Plague antibody Fm3 and application thereof

Abstract

The invention discloses a plague antibody Fm3 and application thereof. The invention provides a brand new murine monoclonal antibody targeting plague bacillus F1 protein for the field, and the antibody can effectively neutralize plague virulent strains and drug-resistant strains, has good affinity and specificity, and can effectively resist attack of the virulent strains and the drug-resistant strains with lethal dose. The invention provides a new strategy for developing treatment drugs for plague virulent strains and drug-resistant strains in the field, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to plague antibody Fm3 and its applications. Background Technology

[0002] Plague is a zoonotic disease, with rodents and fleas being the primary hosts and vectors for its transmission. It can be transmitted from rodents to humans and animals through fleas, physical contact, or respiratory droplets. Human infection is usually fatal without antibiotic treatment. The causative agent of plague is *Yersinia pestis* (Yersinia pestis). Yersinia pestis Yersinia pestis is a non-motile, non-spore-forming, aerobic, Gram-negative bacillus or coccus. Infection with Yersinia pestis can cause several major forms of plague, including bubonic plague, septicemic plague, pneumonic plague, meningeal plague, and pharyngeal plague.

[0003] Although Yersinia pestis is naturally susceptible to antibiotics, the long-term use of antibiotics has led to an increasing number of reports of drug-resistant strains. In 2021, the Qinghai Provincial Institute for Endemic Disease Control and Prevention reported the isolation of a streptomycin-resistant Yersinia pestis strain, S19960127, which exhibited high-level resistance to streptomycin. The drug resistance mechanisms of Yersinia pestis mainly include: resistance plasmid-mediated resistance; efflux pumps that expel antibiotics from the cell; and altering the permeability of the cell membrane to prevent antibiotic entry. Existing antibiotics are ineffective against these resistance mechanisms, thus necessitating the research of new alternative drugs.

[0004] Monoclonal antibodies have demonstrated significant advantages in treating a wide range of diseases, including infectious diseases, autoimmune diseases, cancer, and even degenerative diseases. Most reported plague antibodies are murine monoclonal antibodies derived from murine hybridomas, and these murine monoclonal antibodies have shown good neutralizing effects against multiple virulent strains of plague. In addition, some human monoclonal antibodies have shown good neutralizing effects against virulent strains; however, their protective efficacy against drug-resistant plague bacteria has not yet been reported. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the art with a novel plague antibody that has a neutralizing effect on both highly virulent and drug-resistant plague strains, as well as related products and uses.

[0006] The present invention achieves the above-mentioned objectives by adopting the following technical solution: The first aspect of the present invention provides a plague antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region CDR1 of the antibody or the antigen-binding fragment is shown as SEQ ID NO:1; CDR2 is shown as SEQ ID NO:2; CDR3 is shown as SEQ ID NO:3; and the light chain variable region CDR1 is shown as SEQ ID NO:5; CDR2 is shown as SEQ ID NO:6; and CDR3 is shown as SEQ ID NO:7.

[0007] Furthermore, the heavy chain variable region of the antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 75% identity with SEQ ID NO:4; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with SEQ ID NO:8.

[0008] In this invention, the term "antibody" refers to any form of antibody that exhibits the desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or inhibiting ligand-induced receptor signal transduction). Therefore, "antibody" is used in its broadest sense and is explicitly included, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primate-derived, chimeric antibodies, single-chain antibodies, etc.

[0009] In this invention, the term "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The term "antigen-binding fragment" includes aptamers, mirror isoforms, and bivalent antibodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by forming a complex with a specific antigen.

[0010] In some embodiments, the amino acid sequences corresponding to the heavy chain variable regions CDR1-3 of the present invention are not limited to the amino acid sequences shown in SEQ ID NO:1-3, and the amino acid sequences corresponding to the light chain variable regions CDR1-3 of the present invention are not limited to the amino acid sequences shown in SEQ ID NO:5-7. The amino acid sequences or nucleotide sequences corresponding to the CDRs obtained by defining CDR1, CDR2, and CDR3 in the heavy chain variable region shown in SEQ ID NO:4 and CDR1, CDR2, and CDR3 in the light chain variable region shown in SEQ ID NO:8 using any CDR numbering scheme (existing CDR numbering scheme or new CDR numbering scheme to be generated in the future) are all within the protection scope of the present invention.

[0011] In some implementations, the CDR numbering scheme is any one or more combinations of the IMGT numbering scheme, Chothia numbering scheme, Kabat numbering scheme, Martin (enhanced Chothia) numbering scheme, AbM numbering scheme, and Aho numbering scheme. The sequences corresponding to the heavy chain variable region CDR1-3 or the light chain variable region CDR1-3 obtained by defining any one of the above CDR numbering schemes or by defining any combination of two or more CDR numbering schemes are all included within the protection scope of this invention.

[0012] In this invention, the term "identity" refers to sequence similarity to the amino acid sequence used in this invention. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithm required to achieve optimal alignment across the full-length sequences being compared. Therefore, amino acid sequences that are at least 75%, 80%, 85%, 90%, 95%, or 99% identical to the sequences of this invention are within the scope of protection of this invention.

[0013] In this invention, the modified antibody sequences also fall within the scope of protection. The term "modification" refers to any chemical modification of an amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids with different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to a change in the amino acid sequence resulting in an increase of one or more amino acids compared to a naturally occurring molecule. It should be noted that in the modified antibodies provided by this invention, the modification preferably occurs in regions other than the variable region, such as the frame region or constant region of the antibody, and the modified antibody still retains the desired functional properties of the antibody or its antigen-binding fragment of this invention, or has improved antigen-binding properties.

[0014] A second aspect of the present invention provides a bispecific antibody comprising the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0015] Furthermore, the bispecific antibody also includes a second antibody that specifically binds to other antigens.

[0016] In some embodiments, the bispecific antibody comprises binding specificity against two different antigens (one of which is the Yersinia pestis F1 protein described in this invention, and the other antigen is an antigen other than the Yersinia pestis F1 protein). In other embodiments, the bispecific antibody comprises two different binding specificities against the same antigen (e.g., different binding affinities and / or specific epitopes against the same antigen).

[0017] A third aspect of the present invention provides a nucleic acid molecule that encodes the antibody or antigen-binding fragment thereof described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention.

[0018] In a specific embodiment of the present invention, the nucleotide sequences encoding the heavy chain variable regions CDR1-3 of the antibody or antigen-binding fragment of the first aspect of the present invention are shown in SEQ ID NO:9-11, and the nucleotide sequences encoding the light chain variable regions CDR1-3 of the antibody or antigen-binding fragment of the first aspect of the present invention are shown in SEQ ID NO:13-15.

[0019] In a specific embodiment of the present invention, the nucleotide sequence encoding the heavy chain variable region of the antibody or its antigen-binding fragment described in the first aspect of the present invention is shown in SEQ ID NO:12, and the nucleotide sequence encoding the light chain variable region of the antibody or its antigen-binding fragment described in the first aspect of the present invention is shown in SEQ ID NO:16.

[0020] Those skilled in the art can easily use known methods, such as directed evolution and point mutation, to mutate the nucleotide sequence corresponding to the antibody or its antigen-binding fragment described in this invention. As long as the antibody or its antigen-binding fragment described in the first aspect of this invention or the bispecific antibody described in the second aspect of this invention is derived from the nucleotide sequence of this invention and is equivalent to the sequence of this invention, it also falls within the protection scope of this invention.

[0021] In some embodiments, the nucleic acid molecule is isolated or purified. The sequence of the nucleic acid molecule can be obtained using conventional techniques or hybridoma techniques. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. Alternatively, the relevant sequence can be synthesized artificially, especially when the fragment length is short. Generally, longer fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0022] A fourth aspect of the present invention provides an expression vector comprising the nucleic acid molecule described in the third aspect of the present invention.

[0023] In this invention, a vector refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The vector used in this invention is not limited and can be an expression vector, viral vector, etc. Known vectors or self-constructed vectors can be used. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, etc.

[0024] In some embodiments, the expression vector may contain expression regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, depending on the circumstances and whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0025] In some embodiments, the vector may also contain one or more marker genes that allow selection of host cells for transformation or transfection. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.); prototrophic complementation in auxotrophic hosts, etc. Suitable marker genes for the expression vector of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, and puromycin resistance genes.

[0026] In some implementations, the expression vector can be constructed using methods well known to those skilled in the art. These methods include, but are not limited to, recombinant DNA technology, DNA synthesis technology, etc. DNA encoding the antibody or its antigen-binding fragment can be effectively ligated to a multiple cloning site in the vector to guide mRNA synthesis and thereby protein expression, or for homologous recombination.

[0027] A fifth aspect of the present invention provides a recombinant host cell comprising the expression vector described in the fourth aspect of the present invention.

[0028] In this invention, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, or cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0029] In some embodiments, the recombinant host cells are prepared by introducing the nucleic acid molecules or expression vectors of the present invention as described above into the host cells. The nucleic acid molecules or expression vectors of the present invention as described above can be introduced into the host cells by various suitable methods, not limited to those listed herein, such as calcium phosphate transfection, DEAE-glucan-mediated transfection, microinjection, electroporation, the TALEN method, the ZFN method, non-viral vector-mediated transfection (e.g., liposomes) or viral vector-mediated transfection (e.g., lentiviral infection, retroviral infection, adenovirus infection), and other physical, chemical, or biological means for transfer into cells, such as transposon technology, CRISPR-Cas9, etc.

[0030] The sixth aspect of the present invention provides any of the following products: 1) An antibody conjugate, wherein the antibody conjugate is a complex formed by directly or indirectly conjugating the antibody or its antigen-binding fragment as described in the first aspect of the present invention or the bispecific antibody as described in the second aspect of the present invention to a detectable marker.

[0031] 2) A detection reagent comprising the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the bispecific antibody or the antibody-drug conjugate described in the second aspect of the present invention.

[0032] 3) A detection product comprising the antibody or its antigen-binding fragment as described in the first aspect of the present invention, the bispecific antibody as described in the second aspect of the present invention, the antibody-drug conjugate, or the detection reagent.

[0033] 4) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention or the bispecific antibody as described in the second aspect of the present invention.

[0034] 5) A pharmaceutical preparation comprising the pharmaceutical composition.

[0035] Furthermore, the detectable markers include fluorescent dyes, avidin, paramagnetic atoms, radioactive isotopes, enzyme markers, and colloidal gold.

[0036] In some embodiments, the fluorescent dye includes fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, and polydinoflavin-chlorophyll protein. The avidin includes biotin, avidin, streptavidin, vitellavidin, and avidin-like proteins. The radioactive isotopes include radioactive iodine, radioactive cesium, radioactive iridium, and radioactive cobalt. The enzyme labeling includes horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, lysozyme, and malate dehydrogenase.

[0037] In some implementations, antibodies conjugated to a detectable marker can be used for diagnostic or therapeutic purposes. The detectable marker can be directly attached to or conjugated to the antibody, or indirectly via an intermediate such as a linker known in the art, using techniques known in the art.

[0038] In some embodiments, the diagnostic and / or auxiliary diagnostic uses of the test reagent are achieved by contacting a sample suspected of having a target analyte (Yersinia pestis F1 protein) with the test reagent of the present invention, and analyzing the presence or level of the target analyte in the sample being tested.

[0039] Furthermore, the testing products include reagent kits, chips, and test strips.

[0040] In some embodiments, the kit further includes a container, instructions for use, buffers, etc. In other embodiments, the kit further includes a lysis medium for dissolving the sample to be tested, universal reagents and buffers required for detection, such as various buffer solutions, detection labels, detection substrates, etc. This detection kit can be an in vitro diagnostic device for diagnosing and / or assisting in the diagnosis of plague infection.

[0041] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, which are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used as needed to aid in the stability of the formulation or to help improve the activity or bioavailability of the active substance. In some embodiments, the pharmaceutical composition is used by administering a safe and effective amount of the pharmaceutical composition of the present invention to a human. There are no particular limitations on the dosage and route of administration of the pharmaceutical composition, and a skilled physician can usually readily determine the dosage and effectiveness of the prescription for the desired treatment and / or prevention. The route of administration may include, for example, injection or other treatment methods.

[0042] In some embodiments, the pharmaceutical formulation has a dosage form selected from: solution, suspension, emulsion, tablet, pill, powder, granule, capsule, syrup, sterile aqueous solution, non-aqueous solution, lyophilized formulation, suppository. Furthermore, it can be administered once or multiple times. In this case, the pharmaceutical composition and / or biological agent is administered in the form of a liquid formulation, powder, aerosol, capsule, or suppository. Routes of administration may include, but are not limited to: intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, local, intranasal, intrapulmonary, rectal, etc. When administered orally, it may be formulated with a coating to protect the active ingredient in the pharmaceutical composition from degradation in the stomach. Furthermore, the active ingredient can be administered via any device capable of transfer to the target tissue. In specific embodiments, the pharmaceutical composition provided by the present invention can be formulated into various dosage forms as needed, and the dosage beneficial to the patient can be determined by a clinician based on factors such as the subject's type, age, weight, general disease condition, and route of administration. The route of administration may include, for example, injection or any other suitable route of administration known to those skilled in the art.

[0043] The seventh aspect of the present invention provides any of the following methods: 1) A method for preparing recombinant host cells according to the fifth aspect of the present invention, the method comprising: introducing the expression vector according to the fourth aspect of the present invention into host cells to obtain the recombinant host cells according to the fifth aspect of the present invention.

[0044] 2) A method for producing an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention or a bispecific antibody as described in the second aspect of the present invention, the method comprising: culturing a recombinant host cell as described in the fifth aspect of the present invention, and isolating the antibody or antigen-binding fragment thereof or bispecific antibody from the culture product of the recombinant host cell.

[0045] 3) A method for detecting Yersinia pestis F1 protein in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the test sample with the antibody or its antigen-binding fragment described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the antibody-drug conjugate described in the sixth aspect of the present invention, a detection reagent or a detection product, and detecting the formation of the corresponding immune complex.

[0046] The eighth aspect of the present invention provides any of the following applications: 1) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, or the recombinant host cell described in the fifth aspect of the present invention in the preparation of antibody-drug conjugates for detecting Yersinia pestis F1 protein.

[0047] 2) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, or the antibody conjugate described in the sixth aspect of the present invention in the preparation of a detection reagent for detecting Yersinia pestis F1 protein.

[0048] 3) The application of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate or detection reagent described in the sixth aspect of the present invention in the preparation of a detection product for detecting Yersinia pestis F1 protein.

[0049] 4) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate described in the sixth aspect of the present invention, the detection reagent or detection product in the detection of Yersinia pestis F1 protein for non-diagnostic and non-therapeutic purposes.

[0050] 5) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate described in the sixth aspect of the present invention, the detection reagent or detection product in the preparation of diagnostic products for the diagnosis or auxiliary diagnosis of plague.

[0051] 6) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, or the recombinant host cell described in the fifth aspect of the present invention in the preparation of a medicament for the treatment and / or prevention of plague.

[0052] 7) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention in the preparation of a pharmaceutical formulation for the treatment and / or prevention of plague.

[0053] In this invention, the sample refers to a collection (such as fluid, cells, or tissue) separated from the subject, as well as fluids, cells, or tissues present within the subject's body. Exemplary test samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues or organs, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as culture media (including conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0054] In this invention, the subjects include humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles.

[0055] Furthermore, the plague is a disease caused by infection with Yersinia pestis.

[0056] Furthermore, the plague bacterium is the Yersinia pestis strain, including its highly virulent strains and / or drug-resistant strains.

[0057] Furthermore, the highly virulent strain is 141 strains, and the drug-resistant strain is S19960127 strain.

[0058] Advantages and beneficial effects of the present invention: This invention provides a novel murine monoclonal antibody targeting the F1 protein of Yersinia pestis. The antibody effectively neutralizes virulent and drug-resistant strains of plague, exhibiting good affinity and specificity, and effectively resisting attacks from lethal doses of virulent and drug-resistant strains. This invention provides a new strategy for developing therapeutic drugs against virulent and drug-resistant plague strains, and has promising application prospects. Attached Figure Description

[0059] Figure 1 The titer of F1 antibody in the serum of immunized mice is given.

[0060] Figure 2 To screen for target antibodies in a library for phage ELISA.

[0061] Figure 3 The results show the specificity of Fm3 antibody binding to F1; where A is the ELISA result of Fm3 antibody binding to F1 protein; B is the ELISA result of Fm3 antibody not binding to irrelevant antigen (SARS-CoV-2 NP); and C is the Western Blot result of Fm3 antibody binding to F1 protein.

[0062] Figure 4The affinity of Fm3 antibody to F1 was determined; where A is the dissociation curve of Fm3 antibody to F1; and B is the relevant kinetic parameters of Fm3 antibody to F1.

[0063] Figure 5 The survival curves of mice in the challenge experiment are shown; where A is the highly virulent strain 141 and B is the drug-resistant strain S19960127. Detailed Implementation

[0064] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents.

[0065] The reagents and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are generally performed under conventional conditions in the art or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0066] Example 1: Preparation of Plague Antibody Fm3 I. Experimental Methods 1. Experimental Materials: The Yersinia pestis EV76 vaccine strain was preserved and provided by the Qinghai Provincial Institute for Endemic Disease Control and Prevention. The Yersinia pestis recombinant F1 antigen was preserved and provided by Lanzhou Institute of Biological Products Co., Ltd. BALB / c mice, all female and 6-8 weeks old, were purchased from Xi'an Huishi Biotechnology Co., Ltd. The cloning strain DH5α, the library-constructing strain XL1-Blue, the library-constructing vector pComb3XSS, and the antibody IgG expression vector pGI were preserved by the Jiangsu Provincial Center for Disease Control and Prevention; HEK293F was purchased from ATCC; the reverse transcription kit and PCR reagents were purchased from TaKaRa; SfiI restriction enzyme and T4 ligase were purchased from NEB; all other chemical reagents were of analytical grade.

[0067] 2. Animal immunization This was performed in the biosafety enhanced level 2 laboratory of the Plague Bacteria Specialty Laboratory at the Qinghai Provincial Institute for Endemic Disease Prevention and Control. The specific method is as follows: 0.5 mL containing 5 × 10⁵ bacteria... 7Six mice were immunized with live EV76 strain via subcutaneous injection into the groin on days 0, 10, and 20. Blood was collected from the tail vein of the mice on day 30, and the mice were euthanized. The spleens were harvested. F1 antibodies in the venous blood were detected using an indirect ELISA method. The spleens were ground into individual spleen cells in a biosafety cabinet, and mononuclear cells were separated from the spleen using Ficoll density gradient centrifugation, counted, and then cryopreserved.

[0068] 3. Construction of mouse ScFv antibody library Mouse spleen PBMCs were revived from cryopreservation, and total RNA was extracted using the QIAGEN RNeasy Mini Kit. Reverse transcription was then performed using the Roche Transcriptor First Strand cDNA Synthesis for RT-PCR kit. Specific primers for amplifying mouse VH and VKappa were designed based on the IMGT database, as shown in Table 1. The heavy chain variable region was amplified using MVH1–S2 as upstream primers and a mixture of MSCG1ab-B and MSCG3-B as downstream primers. The light chain variable region was amplified using MVK1–12 as upstream primers and a mixture of MSCJK12-B, MSCJK4-B, and MSCJK5-B as downstream primers. The light and heavy chains were recovered after agarose gel electrophoresis, mixed in equal proportions, and ligated into ScFv fragments by fusion PCR. The ScFv fragment was digested with SfiI and linked with the pComb3XSS vector, which was also digested with SfiI. The phage antibody library was prepared by electroporation of the host bacterium XL1-Blue competent cells. The phage antibody library was packaged with wild-type helper phage VCSM13 and then screened.

[0069] Table 1 Primer Sequences

[0070] 4. Screening for F1-specific phage antibodies The purified Yersinia pestis F1 protein was coated into immunotubes, and a packaged phage library was added to screen for antibody fragments that specifically bind to the F1 protein. The specific steps are as follows: Add the appropriate amount of F1 protein to the immunotubes and coat overnight at 4°C; discard the supernatant, wash the plate 3 times with 0.05% PBST, add 3% BSA and block at 37°C for 2 h; discard the blocking solution, wash the plate 3 times with 0.05% PBST, add the antibody library, and incubate at 37°C with shaking for 1 h, then incubate statically for 1 h; discard the supernatant, wash 10 times with 0.1% PBST, elute with 0.1 M glycine-hydrochloric acid (Gly-HCl) at pH=2.2, neutralize to pH=7.0 with 2 M Tris, take 10 μL to determine the titer, and infect XL1-Blue host bacteria with the remaining phage for amplification. The next day, the phage was precipitated with PEG6000 and screened again for the next round. A total of 3 rounds of enrichment screening were performed.

[0071] 5. Select single clones that specifically bind to F1 cells. The XL1-Blue host bacteriophage was infected with bacteriophages eluted after the third round of screening. The infected bacterial suspension was serially diluted and plated onto ampicillin-resistant agar plates. The next day, 96 single colonies were randomly picked from the plates and incubated in 96-well deep-layer plates at 37°C and 260 rpm for 4 h. Then, the colonies were transferred 1:10 to new deep-layer plates and incubated with shaking for another 4 h. 1 mM IPTG was added for overnight induction at 37°C. The expression of antibodies in the supernatant was then detected by indirect ELISA as follows: Plague F1 antigen was coated at 200 ng / well in a 96-well ELISA plate. 50 μL of 3% skim milk and 50 μL of expression supernatant were added. The plate was incubated at 37°C with shaking for 1 h. The plate was washed three times with PBST. HRP-labeled anti-M13 bacteriophage monoclonal antibody was added, and the plate was incubated at 37°C for 30 min. After washing with PBST, TMB chromogenic solution was added for 10 min. The reaction was terminated with 2 M sulfuric acid solution, and the OD was read. 450 The absorbance value was measured. Positive clones were extracted with plasmids and sent for sequencing. The sequencing results were compared with the gene sequence of the antibody variable region using the IMGT database. Clones with sequence differences were selected for full antibody expression.

[0072] 6. Expressing murine monoclonal antibodies against F1 cells using mammalian cells. Antibodies aligned to the IMGT database were selected. The heavy chain variable region gene was cloned into the pGI-mH vector containing mouse IgG2a via AgeI and SalI restriction sites, and the light chain gene was cloned into the pGI-mK vector containing the mouse Kappa chain constant region via AgeI and BsiwI restriction sites. After successful sequencing, HEK293F cells were co-transfected with PEI transfection reagent. Cell supernatant was collected after 5 days, and the target antibody was purified using a Protein A column.

[0073] II. Experimental Results Six mice, numbered m1, were immunized with the EV76 vaccine strain according to the prescribed procedure. m6 and m7 were serum samples from unimmunized normal mice. Venous blood was collected on day 30, and the antibody titer against the F1 antigen in the serum was measured using an indirect ELISA. Results are as follows: Figure 1 m1 M6 mice immunized with EV76 all produced antibodies against the F1 antigen. Serum diluted 10... 5 Even after dilution, the F1 antigen still showed significant color development. However, in unimmunized m7 mice, the serum did not show a significant reaction to the F1 antigen at any dilution.

[0074] A heavy and light chain variable region of approximately 350 bp was amplified from mouse spleen cDNA. Then, overlapping PCR was used to randomly ligate the heavy and light chains into a ScFv sample of approximately 700 bp. After enzyme digestion, the sample was ligated into a phage vector. The ligation product was electrolyzed four times in XL1-Blue competent cells, and the library size was determined to be 2.0 × 10⁻⁶. 8 CFU was used to randomly select 20 single colonies for sequencing. All 20 colonies contained the complete ScFv antibody fragment, indicating a 100% correct insertion rate in the library. After three rounds of screening with the F1 antigen, 96 single colonies were randomly selected and subjected to IPTG induction and Phage-ELISA. OD was found... 450 There were 28 positive clones with a value >0.5, such as Figure 2 All bacterial cultures were sent for sequencing. After sequencing and comparison and analysis with the IMGT database, a specific monoclonal antibody was obtained, named Fm3. The sequence information is shown in Tables 2 and 3.

[0075] Table 2. Fm3 antibody amino acid sequence information

[0076] Table 3. Nucleotide sequence information of Fm3 antibody

[0077] Example 2: Specific detection of Fm3 antibody I. Experimental Methods 1. ELISA: F1 protein (preserved and provided by Lanzhou Institute of Biological Products Co., Ltd.) or SARS-CoV-2 NP (expressed and preserved by Jiangsu Provincial Center for Disease Control and Prevention) were coated onto 96-well ELISA plates at 1 µg / mL. Purified antibodies were serially diluted starting from 100 ng / mL. Primary antibody was incubated at 37°C for 1 h. After washing with PBST, HRP-labeled anti-mouse IgG was added, followed by incubation at 37°C for 30 min. TMB was then used for color development. OD was read after termination. 450The absorbance value was calculated by taking the average value of three replicates for each sample.

[0078] 2. Western Blot: 10 µg of F1 protein was subjected to SDS-PAGE, and the protein was transferred from the PAGE gel to a PVDF membrane. After blocking with 3% skim milk and incubation with recombinant human F1 monoclonal antibody, HRP-labeled anti-mouse IgG was added after washing. Finally, DAB was used to directly develop the color on the PVDF membrane.

[0079] II. Experimental Results ELISA results as follows Figure 3 A. After Fm3 binds to the F1 antigen, it can be recognized by anti-mouse IgG secondary antibody, and a clear dose-response curve is observed. Through nonlinear fitting, the EC50 of Fm3... 50 The value was 59 ng / µL. Fm3 did not cross-react with irrelevant antigens, namely the SARS-CoV-2 NP antigen, such as... Figure 3 B. In Western blotting experiments, the Fm3 monoclonal antibody recognized linearized F1 protein, showing a distinct band at 15 kDa. This result confirms that this antibody is a typical linear epitope monoclonal antibody. Figure 3 C.

[0080] Example 3: Detection of the affinity between Fm3 antibody and F1 protein I. Experimental Methods The affinity constant and binding kinetic parameters between mouse monoclonal antibody and F1 antigen were determined using the Sartorius Octet R8 molecular interaction analysis system. Utilizing the principle of membrane interference, the monoclonal antibody was immobilized on a Pro A sensor, equilibrated, and then reacted with diluted F1 antigen. Changes in surface optical interference were analyzed to obtain information on intermolecular interactions. Specific steps included: Pro A sensor pre-wetting, sensor equilibration, antibody immobilization, post-immobilization equilibration, F1 antigen binding, antigen dissociation, and sensor regeneration. Molecular interaction and binding kinetic data were acquired and analyzed in real time.

[0081] II. Experimental Results The results showed that Fm3 antibody concentrations from 200 nM to 3.13 nM could produce obvious binding and dissociation curves for F1 antigen, and the curves showed a clear dose-response relationship. Figure 4 A). Figure 4 B represents the relevant kinetic parameters of the reaction between Fm3 and F1. The affinity constant KD value of the Fm3 antibody is 2.1 mM, indicating high affinity.

[0082] Example 4: In vivo challenge experiment of Fm3 antibody against virulent and drug-resistant plague strains. I. Experimental Methods 1. Yersinia pestis: 141 highly virulent strains of Yersinia pestis (standard strain) and 127 streptomycin-resistant strain S19960127 were preserved and provided by the Qinghai Provincial Institute for Endemic Disease Prevention and Control. After resuscitation and culture of both strains, the bacterial flora was ground in physiological saline and turbidity was measured to prepare a 7×10⁻⁶ solution. 8 The original bacterial suspension, at CFU / mL, was serially diluted to 1×10⁻⁶. 7 1×10 6 1×10 5 1.5×10 4 (100MLD), 1×10 4 1×10 3 Concentrations of 300 (2 MLD), 150 (1 MLD), and 75 CFU / mL (0.5 MLD) were used. The experimental group used 1.5 × 10⁻⁶ CFU / mL. 4 Mice were challenged by subcutaneous injection of 0.5 mL of CFU / mL bacterial suspension via the groin; the control group received 1.5 × 10⁻⁶ CFU / mL bacterial suspension. 4 Bacterial suspensions at concentrations of 300, 150, and 75 CFU / mL were challenged using the same method. To verify the viable cell count, 1×10⁻⁶ CFU / mL was used. 4 With 1×10 3 CFU / mL bacterial suspensions were evenly spread on Qingdao Herxheimer's medium, with an inoculation amount of 100 μL / plate. After incubation at 28℃ for 48 h, the bacterial counts were performed, and the actual number of viable challenged bacteria was calculated based on the average colony count.

[0083] 2. Animal Experiments: BALB / c mice were divided into three groups based on the amount of each monoclonal antibody: 100 µg / mouse, 20 µg / mouse, and 4 µg / mouse. Six 6-8 week old female BALB / c mice were immunized in each group. All antibodies were injected intraperitoneally into the mice 24 hours prior to immunization. The following day, the mice were challenged with 100 MLD of the standard strain 141 of Yersinia pestis or streptomycin-resistant strain S19960127. Simultaneously, mice without antibodies were injected with 100 MLD, 2 MLD, 1 MLD, and 0.5 MLD of a virulent strain or strain S19960127 as a control to verify the virulence of the strain. Experimental group information is shown in Table 4. After immunization and challenge, the mice were housed in IVC (in vitro isolation) cages. Mouse weight and mortality were recorded daily. After dissection of the deceased mice, the heart, liver, lungs, and spleen were used for impregnation agar culture of Yersinia pestis. Positive cultures were verified using Yersinia pestis-specific bacteriophage lysis.

[0084] Table 4 Animal Experiment Grouping Information

[0085] II. Experimental Results 100 µg of Fm3 provided 100% protection against challenge with strain 141; in the 20 µg group, the Fm3 antibody showed 25% survival; all animals in the 4 µg group died around day 10, indicating that the 4 µg group had no protective effect. Figure 5 A). In the control MLD group, only one animal in the 0.5 MLD group survived to 21 days. After the experiment, organ and tissue imprint culture showed that the animal had cleared the plague bacteria from its body. All other animals in the control MLD group died within 10 days.

[0086] like Figure 5 Similar results were observed in mice challenged with the drug-resistant strain S19960127. 100 µg of Fm3 provided 75% protection in the mice. All animals in the 20 µg and 4 µg groups died within approximately 10 days, indicating that the two low-dose groups offered no protection. In the control MLD groups, no clear dose-response relationship was observed; all animals in the four control groups died within 10 days. This result suggests that the drug-resistant strain S19960127 challenged was larger than the theoretical value of 100 MLD, resulting in no difference in survival time between the 0.5 MLD and 1 MLD groups and the 100 MLD group.

[0087] Hearts, livers, spleens, and lungs were taken from all the dead mice and imprinted on Herxheimer plates. The next day, bacterial colonies grew on the plates. Verification by lysis of plague-specific bacteriophages showed clear phage bands in all the imprinted bacteria, confirming that the deaths of the mice were caused by plague.

[0088] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A plague antibody or its antigen-binding fragment, characterized in that, The heavy chain variable region CDR1 of the antibody or its antigen-binding fragment is shown in SEQ ID NO:1; CDR2 is shown in SEQ ID NO:2; CDR3 is shown in SEQ ID NO:3; the light chain variable region CDR1 is shown in SEQ ID NO:5; CDR2 is shown in SEQ ID NO:6; CDR3 is shown in SEQ ID NO:

7.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region of the antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 75% identity with SEQ ID NO:4; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with SEQ ID NO:

8.

3. A bispecific antibody, characterized in that, The bispecific antibody comprises the antibody or its antigen-binding fragment as described in any one of claims 1 or 2; Preferably, the bispecific antibody further comprises a second antibody that specifically binds to other antigens.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment as described in claim 1 or 2, or the bispecific antibody as described in claim 3.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4.

6. A recombinant host cell, characterized in that, The recombinant host cell comprises the expression vector of claim 5.

7. Any of the following products: 1) An antibody conjugate, wherein the antibody conjugate is a complex formed by directly or indirectly conjugating the antibody or its antigen-binding fragment as described in any one of claims 1 or 2, or the bispecific antibody as described in claim 3, to a detectable marker; 2) A detection reagent comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, or the antibody-drug conjugate thereof; 3) A detection product comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the antibody-drug conjugate, or the detection reagent; 4) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, or the bispecific antibody as described in claim 3; 5) A pharmaceutical preparation comprising the pharmaceutical composition.

8. The product according to claim 7, characterized in that, The detectable markers include fluorescent dyes, avidin, paramagnetic atoms, radioactive isotopes, enzyme markers, and colloidal gold; Preferably, the detection product includes a reagent kit, a chip, and a test strip.

9. Any of the following methods: 1) A method for preparing recombinant host cells according to claim 6, the method comprising: The expression vector according to claim 5 is introduced into a host cell to obtain the recombinant host cell according to claim 6; 2) A method for producing an antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, or a bispecific antibody as described in claim 3, the method comprising: culturing a recombinant host cell as described in claim 6, and isolating the antibody or antigen-binding fragment thereof or bispecific antibody from the culture product of the recombinant host cell; 3) A method for detecting Yersinia pestis F1 protein in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the test sample with an antibody or its antigen-binding fragment as described in any one of claims 1 or 2, a bispecific antibody as described in claim 3, an antibody-drug conjugate as described in claim 7, a detection reagent or a detection product, and detecting the formation of the corresponding immune complex.

10. Any of the following applications: 1) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, or the recombinant host cell as described in claim 6 in the preparation of antibody-drug conjugates for detecting Yersinia pestis F1 protein; 2) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, the recombinant host cell as described in claim 6, or the antibody conjugate as described in claim 7 in the preparation of a detection reagent for detecting Yersinia pestis F1 protein; 3) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, the recombinant host cell as described in claim 6, the antibody conjugate or detection reagent as described in claim 7 in the preparation of a detection product for detecting Yersinia pestis F1 protein; 4) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, the recombinant host cell as described in claim 6, the antibody conjugate as described in claim 7, the detection reagent or detection product in the detection of Yersinia pestis F1 protein for non-diagnostic and non-therapeutic purposes; 5) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, the recombinant host cell as described in claim 6, the antibody conjugate as described in claim 7, the detection reagent or detection product in the preparation of diagnostic products for the diagnosis or auxiliary diagnosis of plague; 6) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, or the recombinant host cell as described in claim 6 in the preparation of a medicament for the treatment and / or prevention of plague; 7) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, the recombinant host cell as described in claim 6, or the pharmaceutical composition as described in claim 7 in the preparation of a pharmaceutical formulation for the treatment and / or prevention of plague; Preferably, the plague is a disease caused by infection with Yersinia pestis; Preferably, the plague bacterium is Yersinia pestis, and also includes its virulent strains and / or drug-resistant strains; Preferably, the highly virulent strain is 141 strains, and the drug-resistant strain is S19960127 strain.

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