Monkey pox virus A29L antibody and application thereof

Abstract

The invention discloses a monkey pox virus A29L antibody and application thereof. The A29L antibody or an antigen binding fragment of the A29L antibody can be combined with monkey pox virus A29L protein in a high-activity and specific manner. The invention also relates to a coding polynucleotide molecule, a vector and a host cell of the A29L antibody or the antigen binding fragment thereof, and an antibody derivative or other products derived from the A29L antibody or the antigen binding fragment thereof.

Description

Technical Field

[0001] The present invention belongs to the fields of cell biotechnology and immunology, and relates to monkeypox virus A29L antibodies and applications thereof. Background Art

[0002] Monkeypox is a zoonosis caused by the monkeypox virus (MPXV). The monkeypox virus is a double-stranded DNA virus belonging to the genus Orthopoxvirus in the family Poxviridae. The virus's proteins are highly similar to those of other orthopoxviruses, such as vacciniavirus (VACV) and variolavirus (VARV). Monkeypox virus has two forms of infectious viral particles: extracellular enveloped virus (EEV) particles and intracellular mature virus (IMV) particles. Important neutralizing antibody targets for MPXV have been identified for EEV, including A35R and B6R, while important neutralizing antibody targets for IMV have been identified for M1R, A29L, E8L, and H3L.

[0003] Clinically, monkeypox symptoms are similar to those of smallpox, but are milder, with an incubation period of 5-12 days, often 6-13 days. Initial symptoms include fever, headache, swollen lymph nodes, muscle aches, and severe fatigue, followed by a rash on the face and body. Monkeypox is a self-limiting disease, typically resolving within two to three weeks. However, in children, pregnant women, or those with immunosuppression due to other health conditions, monkeypox can lead to secondary infections, such as pneumonia, sepsis, and encephalitis, among other serious illnesses.

[0004] Because the clinical symptoms of monkeypox virus are difficult to distinguish from those of other poxviruses, diagnosis based on clinical presentation is extremely difficult and requires laboratory testing. The WHO recommends PCR detection of viral DNA as the preferred laboratory test for MPXV. However, existing PCR detection methods still have some limitations. Immunoassays offer significant advantages over traditional methods in terms of accuracy and timeliness. However, the performance of immunoassays relies on the performance of antibodies against the monkeypox virus.

[0005] Research has shown that while the smallpox vaccine provides some protection against monkeypox, with the eradication of smallpox and the cessation of vaccination, a large portion of the population (born after 1980) lacks resistance to orthopoxviruses such as monkeypox and variola. Although chemical drugs such as Tecovirimat and Brincidofovir are approved for the treatment of smallpox, and vaccinia immunoglobulin (VIG) is approved for complications associated with smallpox vaccination and for infections caused by orthopoxviruses such as monkeypox, research on their efficacy against monkeypox remains limited. Currently, there are no effective treatments for monkeypox virus.

[0006] Therefore, there is a strong need in the art for antibodies that can effectively bind to and detect monkeypox virus, and antibodies that can treat diseases or conditions caused by monkeypox virus infection or inhibit the activity of monkeypox virus. Summary of the Invention

[0007] In view of this, in order to make up for the deficiencies of the prior art, the present invention is proposed.

[0008] In a first aspect, the present invention provides an A29L antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3;

[0009] Wherein: the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are shown as SEQ ID NO.1, 2 and 3 respectively; the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are shown as SEQ ID NO.4, 5 and 6 respectively.

[0010] In some embodiments, the heavy chain variable region of the A29L antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 90% identical to SEQ ID NO.7, and the light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO.8.

[0011] In a specific embodiment, the heavy chain variable region of the A29L antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO.7, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.8.

[0012] In the present invention, the A29L antibody or its antigen-binding fragment can be a monoclonal antibody, a domain antibody, a single chain (scFv), a Fab fragment, a F(ab')2 fragment, a multispecific antibody, a single domain heavy chain antibody, or a single domain light chain antibody. In some embodiments, such antibodies or their antigen-binding fragments that bind to the monkeypox virus A29L protein are mouse, other rodent, chimeric, humanized, or fully human monoclonal antibodies.

[0013] "Complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" are regions of an antibody variable region that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen-contacting residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes.

[0014] Based on the variable region amino acid sequence of a given A29L antibody or antigen-binding fragment thereof of the present invention, one skilled in the art can routinely determine the CDRs contained therein, for example, by using the Kabat scheme, the AbM scheme, the Chothia scheme, or the Contact scheme to define the CDRs in the variable region amino acid sequence.

[0015] When referring to antibodies defined by specific CDR sequences defined herein, the scope of said antibodies also encompasses antibodies whose variable region sequences comprise said specific CDR sequences, but whose declared CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination).

[0016] The CDRs of the antibodies of the present invention can be manually assessed to determine their boundaries according to any protocol or combination thereof in the art. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways.

[0017] In some embodiments, functional variants of the A29L antibody or antigen-binding fragment thereof described herein are also encompassed by the present invention. "Functional variants" refer to proteins that share significant or substantial sequence identity or similarity with a parent antibody, and retain the biological activity of the parent antibody. Functional variants encompass, for example, variants of the A29L antibody or antigen-binding fragment thereof described herein (parent antibody) that retain the ability to recognize target cells to a similar, equal, or greater degree than the parent antibody. With reference to the parent antibody, a functional variant may, for example, share at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater amino acid sequence identity with the parent antibody.

[0018] In some embodiments, the functional variant may, for example, comprise the amino acid sequence of a parent antibody with at least one conservative amino acid substitution. Alternatively or in addition, the functional variant may comprise the amino acid sequence of a parent antibody with at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is improved compared to the parent antibody.

[0019] In some embodiments, conservative amino acid substitutions are known in the art and include substitutions in which one amino acid having particular physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substitutions of an amino acid with a non-polar side chain for another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), substitutions of a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), substitutions of an uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), substitutions of an amino acid with a β-branched side chain for another amino acid with a β-branched side chain (e.g., He, Thr and Val), and substitutions of an amino acid with an aromatic side chain for another amino acid with an aromatic side chain (e.g., His, Phe, Trp and Tyr).

[0020] Those skilled in the art can readily mutate the nucleotide sequences corresponding to the antibodies of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that exhibit 90% or greater homology to the nucleotide sequences corresponding to the A29L antibodies or antigen-binding fragments thereof of the present invention, as long as they encode the A29L antibodies or antigen-binding fragments thereof, are derived from the nucleotide sequences of the present invention and are equivalent to the sequences of the present invention, and are also encompassed within the scope of protection of the present invention.

[0021] The second aspect of the present invention provides a polynucleotide molecule or a vector comprising the same, wherein the polynucleotide molecule encodes the A29L antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0022] In the present invention, the polynucleotide molecule may comprise natural, non-natural or altered nucleotides; and it may comprise natural, non-natural or altered internucleotide connections, such as phosphoramidate connections or phosphorothioate connections, replacing the phosphodiester present between the nucleotides of the unmodified oligonucleotide. In some embodiments, the nucleotides do not comprise any insertions, deletions, inversions and / or replacements. However, in some cases it may be appropriate for the nucleotides to comprise one or more insertions, deletions, inversions and / or replacements, and therefore, the nucleotides formed by these insertions, deletions, inversions and / or replacements are also within the scope of the present invention.

[0023] The term "encoding" when applied to polynucleotide molecules refers to a polynucleotide that, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA for a polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0024] In some embodiments, the methods for introducing the above-mentioned vector into the host cell include but are not limited to physical methods, chemical methods, and biological methods.

[0025] In some embodiments, the physical method includes but is not limited to calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation.

[0026] In some embodiments, the chemical methods include, but are not limited to, colloidal dispersion systems, lipid-based systems.

[0027] In some embodiments, the biological method includes but is not limited to DNA vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors.

[0028] In some embodiments, examples of vectors that can be used in the present invention include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, and virus-derived vectors.

[0029] Various vectors known in the art can be used, for example, commercially available vectors, and then the polynucleotide encoding the above-mentioned antibody or antigen-binding fragment thereof can be operably linked to an expression regulatory sequence to form an expression vector. In some embodiments, the viral-derived vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxvirus vectors, herpesvirus vectors, baculovirus vectors, papillomavirus vectors, and papovavirus vectors.

[0030] In some embodiments, the expression vector may contain expression control sequences, such as transcriptional and translational initiation and termination codons, that are specific to the type of host cell into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), depending on the circumstances and whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0031] In some embodiments, the vector may also contain one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.); complementation of prototrophy in auxotrophic hosts; and the like. Suitable marker genes for use in the expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, and puromycin resistance genes.

[0032] The third aspect of the present invention provides a modified host cell or a host cell population comprising the same, wherein the modified host cell comprises the polynucleotide molecule or a vector comprising the same according to the second aspect of the present invention, and the A29L antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0033] In some embodiments, the host cell population further comprises host cells other than the engineered host cells.

[0034] In some embodiments, the modified host cells include prokaryotic cells and eukaryotic cells.

[0035] In some embodiments, the prokaryotic cell comprises bacteria, actinomycetes, cyanobacteria, mycoplasmas, chlamydia, or rickettsia.

[0036] In some embodiments, the bacteria include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, Staphylococcus.

[0037] In some embodiments, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0038] The fourth aspect of the present invention provides an antibody derivative of the A29L antibody or its antigen-binding fragment, wherein the antibody derivative comprises a complex formed by directly or indirectly coupling the A29L antibody or its antigen-binding fragment according to the first aspect of the present invention to a detectable marker.

[0039] In the present invention, the term "detectable label" refers to an agent that is detectable, for example, by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Useful detectable labels include, but are not limited to, fluorescent dyes, enzymes, chemiluminescent labels, radioisotopes, electron-dense reagents, colored particles, biotin or digoxigenin. Detectable labels often produce a measurable signal, such as radioactivity, fluorescence, color or enzyme activity. Examples of suitable fluorescent dyes include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin; examples of suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase; chemiluminescent labels include, but are not limited to, luminol and its derivatives, isoluminol and its derivatives, acridinium esters and their derivatives, adamantane, rare earth elements, and ruthenium bipyridine complexes; radioisotopes include, but are not limited to 67 Ga, 68 Ga, 18 F. 52 Fe, 62 Cu, 64 Cu, 67 Cu, 86 Y. 90 Y. 89 Zr, 120 I. 123 I. 13 N. 15 O. 186 Re、 110 In, 111 In.

[0040] Antibodies conjugated to detectable labels can be used for diagnostic or therapeutic purposes.The detectable label can be linked or coupled directly to the antibody, or indirectly through an intermediate such as a linker known in the art, using techniques known in the art.

[0041] The fifth aspect of the present invention provides a detection reagent, which comprises the A29L antibody or antigen-binding fragment thereof according to the first aspect of the present invention or an antibody derivative of the A29L antibody or antigen-binding fragment thereof according to the fourth aspect of the present invention.

[0042] The sixth aspect of the present invention provides a conjugate of an A29L antibody or an antigen-binding fragment thereof, wherein the conjugate comprises a conjugate formed by coupling the A29L antibody or an antigen-binding fragment thereof according to the first aspect of the present invention with a therapeutic agent.

[0043] In some embodiments, the therapeutic agent includes, but is not limited to, a cytotoxic agent, a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, a chemotherapeutic agent, an oncolytic drug, a cytokine, an activator of a co-stimulatory molecule, or an inhibitor of an inhibitory molecule.

[0044] The seventh aspect of the present invention provides a pharmaceutical composition for treating diseases or conditions caused by monkeypox virus infection or inhibiting the activity of monkeypox virus, wherein the pharmaceutical composition comprises the A29L antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or a conjugate of the A29L antibody or antigen-binding fragment thereof according to the sixth aspect of the present invention.

[0045] In the present invention, the term "treating" refers to the improvement, prevention, alleviation or reversal of a disease or condition or at least one distinguishable symptom thereof, including the treatment of patients at risk for or suspected of having the disease and patients who are ill or diagnosed with the disease or condition, and includes the inhibition of clinical relapse. As used herein, the term "preventing" refers to the complete prevention of symptoms of a disease, such as fever, swollen lymph nodes, rash, pneumonia, encephalitis, organ failure, etc. The term "relief" as used herein refers to a reduction in the severity or duration of disease symptoms. Relief includes but does not require complete recovery or complete prevention of the disease or its symptoms.

[0046] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0047] In the present invention, the term "excipient" refers to any additive in a pharmaceutical preparation other than the main drug, also known as an adjuvant. General requirements for excipients include stable properties, no incompatibility with the main drug, no side effects, no effect on efficacy, resistance to deformation, cracking, mildew, or insect damage at room temperature, harmlessness to the human body, no physiological effects, no chemical or physical interaction with the main drug, and no impact on the determination of the main drug content.

[0048] In some embodiments, the excipients include binders, fillers, disintegrants, lubricants, ointments, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, and colorants.

[0049] In some embodiments, the disease or condition caused by the monkeypox virus infection comprises fever, swollen lymph nodes, rash, pneumonia, encephalitis, or organ failure.

[0050] In some embodiments, the pharmaceutical compositions described above can be administered by any suitable route known in the art, including but not limited to oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic and / or cerebrospinal.

[0051] In some embodiments, the above-described compositions can be formulated into pharmaceutical compositions suitable for intravenous injection into humans according to conventional procedures. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. Pharmaceutical compositions may also include a solubilizer and a local anesthetic, such as lidocaine, to relieve pain at the injection site. Generally, the active ingredients are supplied individually or mixed together in unit dosage form, such as as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or pouch) indicating the amount of active agent. If the composition is to be administered by infusion, the composition can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water or saline for injection can be used so that the active ingredients can be mixed prior to administration.

[0052] An eighth aspect of the present invention provides any one of the following methods, comprising:

[0053] (1) A method for preparing the A29L antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the method comprising the following steps: culturing the modified host cell according to the third aspect of the present invention or a host cell population comprising the modified host cell, and isolating the A29L antibody or antigen-binding fragment thereof according to the first aspect of the present invention from the culture;

[0054] (2) A method for detecting A29L or a fragment thereof in a sample to be tested, the method comprising the following steps: contacting the sample to be tested with the A29L antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or contacting the sample to be tested with the A29L antibody or an antibody derivative thereof described in the fourth aspect of the present invention, or contacting the sample to be tested with the product for detecting A29L described in the fifth aspect of the present invention, and detecting the formation of a complex between the A29L antibody or antigen-binding fragment thereof, or the antibody derivative thereof and A29L or a fragment thereof;

[0055] (3) A method for preparing the modified host cell or a host cell population comprising the modified host cell according to the third aspect of the present invention, the method comprising the following steps: introducing the polynucleotide molecule or a vector comprising the polynucleotide molecule according to the second aspect of the present invention into the host cell.

[0056] A ninth aspect of the present invention provides any one of the following applications:

[0057] (1) Use of the A29L antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the antibody derivative of the A29L antibody or antigen-binding fragment thereof described in the fourth aspect of the present invention, or the detection reagent described in the fifth aspect of the present invention in the preparation of a product for detecting A29L or its fragments;

[0058] (2) Use of the A29L antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the antibody derivative of the A29L antibody or antigen-binding fragment thereof according to the fourth aspect of the present invention, or the detection reagent according to the fifth aspect of the present invention in the preparation of a product for diagnosing whether a subject is infected with monkeypox virus or diagnosing a disease or condition caused by monkeypox virus infection;

[0059] (3) Use of the A29L antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or a conjugate of the A29L antibody or antigen-binding fragment thereof described in the sixth aspect of the present invention, in the preparation of a pharmaceutical composition for treating diseases or conditions caused by monkeypox virus infection or inhibiting the activity of monkeypox virus.

[0060] In some embodiments, the product comprises a kit, a chip, or a test strip.

[0061] In some embodiments, the kit includes but is not limited to an ELISA detection kit, an immunofluorescence detection kit, a flow cytometry kit, and an IHC detection kit.

[0062] In some embodiments, the kit may include a container, instructions for use, a buffer, etc. In other embodiments, the kit further includes a lysis medium for dissolving the sample to be tested, general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit may be an in vitro diagnostic device.

[0063] The tenth aspect of the present invention provides any of the following methods:

[0064] (1) A method for diagnosing whether a subject is infected with monkeypox virus, the method comprising contacting a test sample with the A29L antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or contacting the test sample with the A29L antibody or antibody derivative thereof described in the fourth aspect of the present invention, or contacting the test sample with the detection reagent described in the fifth aspect of the present invention, and detecting the formation of a complex between the A29L antibody or antigen-binding fragment thereof, or the antibody derivative thereof and A29L or its fragment; if the formation of the complex is detected, the subject is diagnosed as being infected with monkeypox virus;

[0065] (2) A method for treating monkeypox virus infection, comprising administering to a subject the A29L antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the conjugate of the A29L antibody or antigen-binding fragment thereof described in the sixth aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention.

[0066] The present invention has the following advantages and beneficial effects:

[0067] The present invention discloses monkeypox virus A29L antibodies and their uses. The A29L antibodies or antigen-binding fragments thereof of the present invention can bind to the monkeypox virus A29L protein with high activity and specificity. The present invention also relates to polynucleotide molecules encoding the A29L antibodies or antigen-binding fragments thereof, vectors, host cells, and antibody derivatives or other products derived from the A29L antibodies or antigen-binding fragments thereof of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is the SDS-PAGE result after antibody expression and purification;

[0069] Figure 2 This is the electrophoresis diagram after antibody expression and purification;

[0070] Figure 3 This is a graph showing the antibody binding activity test results. DETAILED DESCRIPTION

[0071] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0072] Example

[0073] 1. Recombinant expression of immunogens

[0074] The recombinant monkeypox virus A29L protein sequence was derived from strain ON563414.3. The monkeypox virus membrane protein A29L protein sequence (sequence shown in SEQ ID NO. 9, sequence information is shown in Table 1) was synthesized and constructed into the pCDNA3.1 vector. The transfection plasmid was extracted and transfected into HEK293 cells, and the cells were cultured for 7 days. The supernatant was harvested and purified using a Ni column. After concentration and buffer exchange, the recombinant monkeypox virus A29L protein was obtained.

[0075] 2. Immunity

[0076] Immunization of mice: Freund's complete adjuvant was used as the adjuvant for the first immunization, with each mouse immunized with 100 μg of protein by intraperitoneal injection. The total dose of Freund's complete adjuvant and protein was 0.5 ml per mouse. The second immunization was performed 3 weeks after the first immunization; Freund's incomplete adjuvant was used for the second immunization, with each mouse immunized with 50 μg of protein. The total dose of Freund's incomplete adjuvant and protein was 0.5 ml per mouse. The third immunization was performed 2 weeks after the third immunization; cell fusion was prepared 10 days after the third immunization.

[0077] SP2 / 0 fusion: Take feeder cells and press 10 5 / well, plate 10 on the day before fusion 5 Mouse immune spleen cells were fused with the prepared myeloma cells using the fusion agent PEG, and plated into a 96-cell culture plate that had been added with feeder cells, 100 μl / well.

[0078] Screening: Positive wells were screened by ELISA. Recombinant monkeypox virus A29L protein was plated onto a 96-well plate overnight. The plate was washed and blocked with skim milk powder at 37°C for 1 hour. The plate was washed and 100 μl of 96-well culture supernatant was added. The plate was incubated at 37°C for 1 hour. The plate was washed and HRP-labeled goat anti-mouse secondary antibody was added. The plate was incubated at 37°C for 30 minutes. The plate was washed and the color development solution was added. The color development solution was developed for 10 minutes. The stop solution was added and the OD was read. 450 The high expression cell lines were screened for subcloning.

[0079] 3. Sequence fishing

[0080] Subcloned cells were collected, RNA was extracted, and reverse transcribed. Amplification primers were designed and used for PCR. The PCR product was ligated into a vector, and the vector was transformed into recombinant host cells. The cells were cultured overnight, and clones were picked the next day for sequencing. The sequence information for the antibody clone 3E5 is shown in Table 1. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8.

[0081] Table 1 Amino acid sequences of monkeypox virus A29L protein and its antibodies

[0082]

[0083] 4. Antibody Expression

[0084] The 3E5 antibody was expressed by transiently transfecting the 3E5 antibody plasmid into HEK293 cells using PEI, and its expression level was detected, which was 328 mg / L.

[0085] 5. Physical and chemical properties testing

[0086] The purity of 3E5 antibody was detected by SDS-PAGE. The experimental results are as follows Figure 1 As shown, the antibody purity is greater than 95%.

[0087] The purity of 3E5 antibody was tested by HPLC. Figure 2 As shown, the antibody purity is greater than 95%.

[0088] 6. Binding activity detection

[0089] Antibody activity was detected by ELISA: A29L protein was diluted in 0.05 M carbonate buffer and coated at a concentration of 1 μg / ml in 100 μl / well at 4°C overnight; the plate was washed, the residual liquid was flicked off, 300 μl / well of 5% skim milk powder was added for blocking, and the plate was incubated at 37°C for 1 h; the plate was washed, the residual liquid was flicked off, the 3E5 antibody was diluted to 10 μg / ml, 11 steps of 4x dilution were performed, 100 μl / well of 3E5 antibody was added, and the plate was incubated at 37°C for 1 h; anti-mouse IgG-HRP-labeled secondary antibody was added at 100 μl / well and incubated at 37°C for 30 min; the plate was washed, the residual liquid was flicked off, and the color development solution was added at 100 μl / well for 10 min; the stop solution was added at 100 μl / well; and the OD was read. 450 values for data analysis.

[0090] The experimental results are as follows Figure 3 As shown, 3E5 antibody can bind well to A29L antibody, EC 50 It is 0.1410ug / ml.

[0091] 7. Specificity detection

[0092] Detect antibody specificity by ELISA: coat monkeypox virus A29L, A35R, B6R, smallpox virus A29L, A35R, B6R, vaccinia virus protein A27, A35R, B6R, add 3E5 antibody, incubate at 37°C for 1 hour; wash the plate, add HRP-labeled secondary antibody, incubate at 37°C for 30 minutes; wash the plate, add color development solution, develop for 10 minutes, add stop solution, and read the OD 450 values for data analysis.

[0093] Experimental results: Only monkeypox virus A29L showed normal color development, and the other antigens had no positive reaction, indicating that the antibodies provided in this application have good specificity.

[0094] In summary, the antibodies provided in this application can bind to the monkeypox virus A29L protein with high activity and specificity.

[0095] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. An A29L antibody or an antigen-binding fragment thereof, characterized in that: The A29L antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3; Wherein: the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are shown as SEQ ID NO.1, 2 and 3 respectively; the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are shown as SEQ ID NO.4, 5 and 6 respectively.

2. The A29L antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region of the A29L antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence that is at least 90% identical to SEQ ID NO.7, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.8 or an amino acid sequence that is at least 90% identical to SEQ ID NO.

8.

3. A polynucleotide molecule or a vector comprising the same, characterized in that: The polynucleotide molecule encodes the A29L antibody or antigen-binding fragment thereof according to any one of claims 1-2.

4. A modified host cell or a host cell population comprising the same, characterized in that The modified host cell comprises the A29L antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, the polynucleotide molecule according to claim 3, or a vector comprising the same; Preferably, the host cell population further comprises host cells other than the modified host cells; Preferably, the modified host cells include prokaryotic cells and eukaryotic cells; Preferably, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasmas, chlamydia, and rickettsia; Preferably, the bacteria include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, Staphylococcus; Preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

5. An antibody derivative of the A29L antibody or its antigen-binding fragment, characterized in that: The antibody derivative comprises a complex formed by directly or indirectly coupling the A29L antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 to a detectable label; Preferably, the detectable label comprises a fluorescent dye, an enzyme, a chemiluminescent label, a radioisotope, an electron-dense reagent, a colored particle, biotin, or digoxigenin; Preferably, the fluorescent dye includes umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine aminofluorescein, dansyl chloride, and phycoerythrin; Preferably, the enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; Preferably, the chemiluminescent markers include luminol and its derivatives, isoluminol and its derivatives, acridinium esters and its derivatives, adamantane, rare earth elements, and bipyridine ruthenium complexes; Preferably, the radioisotope comprises 67 Ga, 68 Ga, 18 F. 52 Fe, 62 Cu, 64 Cu, 67 Cu, 86 Y. 90 Y. 89 Zr, 120 I. 123 I. 13 N. 15 O. 186 Re、 110 In, 111 In.

6. A detection reagent, characterized in that The detection reagent comprises the A29L antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 or an antibody derivative of the A29L antibody or antigen-binding fragment thereof according to claim 5.

7. A conjugate of an A29L antibody or an antigen-binding fragment thereof, characterized in that: The conjugate comprises a conjugate formed by coupling the A29L antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 with a therapeutic agent; Preferably, the therapeutic agent comprises a cytotoxic agent, a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, a chemotherapeutic agent, an oncolytic drug, a cytokine, an activator of a co-stimulatory molecule, or an inhibitor of an inhibitory molecule.

8. A pharmaceutical composition for treating diseases or conditions caused by monkeypox virus infection or inhibiting the activity of monkeypox virus, characterized in that: The pharmaceutical composition comprises the A29L antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, or a conjugate of the A29L antibody or antigen-binding fragment thereof according to claim 7; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients; Preferably, the excipients include adhesives, fillers, disintegrants, lubricants, ointments, preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants; Preferably, the diseases or conditions caused by monkeypox virus infection include fever, lymphadenopathy, rash, pneumonia, encephalitis, and organ failure.

9. Any of the following methods, characterized in that The method comprises: (1) A method for preparing the A29L antibody or antigen-binding fragment thereof according to any one of claims 1-2, characterized in that the method comprises the following steps: culturing the modified host cell according to claim 4 or a host cell population comprising the modified host cell, and isolating the A29L antibody or antigen-binding fragment thereof according to any one of claims 1-2 from the culture; (2) A method for detecting A29L or a fragment thereof in a sample to be tested, characterized in that the method comprises the following steps: contacting the sample to be tested with the A29L antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, or contacting the sample to be tested with the A29L antibody or the antibody derivative of the antigen-binding fragment thereof according to claim 5, or contacting the sample to be tested with the product for detecting A29L according to claim 6, and detecting the formation of a complex between the A29L antibody or the antigen-binding fragment thereof, or the antibody derivative of the A29L antibody or the antigen-binding fragment thereof, and A29L or a fragment thereof; (3) A method for preparing the modified host cell according to claim 4 or a host cell population comprising the modified host cell, characterized in that the method comprises the following steps: introducing the polynucleotide molecule according to claim 3 or a vector comprising the polynucleotide molecule into a host cell.

10. Any of the following applications: (1) Use of the A29L antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, the antibody derivative of the A29L antibody or antigen-binding fragment thereof according to claim 5, or the detection reagent according to claim 6 in the preparation of a product for detecting A29L or its fragment; (2) Use of the A29L antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, the antibody derivative of the A29L antibody or antigen-binding fragment thereof according to claim 5, or the detection reagent according to claim 6 in the preparation of a product for diagnosing whether a subject is infected with monkeypox virus or diagnosing a disease or condition caused by monkeypox virus infection; (3) Use of the A29L antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, or a conjugate of the A29L antibody or antigen-binding fragment thereof according to claim 7, in the preparation of a pharmaceutical composition for treating diseases or conditions caused by monkeypox virus infection or inhibiting monkeypox virus activity; Preferably, the product comprises a kit, a chip or a test strip; Preferably, the kit includes an ELISA detection kit, an immunofluorescence detection kit, a flow cytometry detection kit, and an IHC detection kit.

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