Anti-monkeypox virus antibody or antigen-binding fragment thereof, reagent and kit for detecting monkeypox virus

Abstract

The present invention discloses an anti-monkeypox virus antibody or an antigen-binding fragment thereof, a reagent and a kit for detecting monkeypox virus, and relates to the field of antibodies. The anti-monkeypox virus antibody disclosed in the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region, and the antibody provides an important source of raw materials for the detection of monkeypox virus and has improved affinity or activity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present disclosure claims priority to a Chinese patent application with application number 202211126051.X filed with the Chinese Patent Office on September 16, 2022, and entitled “Anti-monkeypox virus antibodies or functional fragments thereof, reagents and kits for detecting monkeypox virus”, the entire contents of which are incorporated by reference into the present disclosure. Technical Field

[0003] The present invention relates to the field of antibody technology, and in particular to an anti-monkeypox virus antibody or an antigen-binding fragment thereof, and a reagent and a kit for detecting monkeypox virus. Background Art

[0004] Monkeypox is caused by Monkeypox virus (MPXV). Monkeypox virus is a double-stranded DNA virus belonging to the genus Orthopoxvirus of the Poxviridae family. Monkeypox is a viral zoonosis that occurs mainly in the tropical rainforests of Central and West Africa and is occasionally exported to other regions. Current studies have found that the virus invades the human body through mucous membranes and broken skin. People are mainly infected through contact with lesions, blood, and other body fluids of infected animals, or through bites and scratches from infected animals. It is mainly transmitted from person to person through close contact, but can also be transmitted through droplets, contact with objects contaminated with the virus, and vertical transmission through the placenta. Sexual transmission cannot be ruled out.

[0005] In terms of clinical symptoms, the symptoms of monkeypox infection are similar to those of smallpox, but the clinical symptoms of monkeypox are milder, with an incubation period of 5-12 days, mostly 6-13 days. The initial symptoms of the patient are fever, headache, swollen lymph nodes, muscle aches, severe fatigue, and then a rash on the face and body. Monkeypox is a self-limiting disease, and patients usually recover on their own within two to three weeks; but for children, pregnant women, or people who are immunosuppressed due to other health conditions, monkeypox may cause other secondary infections, such as pneumonia, sepsis, and encephalitis, which are serious diseases.

[0006] At present, the detection methods of monkeypox virus are mainly PCR amplification and immunoassay. PCR amplification is the main detection method for monkeypox virus at home and abroad, but it has high requirements for instruments and equipment, detection sites and environmental conditions, and has disadvantages such as long detection time and low throughput, which is not convenient for large-scale detection of the population. The immunoassay based on colloidal gold has the advantages of being fast, convenient, and easy to carry, so it has become a hot spot for research and development, and the detection performance of colloidal gold depends on the performance of antibodies against monkeypox virus. Therefore, there is a strong demand in this field for antibodies that effectively bind to monkeypox virus and detect it. Summary of the invention

[0007] The object of the present invention is to provide an anti-monkeypox virus antibody or an antigen-binding fragment thereof, and a reagent and a kit for detecting monkeypox virus or monkeypox virus antigen.

[0008] The present invention is achieved in that:

[0009] In a first aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1 / HCDR2 / HCDR3 combination is the same as the HCDR1 / HCDR2 / HCDR3 combination contained in the heavy chain variable region shown in any one of SEQ ID NOs:7 to 10, and the LCDR1 / LCDR2 / LCDR3 combination is the same as the LCDR1 / LCDR2 / LCDR3 combination contained in the light chain variable region shown in SEQ ID NO:11.

[0010] In an optional embodiment, the antibody or antigen-binding fragment thereof comprises any one of (a) to (c):

[0011] (a) HCDR1-3 and LCDR1-3; the amino acid sequences of HCDR1, HCDR2 and HCDR3 include SEQ ID Nos: 1-3 or are shown in sequence as SEQ ID Nos: 1-3; the amino acid sequences of LCDR1, LCDR2 and LCDR3 include SEQ ID Nos: 4-6 or are shown in sequence as SEQ ID Nos: 4-6;

[0012] (b) a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 7 to 10; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 11;

[0013] (c) a heavy chain variable region and / or a light chain variable region having an amino acid sequence that is at least 80% identical to the heavy chain variable region and / or light chain variable region sequence shown in (b), and comprising HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence shown in (a).

[0014] In a second aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, wherein the binding epitope of the antibody or the antigen-binding fragment thereof is the same as the binding epitope of the antibody or the antigen-binding fragment thereof described in any one of the above items; or, the antibody or the antigen-binding fragment thereof competitively binds to the same epitope as the antibody or the antigen-binding fragment thereof described in any one of the above items.

[0015] In a third aspect, an embodiment of the present invention provides an antibody conjugate, which includes the antibody or antigen-binding fragment thereof as described in the above embodiments.

[0016] In a fourth aspect, an embodiment of the present invention provides a reagent or a kit, which includes the antibody or antigen-binding fragment thereof as described in the preceding embodiments or the antibody conjugate as described in the preceding embodiments.

[0017] In a fifth aspect, an embodiment of the present invention provides a method for detecting monkeypox virus or monkeypox virus antigen, which comprises: contacting the antibody or antigen-binding fragment thereof, antibody conjugate, reagent or kit as described in the above embodiments with the monkeypox virus or monkeypox virus antigen in the sample to be tested to form an immune complex.

[0018] In a sixth aspect, an embodiment of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof described in the preceding embodiments.

[0019] In a seventh aspect, an embodiment of the present invention provides a vector comprising the isolated nucleic acid described in the preceding embodiment.

[0020] In an eighth aspect, an embodiment of the present invention provides a cell containing the isolated nucleic acid described in the previous embodiment or the vector described in the previous embodiment.

[0021] In a ninth aspect, an embodiment of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof described in the preceding embodiment, comprising: culturing the cells described in the preceding embodiment.

[0022] In a tenth aspect, embodiments of the present invention provide the use of antibodies or antigen-binding fragments thereof, antibody conjugates, reagents or kits as described in the preceding embodiments for detecting monkeypox virus or monkeypox virus antigens or for preparing products for detecting monkeypox virus or monkeypox virus antigens.

[0023] The present invention has the following beneficial effects:

[0024] The anti-monkeypox virus antibody disclosed in the present invention comprises the heavy chain complementary determining region and the light chain complementary determining region. The antibody provides an important source of raw materials for the detection of monkeypox virus or monkeypox virus antigen and has improved affinity or activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0026] Figure 1The results of reducing SDS-PAGE of Anti-MPXV 11D3RMb1 to 4 are shown. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0028] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0029] In a first aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1 / HCDR2 / HCDR3 combination is the same as the HCDR1 / HCDR2 / HCDR3 combination contained in the heavy chain variable region shown in any one of SEQ ID NOs:7 to 10, and the LCDR1 / LCDR2 / LCDR3 combination is the same as the LCDR1 / LCDR2 / LCDR3 combination contained in the light chain variable region shown in SEQ ID NO:11.

[0030] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

[0031] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

[0032] In the present invention, the heavy chain complementary determining region is represented by HCDR, and the three CDRs contained in the heavy chain variable region include HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, and the three CDRs contained in the light chain variable region include LCDR1, LCDR2 and LCDR3.

[0033] In alternative embodiments, the CDRs are defined by the Kabat, Chothia, IMGT or Lesk numbering systems.

[0034] In alternative embodiments, the CDRs may be defined according to the Kabat, Chothia, AbM, Contact or IMGT systems. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely adopted standard for numbering antibody residues. There are other CDRs definitions that may not strictly follow one of the above methods, but will still overlap with at least a portion of the CDRs defined by Kabat, although they may be shortened or extended based on predictions or experimental results for specific residues or groups of residues. As described herein, CDRs may refer to CDRs defined by any method known in the art, including a combination of methods.

[0035] In alternative embodiments, the CDRs may refer to CDRs defined by any method known in the art.

[0036] In an alternative embodiment, the antibody or antigen-binding fragment thereof comprises:

[0037] (a) The amino acid sequences of HCDR1, HCDR2 and HCDR3 include SEQ ID Nos: 1 to 3 or are shown in sequence as SEQ ID Nos: 1 to 3; the amino acid sequences of LCDR1, LCDR2 and LCDR3 include SEQ ID Nos: 4 to 6 or are shown in sequence as SEQ ID Nos: 4 to 6.

[0038] In an optional embodiment, the CDRs are based on the CDRs defined by the Kabat system, with the C-terminus of HCDR3 shortened by 3 amino acids and the C-terminus of LCDR3 shortened by 2 amino acids.

[0039] In a second aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises: (a) the amino acid sequences of HCDR1, HCDR2 and HCDR3 comprise SEQ ID Nos: 1 to 3 in sequence or are shown in sequence as SEQ ID Nos: 1 to 3; the amino acid sequences of LCDR1, LCDR2 and LCDR3 comprise SEQ ID Nos: 4 to 6 in sequence or are shown in sequence as SEQ ID Nos: 4 to 6.

[0040] In an optional embodiment, the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 correspond to the amino acids in the H31-H35, H50-H65, H95-H100B, L24-34, L50-56, L89-95 position segments under Kabat numbering, respectively.

[0041] In an alternative embodiment, the antibody or antigen-binding fragment thereof further comprises a framework region.

[0042] In an optional embodiment, the antibody or antigen-binding fragment thereof described in the first aspect or the second aspect comprises:

[0043] (b) a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 7 to 10, or consists of the amino acid sequence shown in any one of SEQ ID NOs: 7 to 10; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11, or consists of the amino acid sequence shown in SEQ ID NO: 11.

[0044] In a third aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises: (b) a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 7 to 10, or consists of the amino acid sequence shown in any one of SEQ ID NOs: 7 to 10; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11, or consists of the amino acid sequence shown in SEQ ID NO: 11.

[0045] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region other than the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0046] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0047] In an optional embodiment, the antibody or its functional fragment comprises:

[0048] (c) a heavy chain variable region and / or a light chain variable region having an amino acid sequence that is at least 80% identical to the heavy chain variable region and / or light chain variable region sequence shown in (b), and comprising HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence shown in (a).

[0049] In an optional embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region in a heavy chain variable region shown in any one of SEQ ID NOs:7 to 10, and a light chain framework region in a light chain variable region shown in SEQ ID NO:11.

[0050] In an optional embodiment, the framework region amino acid sequence of the antibody or antigen-binding fragment thereof may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned framework region.

[0051] In an alternative embodiment, the antibody or antigen-binding fragment thereof further comprises a constant region.

[0052] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0053] In an optional embodiment, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; and the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0054] In an optional embodiment, the species origin of the constant region is any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese and humans.

[0055] In an alternative embodiment, the heavy chain constant region comprises SEQ ID NO: 12 or an amino acid sequence that is at least 80% identical thereto.

[0056] In an alternative embodiment, the heavy chain constant region consists of SEQ ID NO: 12 or an amino acid sequence that is at least 80% identical thereto.

[0057] In an alternative embodiment, the light chain constant region comprises SEQ ID NO: 13 or an amino acid sequence at least 80% identical thereto.

[0058] In an alternative embodiment, the light chain constant region consists of SEQ ID NO: 13 or an amino acid sequence that is at least 80% identical thereto.

[0059] In an optional embodiment, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain; the heavy chain comprises an amino acid sequence shown in any one of SEQ ID NOs: 14 to 17 or having at least 80% identity therewith; or, consists of an amino acid sequence shown in any one of SEQ ID NOs: 14 to 17 or having at least 80% identity therewith; the light chain comprises SEQ ID NO: 18 or an amino acid sequence having at least 80% identity therewith; or, consists of SEQ ID NO: 18 or an amino acid sequence having at least 80% identity therewith.

[0060] Specifically, the above-mentioned "at least 80% identity" can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, and the variant site of identity is not in the CDRs region.

[0061] On the other hand, an embodiment of the present invention further provides an antibody or an antigen-binding fragment thereof, wherein the binding epitope of the antibody or the antigen-binding fragment thereof is the same as the binding epitope of the antibody or the antigen-binding fragment thereof described in any one of the above items; or, the antibody or the antigen-binding fragment thereof competitively binds to the same epitope as the antibody or the antigen-binding fragment thereof described in any one of the above items.

[0062] In an optional embodiment, the same epitope is located within the amino acid sequence 1 to 110 of the monkeypox virus A29L protein; or, the antibody or antigen-binding fragment thereof competes with any of the above antibodies or antigen-binding fragments for binding to the monkeypox virus A29L protein.

[0063] In an alternative embodiment, the antibody or antigen-binding fragment thereof has a KD of ≤7.36×10 -8 Affinity binding of M to monkeypox virus antigens.

[0064] In an alternative embodiment, the antibody or antigen-binding fragment thereof has a KD≤10 -8 M, KD≤10 -9 M, KD≤10 -10 M, KD≤10 -11 M, KD≤10 -12 Affinity binding of M to monkeypox virus antigens.

[0065] In an alternative embodiment, the antibody or antigen-binding fragment thereof has a KD of ≤1.78×10 -9 Affinity binding of M to monkeypox virus antigens.

[0066] In an optional embodiment, the antigen binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3 described in any of the above embodiments, and the light chain variable region comprises LCDR1 to LCDR3 described in any of the above embodiments.

[0067] In an optional embodiment, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0068] Antigen-binding fragments of the above antibodies generally have the same binding specificity as the antibody from which they are derived.

[0069] In the present invention, the terms "antigen-binding fragment" and "functional fragment" having the same binding specificity as the antibody from which it is derived are used interchangeably.

[0070] Those skilled in the art will readily understand based on the contents described in the present invention that the antigen-binding fragments of the above-mentioned antibodies can be obtained by, for example, enzymatic digestion methods (including pepsin or papain) and / or by chemical reduction methods for splitting disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, those skilled in the art can readily obtain the above-mentioned antigen-binding fragments.

[0071] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0072] On the other hand, an embodiment of the present invention further provides an antibody conjugate, which includes the antibody or antigen-binding fragment thereof as described in the above embodiment.

[0073] In an optional embodiment, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof.

[0074] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody or antigen-binding fragment thereof.

[0075] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0076] In an optional embodiment, the marker is selected from at least one of a fluorescent dye, an enzyme, a radioactive isotope, a chemiluminescent agent and a nanoparticle marker.

[0077] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0078] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0079] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0080] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0081] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0082] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0083] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.

[0084] In an optional embodiment, the colloidal metal includes but is not limited to colloidal gold or colloidal silver.

[0085] In an optional embodiment, the colloidal metal is colloidal gold.

[0086] In an optional embodiment, the antibody conjugate further comprises a solid phase carrier coupled to the antibody or antigen binding fragment thereof. In the antibody conjugate, the antibody is coupled to the solid phase carrier.

[0087] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0088] In an optional embodiment, the solid phase includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

[0089] In an optional embodiment, the solid phase carrier is a nitrocellulose membrane.

[0090] On the other hand, an embodiment of the present invention further provides a reagent or a kit, which includes the antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments or the antibody conjugate as described in any of the foregoing embodiments.

[0091] In an alternative embodiment, the reagent or kit has improved detection sensitivity or specificity.

[0092] On the other hand, an embodiment of the present invention further provides a method for detecting monkeypox virus or monkeypox virus antigen, comprising:

[0093] The antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments, the antibody conjugate as described in any of the foregoing embodiments, or the reagent or kit as described in any of the foregoing embodiments is contacted with the monkeypox virus or monkeypox virus antigen in the sample to be detected to form an immune complex.

[0094] In a preferred embodiment, the immune complex further comprises a second antibody that binds to the antibody or antigen-binding fragment thereof.

[0095] In a preferred embodiment, the immune complex further comprises a second antibody, which binds to the monkeypox virus or a monkeypox virus antigen.

[0096] On the other hand, an embodiment of the present invention also provides the use of the antibody or antigen-binding fragment described in any of the preceding embodiments, or the antibody conjugate described in any of the preceding embodiments, or the reagent or kit described in any of the preceding embodiments in detecting monkeypox virus or monkeypox virus antigen or preparing a product for detecting monkeypox virus or monkeypox virus antigen.

[0097] On the other hand, an embodiment of the present invention also provides the use of the antibody or antigen-binding fragment described in any of the preceding embodiments, or the antibody conjugate described in any of the preceding embodiments, or the reagent or kit described in any of the preceding embodiments in the preparation of a product having at least one of the following uses, wherein the uses include: diagnosing or assisting in diagnosing diseases related to monkeypox virus infection, and predicting or assisting in predicting at least one of the prognosis and efficacy of diseases related to monkeypox virus infection.

[0098] In an optional embodiment, the related diseases caused by the monkeypox virus infection include at least one of fever, headache, swollen lymph nodes, muscle aches, severe fatigue, pneumonia, sepsis and encephalitis.

[0099] In an alternative embodiment, the product comprises a reagent or a kit.

[0100] In an alternative embodiment, the reagent or kit has improved detection sensitivity or specificity.

[0101] On the other hand, an embodiment of the present invention further provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof described in any of the above embodiments.

[0102] On the other hand, an embodiment of the present invention further provides a vector comprising the isolated nucleic acid described in any of the above embodiments.

[0103] On the other hand, an embodiment of the present invention further provides a cell, which contains the isolated nucleic acid described in any of the foregoing embodiments or the vector described in any of the foregoing embodiments.

[0104] On the other hand, an embodiment of the present invention further provides a method for preparing the antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, comprising: culturing the cell as described in any of the preceding embodiments.

[0105] Based on the amino acid sequence of the antibody or its antigen-binding fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its antigen-binding fragment, for example, separating and purifying the antibody or its antigen-binding fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its antigen-binding fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its antigen-binding fragment of the present invention, it belongs to the protection scope of the present invention.

[0106] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0107] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0108] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0109] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0110] Example 1 Preparation of Anti-MPXV 11D3 Monoclonal Antibody

[0111] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by a gene sequencing company. The hybridoma cell line secreting the Anti-MPXV 11D3 monoclonal antibody was prepared in this laboratory and was revived for future use.

[0112] 1. Expression plasmid construction

[0113] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TM RACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by a gene sequencing company.

[0114] 1.1 Anti-MPXV 11D3 antibody gene preparation

[0115] mRNA was extracted from the hybridoma cell line secreting Anti-MPXV 11D3 monoclonal antibody, and the DNA product was obtained by RT-PCR. It was inserted into the pMD-18T vector and transformed into DH5α competent cells. After the colonies grew, 4 positive clones of the Heavy Chain and Light Chain genes were taken and sent to a gene sequencing company for sequencing.

[0116] 1.2 Sequence analysis of the variable region gene of Anti-MPXV 11D3 antibody

[0117] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. In the gene fragment amplified by the Light Chain, the VL gene sequence was 333bp, and there was a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 366bp, belonging to the VH1 gene family, and there was a 57bp leader peptide sequence in front of it.

[0118] 1.3 Construction of recombinant antibody expression plasmid

[0119] pcDNA TM 3.4 vector is a constructed recombinant antibody eukaryotic expression vector, which has been modified to introduce multiple cloning restriction sites and is subsequently referred to as 3.4A expression vector; based on the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the Anti-MPXV 11D3 antibody were designed, with restriction endonuclease sites and protective bases at both ends, respectively, and the 0.71KB Light Chain gene fragment and the 1.41kb Heavy Chain gene fragment were amplified by the PCR amplification method.

[0120] The Heavy Chain and Light Chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was double-digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of the Heavy Chain and Light Chain, respectively.

[0121] 2. Recombinant Antibody Sample Preparation

[0122] Resuscitate HEK293 cells in advance and subculture them to 200 ml system to make the cell density reach 3-5×10 6 cells / ml, cell viability>95%; wash the cells by centrifugation, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6 cells / ml, as cell diluent. Prepare plasmid DNA and transfection reagent diluents with culture medium respectively. Add transfection reagent diluent to plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to cell diluent within 1 minute, mix well and take samples to count, record and observe the viability of cells after transfection, and place them in a 35℃ constant temperature incubator for culture, speed 120rmp, CO2 content 8%, and centrifuge after 13 days. The supernatant of centrifugation was affinity purified using protein A affinity chromatography column. Take 6μg of purified antibody for reducing SDS-PAGE, and the electrophoresis is shown in the figure. After reducing SDS-PAGE, two bands are shown, one Mr is 50KD (heavy chain) and the other Mr is 28KD (light chain).

[0123] 3. Affinity and activity optimization

[0124] Although the Anti-MPXV 11D3 monoclonal antibody prepared above has the ability to bind to monkeypox virus antigens, its affinity and antibody activity are not ideal, so the applicant performs directed mutation on the variable region of the antibody. That is, the antibody variable region structure simulation, antigen and antibody variable region complex structure simulation, antibody key amino acid analysis and mutation design are performed by computer, bidirectional primers covering the mutation site are designed and synthesized according to the mutation scheme, primers at both ends of the target DNA are synthesized, high-fidelity PCR reaction is performed, the PCR product is cloned into the vector, and then the mutant antibody is prepared according to the above method 2. After screening, monoclonal antibodies with significantly improved affinity and antibody activity are obtained, and are named Anti-MPXV 11D3RMb1 to Anti-MPXV 11D3RMb4, and their heavy chain and light chain amino acid sequences are as follows.

[0125] Table 1 Antibody sequences

[0126] Sample name Heavy chain number Light chain number Anti-MPXV 11D3RMb1 SEQ ID NO:14 SEQ ID NO:18 Anti-MPXV 11D3RMb2 SEQ ID NO:15 SEQ ID NO:18 Anti-MPXV 11D3RMb3 SEQ ID NO:16 SEQ ID NO:18 Anti-MPXV 11D3RMb4 SEQ ID NO:17 SEQ ID NO:18

[0127] Example 2 Affinity Analysis

[0128] The purified antibody was diluted in advance, and the monkeypox recombinant antigen (purchased from Feipeng Bio) was diluted in gradients at the same time; the binding and dissociation curves of the antigen and antibody were tested on the Biacore 8K+ device using a CM5 chip that had been pre-coupled with goat anti-mouse IgG, and the instrument automatically fitted to obtain the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., affinity constant; ka represents the binding rate; and kd represents the dissociation rate).

[0129] Table 2 Affinity analysis data

[0130]

[0131]

[0132] Example 3 Activity Identification

[0133] The coating solution (main component NaHCO3) was diluted with recombinant monkeypox recombinant antigen (purchased from Feipeng Bio) to 1ug / ml, 100uL per well, and incubated at 4°C overnight; the next day, the cells were washed twice with washing solution (main component Na2HPO4+Nacl), and patted dry; blocking solution (20% BSA+80% PBS) was added, 120uL per well, incubated at 37°C for 1h, and patted dry; the diluted purified antibody and control antibody were added, 100uL / well, incubated at 37°C for 30min; the cells were washed 5 times with washing solution, and patted dry; goat anti-mouse IgG-HRP was added, 100uL per well, incubated at 37°C for 30min; the cells were washed 5 times with washing solution, and patted dry; color developing solution A (50uL / well) and color developing solution B (50uL / well) were added, and the cells were incubated for 10min; the stop solution was added, 50uL / well; the OD value was read at 450nm (reference 630nm) on the microplate reader.

[0134] Table 3 Activity data

[0135] Concentration (ng / ml) 31.25 15.63 7.81 3.91 1.95 0.00 Control Antibodies 1.324 0.754 0.402 0.233 0.114 0.017 Anti-MPXV 11D3RMb1 2.148 1.757 0.995 0.502 0.165 0.012 Anti-MPXV 11D3RMb2 2.144 1.778 0.972 0.524 0.174 0.013 Anti-MPXV 11D3RMb3 2.158 1.756 0.998 0.558 0.185 0.012 Anti-MPXV 11D3RMb4 2.176 1.732 0.923 0.576 0.177 0.015

[0136] Example 4 Antibody Stability Assessment

[0137] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant temperature box) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and the activity of the 21-day samples was tested. The results showed that there was no obvious protein state change in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the expressed antibodies were stable. The following table shows the OD results of the enzyme immunoassay activity test after 21 days of testing.

[0138] Table 4 Stability data

[0139] Sample concentration (ng / ml) 15.63 7.81 0.00 4℃, 21-day samples 1.756 0.927 0.012 -80℃, 21 days sample 1.738 0.938 0.013 37℃, 21 days sample 1.772 0.991 0.011

[0140] Example 5 Performance Evaluation

[0141] The reactivity of the above-prepared antibodies with monkeypox virus A29L protein was identified by ELISA. The ELISA operation was as follows: 0.5 μg / ml and 0.05 μg / ml of MKPVA29L were used to coat the microtiter plate at 100 ul / well, respectively, and incubated at 37°C for 2 hours. BSA was blocked. The above-prepared antibodies were diluted to 0.5 ug / ml and 0.05 ug / ml with PBS, 50 ul / well, and incubated at 37°C for 30 minutes.

[0142] Negative control: add PBS for incubation.

[0143] Secondary antibody: 100ul / well, add goat anti-mouse IgG-HRP, incubate at 37℃ for 30min.

[0144] Color development: add 50ul of solution A and solution B respectively, add 50ul of stop solution after 10 minutes, and read the results.

[0145] The results showed that the reactivity of the antibodies prepared above with monkeypox virus A29L protein was better than that of the control.

[0146] Table 5 Performance evaluation data

[0147]

[0148] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0149] Some of the amino acid sequences involved in this application are shown in Table 6 below:

[0150] Table 6 Amino acid sequence

[0151]

[0152]

Claims

1. An antibody or antigen-binding fragment thereof against monkeypox virus, wherein the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1, HCDR2, and HCDR3 combination is the same as the HCDR1, HCDR2, and HCDR3 combination contained in the heavy chain variable region shown in any one of SEQ ID NOs: 7 to 10; the LCDR1, LCDR2, and LCDR3 combination is the same as the LCDR1, LCDR2, and LCDR3 combination contained in the light chain variable region shown in SEQ ID NO: 11; and the CDRs are defined by the Kabat, Chothia, AbM, Contact, or IMGT systems.

2. An antibody or antigen-binding fragment thereof against monkeypox virus, the antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The antibody or antigen-binding fragment thereof includes any one of (a) to (c): (a) HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 4 to 6, respectively; (b) a heavy chain variable region and a light chain variable region; and comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the sequence shown in (a); the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 7 to 10, or consists of the amino acid sequence shown in any one of SEQ ID NOs: 7 to 10; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11, or consists of the amino acid sequence shown in SEQ ID NO: 11; (c) a heavy chain variable region and / or a light chain variable region having an amino acid sequence that is at least 80% identical to the heavy chain variable region and / or light chain variable region sequence shown in (b), and comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the sequence shown in (a).

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The constant region includes a heavy chain constant region and / or a light chain constant region.

5. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The heavy chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; and / or; the light chain constant region is selected from κ-type or λ-type light chain constant region.

6. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The species origin of the constant region is any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese and humans.

7. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 12 or having at least 80% identity thereto; or, consists of SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto.

8. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 13 or having at least 80% identity thereto; or, consists of an amino acid sequence as shown in SEQ ID NO: 13 or having at least 80% identity thereto.

9. An anti-monkeypox virus antibody or an antigen-binding fragment thereof, comprising a heavy chain and a light chain, characterized in that: The heavy chain comprises the HCDR1, HCDR2, and HCDR3 combination defined in claim 1, and the light chain comprises the LCDR1, LCDR2, and LCDR3 combination defined in claim 1; the heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 14 to 17 or having at least 80% identity thereto; or, consists of an amino acid sequence as shown in any one of SEQ ID NOs: 14 to 17 or having at least 80% identity thereto; the light chain comprises SEQ ID NO: 18 or an amino acid sequence as shown in any one of SEQ ID NOs: 14 to 17 or having at least 80% identity thereto; or, consists of SEQ ID NO: 18 or an amino acid sequence as shown in any one of SEQ ID NOs: 18 or having at least 80% identity thereto.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 or 4 to 9, characterized in that: The antibody or antigen-binding fragment thereof has a KD of ≤7.36×10 -8 Affinity binding of M to monkeypox virus antigens.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 or 4 to 9, characterized in that: The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

12. An antibody conjugate, characterized in that: The invention comprises the antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 11 and a marker or solid phase carrier coupled thereto.

13. An antibody conjugate, characterized in that: The invention comprises the antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 11 and biotin coupled thereto.

14. The antibody conjugate according to claim 12, characterized in that: The marker is selected from at least one of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents and nanoparticle markers.

15. The antibody conjugate according to claim 12, characterized in that: The label is colloidal gold.

16. A reagent or a kit, characterized in that: It includes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or the antibody conjugate according to any one of claims 12 to 15.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 in the preparation of a product for detecting monkeypox virus or monkeypox virus antigen, characterized in that: The method comprises: contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the antibody conjugate according to any one of claims 12 to 15, or the reagent or kit according to claim 16 with the monkeypox virus or monkeypox virus antigen in the sample to be detected to form an immune complex.

18. The use according to claim 17, characterized in that The immune complex comprises a second antibody, which binds to the antibody or its antigen-binding fragment; or; the second antibody binds to the monkeypox virus or a monkeypox virus antigen.

19. An isolated nucleic acid, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

20. A carrier, characterized in that It contains the nucleic acid according to claim 19.

21. A cell, characterized in that It contains the nucleic acid according to claim 19 or the vector according to claim 20.

22. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, characterized in that: It includes: Cultivate the cell of claim 21.

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the antibody conjugate according to any one of claims 12 to 15, or the reagent or kit according to claim 16 in the preparation of a product for detecting monkeypox virus or monkeypox virus antigen.

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