Monkeypox virus monoclonal neutralizing antibodies and their applications

Abstract

This invention belongs to the field of biomedicine, specifically relating to monoclonal neutralizing antibodies against monkeypox virus B6 and M1 antigens and their applications. This invention provides two monoclonal neutralizing antibodies targeting monkeypox virus B6 antigen and two targeting M1 antigen. These antibodies can broadly bind to monkeypox virus B6 or M1 antigens and proteins homologous to B6 or M1 in vaccinia virus, smallpox virus, and vaccinia virus. They can also neutralize orthopoxvirus infections, represented by vaccinia virus, and can be used for the prevention and treatment of orthopoxvirus infections, including but not limited to monkeypox virus, vaccinia virus, smallpox virus, and vaccinia virus, showing significant application potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to monoclonal neutralizing antibodies against monkeypox virus B6 and M1 antigens and their applications. Background Technology

[0002] Monkeypox virus (MPXV) belongs to the genus Orthopoxvirus, which also includes smallpox virus (VARV), vaccinia virus (VACV), and cowpox virus (CPXV). Infection in humans can cause diseases of varying severity and endanger human health.

[0003] Monkeypox (Mpox) is a zoonotic disease. The first human case was discovered in 1970. It subsequently spread locally in central and west Africa for a long period. However, since the first confirmed case in the UK in May 2022, confirmed cases have appeared in multiple countries worldwide, indicating that the monkeypox epidemic has become a global pandemic. Studies show that although the smallpox vaccine provides some protection against monkeypox, with the eradication of smallpox and the cessation of vaccination, most people (born after 1980) lack immunity to monkeypox virus, smallpox virus, and other orthopox viruses. Currently, although chemical drugs such as Tecovirimat and Brincidofovir have been approved for the treatment of smallpox, and vaccinia immunoglobulin (VIG) has been approved for complications caused by smallpox vaccination and can also be used for orthopox infections such as monkeypox, research on their efficacy against monkeypox and other orthopox virus infections remains limited. Currently, there are no effective drugs specifically for the treatment of monkeypox virus.

[0004] Monkeypoxvirus and other orthopoxviruses are double-stranded DNA viruses with a genome size of approximately 200 kb, encoding up to 200 proteins. They possess two distinct forms of infectious viral particles: intracellular mature virions (IMV) and extracellular enveloped virions (EEV), which have different envelope structures, modes of infection, and surface antigens. The M1, H3, A29, and E8 markers on the surface of IMV and the B6 and A35 markers on the surface of EEV have been shown to induce the production of neutralizing antibodies, thus making them important targets for research in therapeutic antibodies and vaccines.

[0005] The purpose of this invention is to screen monoclonal antibodies with neutralizing activity against the B6 antigen on the surface of monkeypox virus EEV and the M1 antigen on the surface of monkeypox virus IMV. Summary of the Invention

[0006] This invention uses MPXV B6 expressed in mammalian cells as an antigen. Memory B cells that specifically bind to the MPXV B6 antigen were screened from PBMCs of smallpox-vaccinated individuals using flow cytometry. The sorted individual B cells were then subjected to reverse transcription PCR and nested PCR to amplify the variable region sequence of the antibody using specific primers, and further ligated to the antibody constant region into an expression vector. After expression and purification of the antibodies in mammalian cells, a series of functional tests were performed, including binding capacity and neutralizing activity, resulting in two human monoclonal antibodies, D21 and D68, which exhibit broad-spectrum binding to MPXV, VAV, VARV, and CPXV and can neutralize VAV infection. Simultaneously, this invention uses MPXV M1 expressed in mammalian cells as an antigen. Through flow cytometry sorting, B cells specifically binding to the MPXV M1 antigen were screened from lymph nodes of mice that had survived challenge after being vaccinated with monkeypox virus mRNA vaccine. The sorted B cells were then subjected to 10×genomic sequencing to obtain the antibody variable region sequence. The variable region sequence was further synthesized and ligated into an expression vector along with the antibody constant region. After expression and purification of the antibodies in mammalian cells, a series of functional tests were performed, including binding capacity and neutralizing activity. Two murine monoclonal antibodies, 39# and 40#, were obtained, exhibiting broad-spectrum binding to MPXV, VAV, VARV, and CPXV, and capable of neutralizing VAV infection. This completes the invention.

[0007] This invention provides neutralizing antibodies against monkeypox virus B6 and M1 antigens, specifically neutralizing antibodies D21 and D68 against monkeypox virus B6 antigen or their antigen-binding fragments, or neutralizing antibodies 39# and 40# against monkeypox virus M1 antigen or their antigen-binding fragments.

[0008] Its three complementarity-determining regions (CDRs) of the D21 heavy chain variable region have amino acid sequences selected from the following group:

[0009] CDR1 as shown in SEQ ID NO:1

[0010] CDR2 as shown in SEQ ID NO:2, and

[0011] CDR3 as shown in SEQ ID NO:3;

[0012] Its three complementarity-determining regions (CDRs) of the D21 light chain variable region have amino acid sequences selected from the following group:

[0013] As shown in SEQ ID NO:4, CDR1,

[0014] CDR2 as shown in SEQ ID NO:5, and

[0015] CDR3 as shown in SEQ ID NO:6.

[0016] Its three complementarity-determining regions (CDRs) of the D68 heavy chain variable region have amino acid sequences selected from the following group:

[0017] As shown in SEQ ID NO:7, CDR1,

[0018] CDR2 as shown in SEQ ID NO:8, and

[0019] CDR3 as shown in SEQ ID NO:9;

[0020] Its three complementarity-determining regions (CDRs) of the D68 light chain variable region have amino acid sequences selected from the following group:

[0021] CDR1 as shown in SEQ ID NO:10

[0022] CDR2 as shown in SEQ ID NO:11, and

[0023] CDR3 as shown in SEQ ID NO:12.

[0024] Its 39# heavy chain variable region has three complementarity-determining regions (CDRs) with amino acid sequences selected from the following group:

[0025] CDR1 as shown in SEQ ID NO:13

[0026] CDR2 as shown in SEQ ID NO:14, and

[0027] CDR3 as shown in SEQ ID NO:15;

[0028] Its 39# light chain variable region has three complementarity-determining regions (CDRs) with amino acid sequences selected from the following group:

[0029] CDR1 as shown in SEQ ID NO:16

[0030] CDR2 as shown in SEQ ID NO:17, and

[0031] CDR3 as shown in SEQ ID NO:18.

[0032] Its 40# heavy chain variable region has three complementarity-determining regions (CDRs) with amino acid sequences selected from the following group:

[0033] CDR1, as shown in SEQ ID NO:19,

[0034] CDR2 as shown in SEQ ID NO:20, and

[0035] CDR3 as shown in SEQ ID NO:21;

[0036] Its 40# light chain variable region has three complementarity-determining regions (CDRs) with amino acid sequences selected from the following group:

[0037] As shown in SEQ ID NO:22, CDR1,

[0038] CDR2 as shown in SEQ ID NO:23, and

[0039] CDR3 as shown in SEQ ID NO:24.

[0040] Here, "antigen-binding fragment" refers to the antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody.

[0041] In one embodiment, the D21 antibody or its antigen-binding fragment contains:

[0042] As shown in SEQ ID NO:25, the heavy chain variable region, and

[0043] The light chain variable region is shown in SEQ ID NO:26.

[0044] In one embodiment, the D68 antibody or its antigen-binding fragment contains:

[0045] As shown in SEQ ID NO:27, the heavy chain variable region, and

[0046] The light chain variable region is shown in SEQ ID NO:28.

[0047] In one embodiment, the 39# antibody or its antigen-binding fragment contains:

[0048] As shown in SEQ ID NO:29, the heavy chain variable region, and

[0049] The light chain variable region as shown in SEQ ID NO:30.

[0050] In one embodiment, the 40# antibody or its antigen-binding fragment contains:

[0051] As shown in SEQ ID NO:31, the heavy chain variable region, and

[0052] The light chain variable region as shown in SEQ ID NO:32.

[0053] In one embodiment, the antibody or its antigen-binding fragment contains:

[0054] As shown in SEQ ID NO:33, the heavy chain constant region, and

[0055] As shown in SEQ ID NO:34, the light chain constant region.

[0056] In one embodiment, the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, biantibody, and antibody composition.

[0057] The “Fab” consists of a light chain, a heavy chain, a variable region, and CH1.

[0058] The “Fab′ fragment” contains a light chain and a heavy chain portion that includes the variable region and the region between the CH1 or CH1 and CH2 domains. Interchain disulfide bonds are formed between the two heavy chains of the two Fab′ fragments to form the F(ab′)2 molecule.

[0059] The “F(ab′)2 segment” contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab′)2 segment consists of two Fab′ segments held together by disulfide bonds between the two heavy chains.

[0060] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0061] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment containing antibody variable regions, which are encapsulated within a single polypeptide chain. Generally, scFv contains a polypeptide linker between the heavy chain variable region and the light chain variable region, which allows the scFv to form the desired structure for antigen binding.

[0062] A "dual antibody" is an antigen-binding fragment having two antigen-binding sites. The fragment contains a VH (VH-VL or VL-VH) linked to a VL in the same polypeptide chain. By using a linker short enough not to pair between two domains on the same chain, the domain pairs with a complementary domain of the other chain to form two antigen-binding sites.

[0063] "Antibody composition" refers to a mixture of multiple antibodies.

[0064] The present invention provides a polynucleotide that encodes any of the aforementioned neutralizing antibodies or their antigen-binding fragments or polypeptides.

[0065] The present invention provides an expression vector comprising the above-mentioned polynucleotides.

[0066] The present invention provides a host cell comprising the above-described expression vector.

[0067] This invention provides the use of any of the above-mentioned neutralizing antibodies or their antigen-binding fragments in the preparation of medicaments for the treatment and prevention of orthopox virus infections, including but not limited to monkeypox virus, vaccinia virus, smallpox virus, and cowpox virus.

[0068] The present invention provides a pharmaceutical composition comprising any of the aforementioned neutralizing antibodies or their antigen-binding fragments and a pharmaceutical carrier.

[0069] This invention also provides pharmaceutical compositions containing the monkeypox virus neutralizing antibody or its antigen-binding fragment of the present invention. To prepare the pharmaceutical compositions, various desired dosage forms can be prepared by mixing the antibody or its antigen-binding fragment with a pharmaceutical carrier or excipient. Examples of dosage forms for the pharmaceutical compositions of this invention include, for example, oral dosage forms such as tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated tablets, small pellets, sublingual tablets, and ointments; and non-oral dosage forms such as injections, suppositories, transdermal preparations, ointments, plasters, and topical liquids. Those skilled in the art can select appropriate dosage forms based on the route of administration and the target population.

[0070] The dosage of the active ingredient in the pharmaceutical composition of the present invention varies depending on the target patient, the target organ, symptoms, method of administration, etc. It can be determined based on the doctor's judgment, taking into account the type of dosage form, method of administration, patient's age and weight, patient's symptoms, etc.

[0071] The beneficial effects of this invention are:

[0072] This invention screened and obtained two monoclonal neutralizing antibodies targeting the B6 antigen of monkeypox virus and two targeting the M1 antigen. They can broadly bind to the B6 or M1 antigen of monkeypox virus and proteins homologous to B6 or M1 in vaccinia virus, smallpox virus, and cowpox virus. They can also neutralize orthopoxvirus infections represented by vaccinia virus and have the potential to treat and prevent orthopoxvirus infections including but not limited to monkeypox virus, vaccinia virus, smallpox virus, and cowpox virus. Attached Figure Description

[0073] Figure 1 : Kinetic curves of broad-spectrum binding of D21 antibody to MPXV B6 and homologous antigens of B6 in VCV, VARV, and CPXV;

[0074] Figure 2 : Kinetic curves of broad-spectrum binding of D68 antibody to MPXV B6 and homologous antigens of B6 in VCV, VARV, and CPXV;

[0075] Figure 3: Kinetic curves of antibody 39# broadly binding to MPXV M1 and homologous antigens of M1 in VCV, VARV, and CPXV;

[0076] Figure 4 : Kinetic curves of the broad-spectrum binding of antibody 40# to MPXV M1 and the homologous antigens of M1 in VAV, VARV, and CPXV;

[0077] Figure 5 The effect of D21 and D68 on neutralizing orthopoxvirus infection, represented by VCV.

[0078] Figure 6 39# and 40# have the effect of neutralizing vaccinia virus infection, represented by VCV.

[0079] The specific information of each sequence in this invention is as follows:

[0080] SEQ ID NO:1: GFSISTYP

[0081] SEQ ID NO:2: ISHDGRNK

[0082] SEQ ID NO:3:ARAYPYAFDVSEQ ID NO:4:QSVRND

[0083] SEQ ID NO:5: GAS

[0084] SEQ ID NO:6:QQYKDWPPWT

[0085] SEQ ID NO:7: GGTFSDYA

[0086] SEQ ID NO:8: ILPIVGVP

[0087] SEQ ID NO:9: ARRSGINGHGLDV

[0088] SEQ ID NO:10: QSLLNTNGYNY

[0089] SEQ ID NO:11: LGS

[0090] SEQ ID NO:12:MQTLQTQGYT

[0091] SEQ ID NO:13:GYTFTSYW

[0092] SEQ ID NO:14:INPSTGYT

[0093] SEQ ID NO:15: TRSDSTNYLFVY

[0094] SEQ ID NO:16: QSLLNSRTRKNH

[0095] SEQ ID NO:17: WAS

[0096] SEQ ID NO:18: KQSYNLMYT

[0097] SEQ ID NO:19: GYIFTRYW

[0098] SEQ ID NO:20:INPSTGYT

[0099] SEQ ID NO:21: ARSDYTNYVFEY

[0100] SEQ ID NO:22: QSLVHSNGNTY

[0101] SEQ ID NO:23: KVS

[0102] SEQ ID NO:24: SQSTHVPYT

[0103] SEQ ID NO:25:QVQLVQSGGGVVQPGRSLRLSCVASGFSISTYPLHWVRQAPGKGLE WVAVISHDGRNKYYADSVKGRFSISRDNSKNTVYLQMNSLRVEDTAVYYCARAYPYAFDV WGQGTLVTVSS

[0104] SEQ ID NO:26:EIVLTQSPATLSVSPGETATLSCRASQSVRNDLAWYQQKPGQAPRLLI YGASTRASGIPARFSGSGSGTEFTLTISSLQSEDFAIYYCQQYKDWPPWTFGQGTKVEIK

[0105] SEQ ID NO:27:QVQLVQSGAEVKKPGSSVKVSCKSSGGTFSDYAINWVRQAPGQGL EWMGRILPIVGVPNYAQKFQGRVTITADKSSSTAYKEVSGLRSEDTAVYYCARRSGINGHGL DVWGQGTTVIVSS

[0106] SEQ ID NO:28:DIVMTQSPLSLPVTPGEPASISCRSSQSLLNTNGYNYLEWYLHKPGQ SPQLLIYLGSHRASGVPDRFSGSGSGTDFTLKISRAEPEDVGVYYCMQTLQTQGYTFGQGT KLEIK

[0107] SEQ ID NO:29:QVQLQQSGAELAKPGASVKMSCKASGYTFTSYWMHWVKQRPGQG LEWIGYINPSTGYTEYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCTRSDSTNYLF VYWGQGTLVTVSS

[0108] SEQ ID NO:30:DIVMSQSPSSLAVSAGEKVTMRCKSSQSLLNSRTRKNHLAWYQQKP GQSPTLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLMYTFGGG TKLEIK

[0109] SEQ ID NO:31:QVQLQQSGAEVAKPGASVRMSCKASGYIFTRYWMHWVKQRPGQG LEWIGYINPSTGYTEYNQKFKDKAALTADKSSGTAYMQLSSLTSEDSAVYYCARSDYTNYV FEYWGQGTLVTVSS

[0110] SEQ ID NO:32:DVVMTQTPLSLPVSLGDHASISCRSSQSLVHSNGNTYLHWSLQKPG QSPKLLIFKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTEL EIK

[0111] SEQ ID NO:33:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0112] SEQ ID NO:34: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECS. Detailed Implementation

[0113] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0114] Example 1: Isolation of MPXV B6 protein-specific memory B cells

[0115] With informed consent from the volunteers, approximately 15 mL of blood was collected, and PBMCs were separated using lymphocyte separation tubes (purchased from Dakota). The separated PBMCs were incubated with MPXV B6 protein (final concentration 400 nM) on ice for 30 min, then washed twice with PBS, and subsequently incubated with the following antibodies (purchased from BD or Miltenyi): anti-human CD3 / PE-Cy5, anti-human CD16 / PE-Cy5, anti-human CD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / Pacific Blue, anti-human IgG / FITC, and anti-His / PE. After incubation with the antibodies on ice for 30 min, the PBMCs were washed twice with PBS and transferred to flow cytometry tubes. PE-Cy5 was collected after FACSAria III sorting.- APC-Cy7 + Pacific Blue + FITC + PE + The cell population, namely antigen-specific memory B cells, was directly collected into a 96-well plate, with one cell per well.

[0116] Example 2: Amplification of BCR sequence in a single memory B cell and construction of an IgG all-antibody expression vector

[0117] The memory B cells obtained in Example 1 were reverse transcribed using thermostable M-MVL reverse transcriptase (purchased from Beijing Jialan), and template conversion and adapter addition were performed using TSO primers at 42°C for 90 min; then at 50°C for 2 min, 42°C for 2 min, for 10 cycles; and at 70°C for 15 min to obtain cDNA.

[0118] The cDNA of this reverse transcription product was used as a template for dsDNA amplification and enrichment using HotStar Tap Plus enzyme (QIAgen). The reaction conditions were as follows: 95℃, 5 min; 95℃, 30 s, 60℃, 30 s, 72℃, 90 s; 30 cycles; 72℃, 10 min.

[0119] Using the above PCR product as a template, nested PCR was performed to specifically amplify the variable region sequence of the antibody. The first round of PCR (PCRa) reaction conditions were as follows: 95℃, 5 min; 95℃, 30 s, 55℃ (H chain / κ chain) or 50℃ (λ chain), 30 s, 72℃, 90 s, 35 cycles; 72℃, 7 min. This product was then used as a template for the second round of PCR (PCRb), with the following reaction conditions: 95℃, 5 min; 95℃, 30 s, 58℃ (H chain) or 60℃ (κ chain) or 64℃ (λ chain), 30 s, 72℃, 90 s, 35 cycles; 72℃, 7 min, yielding the PCR product.

[0120] PCR products were separated by 1.2% agarose gel electrophoresis. Bands with a size of ~400 bp were excised, recovered, and sequenced. The sequences were analyzed using IgBlast or IMGT online databases.

[0121] The analyzed antibody variable region sequence was ligated to the corresponding heavy / light chain constant regions via homologous recombination and cloned into the expression vector pCAGGS (laboratory-preserved) to obtain IgG full-antibody light and heavy chain recombinant expression plasmids. The antibody light and heavy chain plasmids were co-transfected into HEK293T cells for antibody expression. The protein was then purified sequentially using a protein A affinity chromatography column and a molecular sieve chromatography column, and identified by SDS-PAGE to obtain high-purity antibody protein.

[0122] Among them, the amino acid sequence of the D21 heavy chain variable region is shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:26; the amino acid sequence of the D68 heavy chain variable region is shown in SEQ ID NO:27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:28.

[0123] Analysis revealed that the three complementarity-determining regions (CDRs) of the D21 heavy chain variable region have amino acid sequences selected from the following group: CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3; the three complementarity-determining regions (CDRs) of the D21 light chain variable region have amino acid sequences selected from the following group: CDR1 as shown in SEQ ID NO:4, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6.

[0124] The three complementarity-determining regions (CDRs) of the D68 heavy chain variable region have amino acid sequences selected from the group consisting of: CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:8, and CDR3 as shown in SEQ ID NO:9; the three complementarity-determining regions (CDRs) of the D68 light chain variable region have amino acid sequences selected from the group consisting of: CDR1 as shown in SEQ ID NO:10, CDR2 as shown in SEQ ID NO:11, and CDR3 as shown in SEQ ID NO:12.

[0125] Example 3: Isolation of MPXV M1 protein-specific B cells, 10×genomic sequencing, and construction of IgG all-antibody expression vector

[0126] Lymph nodes were collected from mice that had been vaccinated with monkeypox mRNA vaccine and survived challenge. The cells were ground into a single-cell suspension, and monkeypox virus M1 antigen protein at a final concentration of 400 nM was added and the cells were incubated on ice for 30 min. After washing twice with PBS, the following antibodies were added for staining: Anti-mouse CD138 / APC, Anti-mouse CD93 / APC, Anti-mouse CD38 / PE-Cy7, Anti-mouse IgD / BV510, Anti-mouse GL-7 / FITC, Anti-mouse B220 / BV421, and Anti-His / PE. The cells were incubated on ice in the dark for 30 min, washed twice with PBS, and then transferred to flow cytometry tubes. The CD93-CD138-CD38- / +IgD-GL-7+B220+His+ cell population was sorted into flow cytometry tubes using a BDAria III flow cytometer. Immediately after sorting, a 10×Genomics single-cell sequencing process (performed by the company) was performed. After obtaining the antibody sequence, the variable region locus characteristics were analyzed. The variable region sequence of the antibody was synthesized and linked to the constant regions of IgG1 / Igκ / Igλ to form a complete antibody expression plasmid (pCAGGS vector). The antibody light and heavy chain plasmids were co-transfected into HEK293T cells for antibody expression. The protein was then purified sequentially using a protein A affinity chromatography column and a molecular sieve chromatography column, and identified by SDS-PAGE to obtain high-purity antibody protein.

[0127] Among them, the amino acid sequence of the 39# heavy chain variable region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:30; the amino acid sequence of the 40# heavy chain variable region is shown in SEQ ID NO:31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:32.

[0128] Analysis revealed that the three complementarity-determining regions (CDRs) of the 39# heavy chain variable region have amino acid sequences selected from the following group: CDR1 as shown in SEQ ID NO:13, CDR2 as shown in SEQ ID NO:14, and CDR3 as shown in SEQ ID NO:15; the three complementarity-determining regions (CDRs) of the 39# light chain variable region have amino acid sequences selected from the following group: CDR1 as shown in SEQ ID NO:16, CDR2 as shown in SEQ ID NO:17, and CDR3 as shown in SEQ ID NO:18.

[0129] The three complementarity-determining regions (CDRs) of the 40# heavy chain variable region have amino acid sequences selected from the group consisting of: CDR1 as shown in SEQ ID NO:19, CDR2 as shown in SEQ ID NO:20, and CDR3 as shown in SEQ ID NO:21; the three complementarity-determining regions (CDRs) of the 40# light chain variable region have amino acid sequences selected from the group consisting of: CDR1 as shown in SEQ ID NO:22, CDR2 as shown in SEQ ID NO:23, and CDR3 as shown in SEQ ID NO:24.

[0130] Example 4: Detection of antibody-antigen binding ability using surface plasmon resonance technology

[0131] Surface plasmon resonance analysis was performed using a Biacore 8K (GE Healthcare). The specific steps are as follows:

[0132] A protein A microarray (GE Healthcare) was used. The antibody protein (approximately 500 RU) was immobilized on the microarray by binding to the Fc layer of the protein A. The antigen protein was serially diluted with PBST solution (pH 7.4), loaded onto the microarray surface, and the changes in response values ​​were recorded. The kinetic curves of the antibody-antigen binding were analyzed using BIAevaluation software 8K (GE Healthcare). Figure 1-4 As shown in Tables 1 and 2, the kinetic constants of antibody-antigen binding are as follows. The results show that antibodies D21 and D68 have high affinity for MPXV B6, ​​at 1.9 nM and 6.2 nM respectively. D21 can bind a broad spectrum of VAV, VARV, and CPXV homologs with an affinity comparable to MPXV B6. D68's affinity for VAV homologs is comparable to its affinity for MPXV B6, ​​but its affinity for VARV and CPXV homologs is slightly lower. Antibody 39# has a high affinity for MPXV M1, at 1.2 nM, and binds VAV, VARV, and CPXV with comparable or slightly higher affinity. Compared to 39#, antibody 40# binds VAV and CPXV with higher affinity, at 0.36 nM and 0.17 nM respectively, and binds MPXV and CPXV in a non-dissociative manner (kd < 1.0e-5). In summary, all four antibodies bind to the antigen with high affinity and exhibit excellent broad-spectrum activity.

[0133] Table 1. Kinetic constants of the binding between D21, D68 antibodies and orthopoxvirus homologous proteins such as MPXV B6.

[0134]

[0135] Note: The data in the table are the mean ± standard deviation of three independent trials.

[0136] Table 2 shows the kinetic constants of the binding between antibodies #39 and #40 and orthopoxvirus homologous proteins such as MPXV M1.

[0137]

[0138] Example 5: Plaque assay for detecting the neutralizing effect of antibodies against VCV virus

[0139] VACV EEV Neutralization: D21 and D68 antibodies were serially diluted 3-fold starting at 100 μg / mL, for a total of 10 dilutions. Complement was added to each antibody dilution. Then, antibodies for blocking IMV were added to each antibody dilution. Diluted virus solution (approximately 120 PFU / well) was added to each well. The mixture was incubated at 37°C for 2 hours. The incubated antibody-virus mixture was added to cells, and cells were infected at 37°C for 1 hour. The mixture was discarded, and cells were washed once with PBS in each well. Then, 1 mL of a 1:1 mixture of 2x DMEM and carboxymethyl cellulose was added, and the cells were cultured for another 48 hours. 4% paraformaldehyde fixative was added to each well, and fixation was performed for at least 2 hours. The paraformaldehyde was discarded, and crystal violet staining solution was added to each well. After staining for 2 hours, the results could be observed. The neutralizing activity value (PRNT) of the antibody was calculated based on the number of plaques. 50 The result is as follows: Figure 5 As shown, antibodies D21 and D68 can effectively neutralize VCV virus infection, with activities of 0.075 μg / mL and 0.06 μg / mL, respectively. Based on the broad-spectrum characteristics of the antibodies, it can be seen that they can also effectively neutralize other orthopoxvirus infections such as monkeypox virus.

[0140] VACV IMV Neutralization: Antibodies #39 and #40 were serially diluted 2-fold starting at 100 μg / mL, for a total of 10 dilutions. The diluted virus solution (approximately 120 PFU / well) was added to each well. The mixture was incubated at 37°C for 2 hours. The incubated antibody-virus mixture was then added to cells, and the cells were infected at 37°C for 1 hour. The mixture was discarded, and cells were washed once with PBS in each well. Then, 1 mL of a 1:1 mixture of 2x DMEM and carboxymethyl cellulose was added, and the cells were incubated for another 48 hours. 4% paraformaldehyde fixative was added to each well, and fixation was performed for at least 2 hours. The paraformaldehyde was discarded, and crystal violet staining solution was added to each well. Results were observed after staining for 2 hours. The neutralizing activity value (PRNT) of the antibody was calculated based on the number of plaques. 50 The result is as follows: Figure 6 As shown, antibodies 39# and 40# can effectively neutralize VCV virus infection, with activities of 0.075 μg / mL and 0.06 μg / mL, respectively. Based on the broad-spectrum characteristics of the antibodies, it can be seen that they can also effectively neutralize other orthopoxvirus infections such as monkeypox virus.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that neutralizes monkeypox virus, characterized in that, It is a neutralizing antibody against the monkeypox virus B6 antigen. The amino acid sequences of the three complementarity-determining regions (CDRs) of its heavy chain variable region are as follows: CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3; the amino acid sequences of the three complementarity-determining regions (CDRs) of its light chain variable region are as follows: CDR1 as shown in SEQ ID NO:4, CDR2 of the amino acid sequence GAS, and CDR3 as shown in SEQ ID NO:

6. Alternatively, the amino acid sequences of the three complementarity-determining regions (CDRs) of its heavy chain variable region are: CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:8, and CDR3 as shown in SEQ ID NO:9; the amino acid sequences of the three complementarity-determining regions (CDRs) of its light chain variable region are: CDR1 as shown in SEQ ID NO:10, CDR2 of amino acid sequence LGS, and CDR3 as shown in SEQ ID NO:

12.

2. The monkeypox virus neutralizing antibody or its antigen-binding fragment as described in claim 1, characterized in that, The antibody against monkeypox virus B6 antigen contains a heavy chain variable region as shown in SEQ ID NO:25 and a light chain variable region as shown in SEQ ID NO:

26. Alternatively, it may contain a heavy chain variable region as shown in SEQ ID NO:27 and a light chain variable region as shown in SEQ ID NO:

28.

3. The monkeypox virus neutralizing antibody or its antigen-binding fragment as described in claim 1 or 2, characterized in that, The antibody type is IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

4. The monkeypox virus neutralizing antibody or its antigen-binding fragment as described in claim 3, characterized in that, The antibody also includes a heavy chain constant region and a light chain constant region.

5. The monkeypox virus neutralizing antibody or its antigen-binding fragment as described in claim 4, characterized in that, The antibody contains a sequence of a constant region of one of the human antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

6. The monkeypox virus neutralizing antibody or its antigen-binding fragment as described in claim 5, characterized in that, The amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO:33; the amino acid sequence of the light chain constant region of the antibody is shown in SEQ ID NO:

34.

7. The monkeypox virus neutralizing antibody or its antigen-binding fragment as described in any one of claims 1 to 6, characterized in that, It is Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2.

8. A polynucleotide encoding a monkeypox virus neutralizing antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7.

9. An expression vector comprising the polynucleotide as described in claim 8.

10. A host cell comprising the expression vector as described in claim 9.

11. Use of the monkeypox virus neutralizing antibody or its antigen-binding fragment as described in any one of claims 1 to 7 in the preparation of a medicament for treating and preventing orthopox virus infection, wherein the orthopox virus is selected from monkeypox virus, vaccinia virus, smallpox virus, and cowpox virus.

12. A pharmaceutical composition comprising a monkeypox virus neutralizing antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7 and a pharmaceutical carrier, optionally further comprising an excipient.

13. The pharmaceutical composition according to claim 12, characterized in that, The dosage form of the pharmaceutical composition is an oral dosage form or a non-oral dosage form; the oral dosage form is a tablet, pill, powder, granule, soft / hard capsule, or ointment; the non-oral dosage form is an injection, suppository, transdermal preparation, ointment, plaster, or topical liquid.

14. The pharmaceutical composition according to claim 13, characterized in that, The tablets are film-coated tablets or sublingual tablets; the pills are small pills.

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