Anti-orthopoxvirus monoclonal antibody and application thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses an anti-orthopoxvirus monoclonal antibody and application thereof. The monoclonal antibody for resisting orthopoxvirus provided by the invention is 4H4, 12C3 or 11C9. The anti-orthopoxvirus monoclonal antibody provided by the invention can specifically recognize and target orthopoxviruses such as monkey pox, vaccinia and the like, shows efficient virus neutralizing capacity, and can be used for preparing medicines for preventing and / or treating orthopoxvirus infection and / or diseases related to orthopoxvirus infection; or preparing a detection product for detecting orthopoxvirus and diagnosing diseases related to orthopoxvirus infection.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to an anti-orthopoxvirus monoclonal antibody and its application. Background Art

[0002] Monkeypox virus (MPXV) is a zoonotic virus belonging to the genus Orthopoxvirus in the Poxviridae family. Monoclonal antibodies against orthopoxviruses have important application value in the biomedical field, especially in the prevention and treatment of diseases caused by orthopoxviruses. With the development of biotechnology, monoclonal antibody technology has become an important means to develop new treatment methods and diagnostic tools. Monoclonal antibodies can specifically bind to target antigens and can not only be used as efficient therapeutic drugs, but also can be used to prepare test strips, kits and other products for detecting related diseases, which has greatly promoted the effective prevention and control of various viral diseases.

[0003] At present, in order to solve the diseases caused by orthopoxvirus, the existing technical solutions usually use polyclonal antibodies or traditional antiviral drugs against orthopoxvirus. Among them, although polyclonal antibodies have strong immune response ability, due to their different antibody compositions, the differences between batches and stability problems are difficult to control; and although traditional antiviral drugs can inhibit the proliferation of viruses to a certain extent, they have large side effects and are prone to drug resistance. Therefore, the above methods still have certain limitations in practical applications.

[0004] The use of polyclonal antibodies and traditional antiviral drugs in the existing technology has failed to provide antibodies against orthopoxviruses with extremely high consistency and specificity. In addition, there is still a large gap in the relevant research on the antiviral mechanism of highly efficient neutralizing monoclonal antibodies and bispecific antibodies against monkeypox virus. There is an urgent need for a new type of highly efficient and stable monoclonal antibody to make up for the shortcomings of traditional solutions. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the present application provides a novel, highly effective and stable monoclonal antibody against orthopoxvirus and its application.

[0006] In the first aspect, the present application provides a monoclonal antibody against orthopoxvirus, using the following technical solution: A monoclonal antibody against orthopoxvirus, wherein the antibody is 4H4, 12C3 or 11C9; The heavy chain variable region of the 4H4 antibody comprises three complementary determining regions: CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NOs. 1 to 3; The light chain variable region of the 4H4 antibody comprises three complementary determining regions: CDR-L1, CDR-L2 and CDR-L3, the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NOs. 4-5; the amino acid sequence of CDR-L2 is KVS; The heavy chain variable region of the 12C3 antibody comprises three complementary determining regions: CDR-H4, CDR-H5 and CDR-H6, ​​and the amino acid sequences of CDR-H4, CDR-H5 and CDR-H6 are shown in SEQ ID NOs. 8 to 10; The light chain variable region of the 12C3 antibody comprises three complementary determining regions: CDR-L4, CDR-L5 and CDR-L6, the amino acid sequences of CDR-L4 and CDR-L6 are shown in SEQ ID NOs. 11-12; the amino acid sequence of CDR-L2 is YTS; The heavy chain variable region of the 11C9 antibody comprises three complementary determining regions: CDR-H7, CDR-H8 and CDR-H9, and the amino acid sequences of CDR-H7, CDR-H8 and CDR-H9 are shown in SEQ ID NOs. 15 to 17; The light chain variable region of the 11C9 antibody comprises three complementary determining regions: CDR-L7, CDR-L8 and CDR-L9, the amino acid sequences of CDR-L7 and CDR-L9 are shown in SEQ ID NOs. 18-19; the amino acid sequence of CDR-L2 is YAS.

[0007] In the present application, the monoclonal antibody comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, a bispecific antibody, and a multispecific antibody; and the light chain of the antibody is of κ type.

[0008] In the present application, the 4H4, 12C3 and 11C9 can specifically target and bind to the IMV form of the monkeypox virus; the 4H4 and 11C9 can target the surface protein A29L of the IMV form of the monkeypox virus, and the 12C3 can target the surface protein E8L of the IMV form of the monkeypox virus.

[0009] Optionally, the amino acid sequence of the heavy chain variable region of the 4H4 antibody comprises: (a1) the amino acid sequence shown in SEQ ID NO.6; or (a2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO. 6 and which has the same function as the protein shown in SEQ ID NO. 6; or (a3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homologous to SEQ ID NO.6 and has the same function as the protein shown in SEQ ID NO.6; The amino acid sequence of the light chain variable region of the 4H4 antibody comprises: (a4) the amino acid sequence shown in SEQ ID NO.7; or (a5) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO. 7 and which has the same function as the protein shown in SEQ ID NO. 7; or (a6) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology with SEQ ID NO.7 and having the same function as the protein shown in SEQ ID NO.7; The amino acid sequence of the heavy chain variable region of the 12C3 antibody comprises: (b1) the amino acid sequence shown in SEQ ID NO.13; or (b2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO. 13 and which has the same function as the protein shown in SEQ ID NO. 13; or (b3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homologous to SEQ ID NO.13 and has the same function as the protein shown in SEQ ID NO.13; The amino acid sequence of the light chain variable region of the 12C3 antibody comprises: (b4) the amino acid sequence shown in SEQ ID NO.14; or (b5) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO. 14 and which has the same function as the protein shown in SEQ ID NO. 14; or (b6) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology with SEQ ID NO.14, and having the same function as the protein shown in SEQ ID NO.14; The amino acid sequence of the heavy chain variable region of the 11C9 antibody comprises: (c1) the amino acid sequence shown in SEQ ID NO.20; or (c2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO. 20 and which has the same function as the protein shown in SEQ ID NO. 20; or (c3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homologous to SEQ ID NO.20 and has the same function as the protein shown in SEQ ID NO.20; The amino acid sequence of the light chain variable region of the 11C9 antibody comprises: (c4) the amino acid sequence shown in SEQ ID NO.21; or (c5) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO. 21 and which has the same function as the protein shown in SEQ ID NO. 21; or (c6) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homologous to SEQ ID NO.21 and has the same function as the protein shown in SEQ ID NO.21.

[0010] In a second aspect, the present application provides a recombinant protein comprising the anti-orthopoxvirus monoclonal antibody and a tag sequence that assists in expression and / or purification.

[0011] In the present application, the tag sequence is selected from at least one of a His tag, a GGGS sequence and a FLAG tag.

[0012] In a third aspect, the present application provides a biomaterial, wherein the biomaterial comprises at least one of d1-d16; (d1) a nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 2 or the recombinant protein according to claim 3; (d2) an expression cassette comprising the nucleic acid molecule described in (d1); (d3) a vector comprising the nucleic acid molecule described in (d1); (d4) a vector comprising the expression cassette described in (d2); (d5) a transgenic cell line comprising the nucleic acid molecule described in (d1); (d6) a transgenic cell line comprising the expression cassette described in (d2); (d7) a transgenic cell line comprising the vector described in (d3); (d8) a transgenic cell line comprising the vector described in (d4); (d9) a microorganism comprising the nucleic acid molecule described in (d1); (d10) a microorganism comprising the expression cassette described in (d2); (d11) a microorganism comprising the vector described in (d3); (d13) a microorganism comprising the vector described in (d4); (d14) A virus comprising the nucleic acid molecule described in (d1); (d15) a virus comprising the expression cassette described in (d2); (d16) A virus comprising the vector described in (d3); (d17) A virus comprising the vector described in (d4).

[0013] In the present application, the transgenic cell line does not contain reproductive material.

[0014] In a fourth aspect, the present application provides a conjugate, comprising the monoclonal antibody or the recombinant protein, and a conjugated part; The coupling portion comprises at least one of a detectable label, a drug, a toxin, an electron-dense label, biotin / avidin, a spin label, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, and a viral coat protein.

[0015] Optionally, the detectable label is a fluorescent or luminescent label.

[0016] In a fifth aspect, the present application provides the use of monoclonal antibodies, recombinant proteins, biomaterials, and conjugates in the preparation of drugs, reagents, test plates, test kits, or test chips.

[0017] Optionally, the drug is a drug for preventing and / or treating orthopoxvirus infection and / or a disease associated with orthopoxvirus infection.

[0018] In the present application, the orthopoxvirus comprises monkeypox virus and / or a virus with high homology to monkeypox virus.

[0019] Optionally, the diseases associated with orthopoxvirus infection include: skin lesions, pneumonia, corneal infection, sepsis, encephalitis, etc.

[0020] Optionally, the virus with high homology to the monkeypox virus includes vaccinia virus; high homology means homology ≥ 90%.

[0021] Optionally, the drug has at least one of the functions of e1-e2: e1 Prevention and / or treatment of orthopoxvirus infection, e2 Prevention and / or treatment of diseases associated with orthopoxviruses; The reagent, detection plate, kit or detection chip has at least one function among f1-f2: f1 detects orthopoxvirus, f2Diagnosis of diseases associated with orthopoxvirus infection.

[0022] In a sixth aspect, the present application provides a pharmaceutical composition comprising the monoclonal antibody, the recombinant protein, the biomaterial or the conjugate, and a pharmacologically acceptable carrier; The pharmaceutical composition has at least one of the functions e1-e2: e1 Prevention and / or treatment of orthopoxvirus infection, e2Prevention and / or treatment of diseases associated with orthopoxviruses.

[0023] In a seventh aspect, the present application provides a detection product, comprising the monoclonal antibody, the recombinant protein, the biological material or the conjugate; The detection product has at least one of the functions f1-f2: f1 detects orthopoxvirus, f2Diagnosis of diseases associated with orthopoxvirus infection.

[0024] Optionally, the detection product is a reagent, a detection plate, a detection chip or a test kit.

[0025] In summary, this application has the following beneficial effects: The present application provides an orthopoxvirus monoclonal antibody 4H4, 12C3 or 11C9, which can specifically recognize and target orthopoxviruses such as monkeypox and vaccinia, and shows the ability to efficiently neutralize viruses, and has good clinical application value in preventing and treating orthopoxvirus infections. Therefore, the anti-orthopoxvirus monoclonal antibodies provided in the present application can be used to prepare drugs for preventing and / or treating orthopoxvirus infections and / or diseases associated with orthopoxvirus infections, or to prepare detection products for detecting orthopoxviruses and diagnosing diseases associated with orthopoxvirus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The purified monkeypox virus protein E8L ( Figure 1 A) and A29L ( Figure 1 B) SDS-PAGE results; Figure 2 The SDS-PAGE results of purified monoclonal antibodies 4H4, 12C3, and 11C9; Figure 3 A is the ELISA binding activity of monoclonal antibody 4H4 and monkeypox virus protein A29L; Figure 3 B is the ELISA binding activity of monoclonal antibody 11C9 and monkeypox virus protein A29L; Figure 3 C is the ELISA binding activity of monoclonal antibody 12C3 and monkeypox virus protein E8L; Figure 3 E is the binding activity of monoclonal antibodies 4H4 and 11C9 with inactivated vaccinia virus (Tiantan strain); Figure 3 F is the binding activity of monoclonal antibody 12C3 with inactivated vaccinia virus (Tiantan strain); Figure 4 A is the results of the in vitro neutralization experiment of monoclonal antibodies 4H4 and 11C9 against vaccinia virus (Tiantan strain); Figure 4 B is the result of the neutralization experiment of monoclonal antibody 12C3 against vaccinia virus (Tiantan strain) in vitro; Figure 4 C shows the results of the neutralization experiment of monoclonal antibodies 4H4, 11C9, and 12C3 against monkeypox virus in vitro. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0028] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, medical microbiology, and immunology laboratory operation procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0029] As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The crystallizable fragment (Fc segment) is equivalent to the CH2 and CH3 functional regions of the antibody, which can form crystals. It has no antigen binding activity, but has functions such as fixing complement and binding to Fc receptors. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain variable region VH and the light chain variable region VL region can also be subdivided into highly variable regions, called complementary determining regions (CDRs), with more conservative framework regions (FRs) interspersed between them. The variable regions (VH and VL) of each heavy chain / light chain pair form antibody binding sites, respectively. The term "antibody" is not limited to any particular method for producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0030] As used herein, the term "monoclonal antibody" refers to an antibody or a fragment of an antibody from a group of extremely homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies and generally contain at least 2 or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using the hybridoma technology first reported by Kohler et al., but can also be obtained using recombinant DNA technology.

[0031] As used herein, the term "single-chain antibody" refers to a single-chain variable region fragment, which is a genetically engineered fusion protein and a small molecule composed of the variable region of the antibody heavy chain and the variable region of the light chain connected by a peptide chain.

[0032] As used herein, "neutralizing antibody" refers to an antibody or antibody fragment that can eliminate or significantly reduce the virulence of a target pathogen.

[0033] As used herein, the term "neutralizing activity" refers to the functional activity of antibodies or antibody fragments that bind to antigenic proteins on viruses, thereby preventing virus-infected cells and / or the maturation of viral progeny and / or the release of viral progeny. Antibodies or antibody fragments with neutralizing activity can prevent the proliferation of viruses, thereby inhibiting or eliminating viral infection.

[0034] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.

[0035] As used herein, the term "homology" refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules. It reflects the relationship between branches originating from the same ancestor in the evolutionary process, and the essential similarity presented by the common origin in evolution or individual development, but their functions are not necessarily the same.

[0036] As used herein, the term "E. coli expression system" refers to an expression system consisting of E. coli (strain) and a vector, wherein the E. coli (strain) is derived from commercially available strains, such as but not limited to: GI698, ER2566, BL21 (DE3), B834 (DE3), BLR (DE3).

[0037] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0038] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0039] As used herein, the term "vaccinia virus (Tiantan strain)" refers to an attenuated strain isolated from a smallpox patient in my country and passaged through inoculation in monkeys, rabbits, cattle and other animals.

[0040] As used herein, the term "TCID 50 ", is the half tissue culture infection dose, also known as the 50% tissue cell infection dose. It refers to the amount of virus required to cause half of the cells in the culture plate wells or test tubes to be damaged or killed, and is used to characterize the titer of the virus.

[0041] As used herein, the term "ELISA" is an enzyme-linked immunosorbent assay, which is an immunoassay method that combines an antigen or antibody on the surface of a solid carrier and utilizes the specific binding of the antigen and antibody and the color reaction of a specific substrate catalyzed by the enzyme labeled on the antibody or antigen to achieve target detection.

[0042] As used herein, the term “IC 50 "" refers to the half-inhibitory concentration of the measured antagonist. In this article, it means the antibody concentration at which the antibody neutralizes the virus to 50% of the maximum effect in the in vitro neutralization experiment.

[0043] In order to obtain neutralizing antibodies with protective effects, the inventors of the present application first used monkeypox virus recombinant proteins A29L (GenBank: QJQ40281.1) and E8L (GenBank: QJQ40248.1) as antigens, and directly subcutaneously immunized BALB / c mice (50ug / mouse) through a sequential immunization strategy, with an interval of two weeks each time. Three days after the impact immunization, the mouse spleen cells were fused with mouse myeloma cells using cell fusion technology, and the recombinant A29L and E8L proteins were coated as antigens to screen and obtain high-titer single monoclonal hybridoma cells. Then, RT-PCR was performed on the single monoclonal hybridoma cells obtained by screening to obtain the sequence encoding the variable region of the antibody. Furthermore, the single monoclonal hybridoma cells were mass-produced in mammalian ascites, and the antibodies were captured by Protein A / G prefabricated gravity columns. A series of functional tests were performed on antibodies 4H4, 11C9, and 12C3, and the results showed that antibodies 4H4, 11C9, and 12C3 could specifically bind to orthopoxviruses and neutralize vaccinia viruses in vitro.

[0044] The raw materials, reagents, solvents, etc. used in the examples of this application can all be obtained commercially. Example 1 Expression and purification of recombinant proteins A29L and E8L of laryngeal poxvirus

[0045] Using EcoRI and XhoI enzymes, DNA fragments encoding monkeypox virus proteins A29L (whose amino acid sequence is shown in SEQ ID NO.22) and E8L (whose amino acid sequence is shown in SEQ ID NO.23) were ligated to the pET25b vector. The ligation product was transformed into BL21 Escherichia coli competent cells, and a single clone was picked and inoculated into 10mL LB medium and cultured at 37°C for 6-8 hours; then it was inoculated into 2L LB medium and cultured at 37°C until OD600 = 0.4-0.6. Subsequently, IPTG was added to the culture to a final concentration of 0.5mM, and the culture was continued at 16°C for 16-20 hours. After the culture was completed, the bacterial precipitate was harvested by high-speed centrifugation. After washing the bacterial precipitate twice with 1×PBS, the cells were ultrasonically broken in an ice water bath. Subsequently, ultracentrifugation was performed, the precipitate was discarded, and the protein in the supernatant solution was purified by Ni-NTA affinity chromatography. After purification, the purity of the target protein was identified by SDS-PAGE, and the results were as follows Figure 1 shown.

[0046] Figure 1 The results showed that Example 1 obtained highly pure A29L and E8L proteins, whose sizes were 11 kDa and 30 kDa, respectively. Example 2 Preparation of monoclonal antibodies 4H4, 12C3, and 11C9

[0047] Using monkeypox virus recombinant proteins A29L (GenBank: QJQ40281.1) and E8L (GenBank: QJQ40248.1) as antigens, BALB / c mice were directly subcutaneously immunized (50ug / mouse) through a sequential immunization strategy, with an interval of two weeks each time. Three days after the impact immunization, the mouse spleen cells were fused with mouse myeloma cells using cell fusion technology, and the recombinant A29L or E8L protein was coated as an antigen to screen for high-titer single monoclonal hybridoma cells. Then, RT-PCR was performed on the single monoclonal hybridoma cells obtained by screening to obtain the sequence encoding the variable region of the antibody (as shown in Table 1 below). Furthermore, the single monoclonal hybridoma cells were mass-produced in mammalian ascites, and the antibodies were captured by Protein A / G prefabricated gravity columns.

[0048] After a large number of experimental studies, two neutralizing monoclonal antibodies targeting A29L (4H4, 11C9) and one neutralizing monoclonal antibody targeting E8L (12C3) were discovered. Figure 2 The SDS-PAGE results showed that three highly pure monoclonal antibodies were obtained, with the molecular weights of the heavy chain and light chain being around 50 kDa and 25 kDa, respectively.

[0049] Table 1 Sequence information encoding antibody variable regions

[0050] Example 3 To investigate the ability of 4H4, 12C3, and 11C9 antibodies to specifically bind to pea sprout virus (Tiantan strain)

[0051] (I) Detecting the binding ability of monoclonal antibodies and antigen proteins using ELISA: The monkeypox proteins A29L and E8L were coated on the ELISA plate at a concentration of 1 μg / ml respectively; then the 96-well plate was blocked with 5% skim milk powder. The 4H4, 12C3, and 11C9 monoclonal antibodies to be tested were diluted gradiently and added to the above-mentioned ELISA plate at a volume of 100 μL per well. The control group was an irrelevant IgG and incubated at 37°C for 30 minutes. The ELISA plate was washed three times with ELISA washing solution (PBST), and then 100 μL of diluted horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody was added and incubated at 37°C for 30 minutes. After washing the ELISA plate three times with PBST, the color developer was added and the reaction was terminated after 15 minutes of color development. The absorbance value of A450nm was then read on the ELISA reader, and the results are as follows: Figure 3 As shown in A~3C.

[0052] Depend on Figure 3As shown in A~3C, antibodies 4H4 and 11C9 can specifically target and bind to monkeypox virus protein A29L, antibody 12C3 can specifically target and bind to monkeypox virus protein E8L, while the PBS control does not show specific binding activity.

[0053] (II) Detection of the binding ability of monoclonal antibodies and vaccinia virus (Tiantan strain) using ELISA: Remove the supernatant from BHK-21 cells infected with vaccinia virus (Tiantan strain) for 48 hours, add an appropriate amount of 1×PBS, and freeze and thaw three times at -80℃. Discard the precipitate by high-speed centrifugation to obtain the supernatant virus liquid. Inactivate the virus liquid at 56℃ for 30 minutes and store it at -80℃ for a long time. Dilute the inactivated virus liquid prepared above 20 times and coat the ELISA plate. Subsequent operations are the same as before. The results are as follows Figure 3 As shown in E~3F.

[0054] Depend on Figure 3 From E to 3F, it can be seen that antibodies 4H4, 11C9, and 12C3 can specifically target and bind to vaccinia virus, among which 12C3 has the strongest ability to bind to inactivated virus.

[0055] Example 4 Evaluation of the ability of monoclonal antibodies 4H4, 12C3, and 11C9 to neutralize vaccinia virus and poxvirus in vitro

[0056] (I) Neutralization test of vaccinia virus (Tiantan strain): The neutralizing activity of the antibody was determined by microneutralization test (MRNT). Monoclonal antibodies 4H4, 11C9, and 12C3 were diluted in series and then mixed with an equal volume of 100TCID50 of virus at 37°C for 1 hour. After incubation, the virus was added to a 96-well plate pre-inoculated with BHK-21 cells and adsorbed in a 37°C, 5% CO2 incubator for 1 hour before the supernatant was removed. After washing, maintenance medium (containing 2% fetal bovine serum) was added and cultured for another 4 days; after 4 days, the cytopathic effect (CPE) was observed and the neutralizing titer of the antibody was calculated (half inhibitory concentration, IC 50 ), the results are as follows Figure 4 A, as shown in 4B.

[0057] From 4A and 4B, we can see that the IC values ​​of 4H4, 11C9, and 12C3 against vaccinia virus (Tiantan strain) are 50 They were 7.78ug / mL, 29.26μg / mL and 13.80μg / mL respectively, indicating that monoclonal antibodies 4H4, 11C9 and 12C3 have the ability to neutralize vaccinia virus to varying degrees, among which 4H4 has the strongest neutralizing activity, followed by 12C3.

[0058] 2. Neutralization test of monkeypox virus: The neutralizing activity of antibodies was determined by the focus reduction neutralization test (FRNT) performed in a biosafety level 3 laboratory. The antibodies were serially diluted and mixed with an equal volume of monkeypox virus for incubation at 37°C for 1 hour; the mixture (containing 200 infectious units of live virus) was then transferred to a 96-well plate pre-seeded with Vero E6 cells and adsorbed in a 37°C, 5% CO2 incubator for 1 hour before removal; after washing, maintenance medium (containing 1.6% carboxymethyl cellulose and 2% fetal bovine serum) was added, and then incubated at 37°C, 5% CO2 for 24 hours; after 24 hours, the supernatant was removed, and the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then incubated with the corresponding monoclonal antibody conjugated to HRP; the reaction was developed using KPL TrueBlue peroxidase substrate; the number of monkeypox virus infection foci was counted using ELISpot, and the results are shown in Figure 2. Figure 4 As shown in C.

[0059] Depend on Figure 4 C shows that the IC50 of 4H4, 11C9 and 12C3 against monkeypox virus are 8.19ug / mL, 22.81μg / mL and 13.86μg / mL, respectively, indicating that monoclonal antibodies 4H4, 11C9 and 12C3 have the ability to neutralize monkeypox virus to varying degrees, among which 4H4 has the strongest neutralizing activity, followed by 12C3.

[0060] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A monoclonal antibody against orthopoxvirus, characterized in that: The antibody is 4H4, 12C3 or 11C9; The heavy chain variable region of the 4H4 antibody comprises three complementary determining regions: CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NOs. 1 to 3; The light chain variable region of the 4H4 antibody comprises three complementary determining regions: CDR-L1, CDR-L2 and CDR-L3, the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NOs. 4-5; the amino acid sequence of CDR-L2 is KVS; The heavy chain variable region of the 12C3 antibody comprises three complementary determining regions: CDR-H4, CDR-H5 and CDR-H6, ​​and the amino acid sequences of CDR-H4, CDR-H5 and CDR-H6 are shown in SEQ ID NOs. 8 to 10; The light chain variable region of the 12C3 antibody comprises three complementary determining regions: CDR-L4, CDR-L5 and CDR-L6, the amino acid sequences of CDR-L4 and CDR-L6 are shown in SEQ ID NOs. 11-12; the amino acid sequence of CDR-L2 is YTS; The heavy chain variable region of the 11C9 antibody comprises three complementary determining regions: CDR-H7, CDR-H8 and CDR-H9, and the amino acid sequences of CDR-H7, CDR-H8 and CDR-H9 are shown in SEQ ID NOs. 15 to 17; The light chain variable region of the 11C9 antibody comprises three complementary determining regions: CDR-L7, CDR-L8 and CDR-L9, the amino acid sequences of CDR-L7 and CDR-L9 are shown in SEQ ID NOs. 18-19; the amino acid sequence of CDR-L2 is YAS.

2. The anti-orthopoxvirus monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the 4H4 antibody comprises: (a1) the amino acid sequence shown in SEQ ID NO.6; or (a2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO. 6 and which has the same function as the protein shown in SEQ ID NO. 6; or (a3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homologous to SEQ ID NO.6 and has the same function as the protein shown in SEQ ID NO.6; The amino acid sequence of the light chain variable region of the 4H4 antibody comprises: (a4) the amino acid sequence shown in SEQ ID NO.7; or (a5) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO. 7 and which has the same function as the protein shown in SEQ ID NO. 7; or (a6) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology with SEQ ID NO.7 and having the same function as the protein shown in SEQ ID NO.7; The amino acid sequence of the heavy chain variable region of the 12C3 antibody comprises: (b1) the amino acid sequence shown in SEQ ID NO.13; or (b2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO. 13 and which has the same function as the protein shown in SEQ ID NO. 13; or (b3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homologous to SEQ ID NO.13 and has the same function as the protein shown in SEQ ID NO.13; The amino acid sequence of the light chain variable region of the 12C3 antibody comprises: (b4) the amino acid sequence shown in SEQ ID NO.14; or (b5) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO. 14 and which has the same function as the protein shown in SEQ ID NO. 14; or (b6) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology with SEQ ID NO.14, and having the same function as the protein shown in SEQ ID NO.14; The amino acid sequence of the heavy chain variable region of the 11C9 antibody comprises: (c1) the amino acid sequence shown in SEQ ID NO.20; or (c2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO. 20 and which has the same function as the protein shown in SEQ ID NO. 20; or (c3) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homologous to SEQ ID NO.20 and has the same function as the protein shown in SEQ ID NO.20; The amino acid sequence of the light chain variable region of the 11C9 antibody comprises: (c4) the amino acid sequence shown in SEQ ID NO.21; or (c5) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO. 21 and which has the same function as the protein shown in SEQ ID NO. 21; or (c6) an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homologous to SEQ ID NO.21 and has the same function as the protein shown in SEQ ID NO.

21.

3. A recombinant protein, characterized in that The invention comprises the anti-orthopoxvirus monoclonal antibody according to claim 1 or 2 and a tag sequence for assisting expression and / or purification.

4. A biomaterial, characterized in that: The biological material comprises at least one of d1-d16; (d1) a nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 2 or the recombinant protein according to claim 3; (d2) an expression cassette comprising the nucleic acid molecule described in (d1); (d3) a vector comprising the nucleic acid molecule described in (d1); (d4) a vector comprising the expression cassette described in (d2); (d5) a transgenic cell line comprising the nucleic acid molecule described in (d1); (d6) a transgenic cell line comprising the expression cassette described in (d2); (d7) a transgenic cell line comprising the vector described in (d3); (d8) a transgenic cell line comprising the vector described in (d4); (d9) a microorganism comprising the nucleic acid molecule described in (d1); (d10) a microorganism comprising the expression cassette described in (d2); (d11) a microorganism comprising the vector described in (d3); (d13) a microorganism comprising the vector described in (d4); (d14) A virus comprising the nucleic acid molecule described in (d1); (d15) a virus comprising the expression cassette described in (d2); (d16) A virus comprising the vector described in (d3); (d17) A virus comprising the vector described in (d4).

5. A conjugate, characterized in that: Comprising the monoclonal antibody according to claim 1 or 2 or the recombinant protein according to claim 3, and a coupling portion; The coupling portion comprises at least one of a detectable label, a drug, a toxin, an electron-dense label, biotin / avidin, a spin label, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, and a viral coat protein.

6. Use of the monoclonal antibody according to claim 1 or 2, the recombinant protein according to claim 3, the biomaterial according to claim 4, or the conjugate according to claim 5 in the preparation of drugs, reagents, test plates, test kits or test chips.

7. The use according to claim 6, characterized in that The drug has at least one of the functions of e1-e2: e1 Prevention and / or treatment of orthopoxvirus infection, e2Prevention and / or treatment of diseases associated with orthopoxviruses; The reagent, detection plate, kit or detection chip has at least one function among f1-f2: f1 detects orthopoxvirus, f2Diagnosis of diseases associated with orthopoxvirus infection.

8. A pharmaceutical composition, characterized in that Comprising the monoclonal antibody according to claim 1 or 2, the recombinant protein according to claim 3, the biomaterial according to claim 4 or the conjugate according to claim 5, and a pharmacologically acceptable carrier; The pharmaceutical composition has at least one of the functions e1-e2: e1 Prevention and / or treatment of orthopoxvirus infection, e2Prevention and / or treatment of diseases associated with orthopoxviruses.

9. A detection product, characterized in that: Comprising the monoclonal antibody according to claim 1 or 2, the recombinant protein according to claim 3, the biological material according to claim 4 or the conjugate according to claim 5; The detection product has at least one of the functions f1-f2: f1 detects orthopoxvirus, f2Diagnosis of diseases associated with orthopoxvirus infection.

10. The detection product according to claim 9, characterized in that: The detection product is a reagent, a detection plate, a detection chip or a test kit.

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