virus is shown in SEQ ID NO:

14.

4. The monoclonal antibody 17H1 for neutralizing monkeypox virus as described in claim 1, characterized in that, The nucleotide sequence of the heavy chain of the monoclonal antibody encoding the monkeypox virus neutralizing antibody is shown in SEQ ID NO:15, and the nucleotide sequence of the light chain of the monoclonal antibody encoding the monkeypox virus neutralizing antibody is shown in SEQ ID NO:

16.

5. The use of the monoclonal antibody 17H1 for neutralizing monkeypox virus as described in any one of claims 1-4 in the preparation of products for detecting monkeypox virus.

6. The use of the monoclonal antibody 17H1 for neutralizing monkeypox virus as described in any one of claims 1-4 in the preparation of a medicament for the prevention and treatment of monkeypox virus.

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