Plague antibody Fm25 and application thereof

Abstract

The invention discloses a plague antibody Fm25 and application thereof.The plague antibody Fm25 is an antibody targeting plague bacillus F1 protein, HCDR1-3 in a heavy chain variable region of the plague antibody Fm25 is shown as SEQ ID NO: 1-3, LCDR1-3 in a light chain variable region of the plague antibody Fm25 is shown as SEQ ID NO: 5-7, the plague antibody Fm25 has high binding activity and good binding specificity on F1 protein, and the plague antibody Fm25 can be used for preparing the plague antibody Fm25. The kit can be used for detecting and treating plague bacillus infection, and has important significance on plague prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the plague antibody Fm25 and its applications. Background Technology

[0002] The causative agent of plague is *Yersinia pestis*. Due to variations in virulence, site of infection, route of transmission, and the body's resistance, plague can manifest in various forms, primarily including bubonic plague, pneumonic plague, septicemic plague, cutaneous plague, intestinal plague, ocular plague, and meningitic plague. All clinical types of plague are characterized by rapid onset, rapid progression, and short duration; therefore, timely and effective treatment can reduce mortality. The traditional treatment for plague patients involves the first-line drug streptomycin, combined with other antibiotics to improve the cure rate. However, with the widespread use of anti-infective drugs both domestically and internationally, drug-resistant strains are increasingly emerging, and drug-resistant plague strains are also appearing.

[0003] Neutralizing monoclonal antibodies are considered promising specific therapeutic agents because their targets and mechanisms of action are well-defined, their development is relatively easy, and they represent a practical approach. Studies show that the F1 (Fraction1) antigen, located on the capsule surface of the plague bacterium and encoded by a 100 kbp pFra plasmid, is one of the specific antigens of the plague bacterium. In the immune protection mechanism against plague infection, F1 antiserum has shown a significant protective effect in animals, thus humoral immunity plays a crucial role. This suggests that screening for F1 antigen monoclonal antibodies could potentially yield protective neutralizing monoclonal antibodies for specific treatment of plague. Currently, there are no reports of antibodies against drug-resistant plague strains. Summary of the Invention

[0004] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0005] The first aspect of the present invention provides a plague antibody or its antigen-binding fragment thereof, the plague antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3.

[0006] Furthermore, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:1, 2 and 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:5, 6 and 7, respectively.

[0007] Furthermore, the heavy chain variable region of the plague 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, and 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 plague antibody or its antigen-binding fragment can be a monoclonal antibody, a domain antibody, a single-chain (scFv), a Fab fragment, an F(ab')2 fragment 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 Yersinia pestis F1 protein are mouse, other rodent, chimeric, humanized, or fully human monoclonal antibodies.

[0009] The "complementarity-determining region" (CDR) or "hypervariant region" is a region in the antibody's variable region that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes.

[0010] Based on the variable region amino acid sequence contained in the given plague antibody or its antigen-binding fragment according to this invention, those skilled in the art can routinely determine the CDR contained therein. For example, the Kabat, AbM, Chothia, or Contact protocols can be used to define the CDR in the variable region amino acid sequence.

[0011] When referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0012] The boundaries of the CDR of the antibody of the present invention can be determined artificially according to any method or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers the CDR sequence determined in any of the foregoing methods.

[0013] In some embodiments, functional variants of the plague antibody or its antigen-binding fragment described in this invention are also included within the scope of protection of this invention. A "functional variant" refers to a protein that has significant or marked sequence identity or similarity to the parent antibody, and that retains the biological activity of the parent antibody. Functional variants encompass, for example, the following variants of the plague antibody or its antigen-binding fragment (parent antibody) described herein, which retain the ability to recognize target cells to a similar, equal, or greater extent than the parent antibody. Referring to the parent antibody, the functional variant may, for example, have at least about 75%, 80%, 85%, 90%, 95%, or higher identity with the parent antibody in terms of amino acid sequence.

[0014] In some embodiments, the functional variant may, for example, comprise the amino acid sequence of a parent antibody having at least one conserved amino acid substitution. Alternatively or supplementally, the functional variant may comprise the amino acid sequence of a parent antibody having at least one non-conserved amino acid substitution. In this case, the non-conserved amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity of the functional variant compared to the parent antibody.

[0015] In some embodiments, conservative amino acid substitution is known in the art and includes the substitution of one amino acid having a particular physical and / or chemical property with another amino acid having the same or similar chemical or physical property. For example, conservative amino acid substitutions can be: an acidic / negatively charged polar amino acid replacing another acidic / negatively charged polar amino acid (e.g., Asp or Glu); an amino acid with a nonpolar side chain replacing another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.); a basic / positively charged polar amino acid replacing another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.); an uncharged amino acid with a polar side chain replacing another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.); an amino acid with a β-branched side chain replacing another amino acid with a β-branched side chain (e.g., He, Thr, and Val); and an amino acid with an aromatic side chain replacing another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr).

[0016] A second aspect of the present invention provides a bispecific antibody comprising the plague antibody or its antigen-binding fragment described in the first aspect of the present invention.

[0017] Furthermore, the bispecific antibody also includes a second antibody that specifically binds to other antigens.

[0018] In some implementations, the second antibody that specifically binds to other antigens is not particularly limited. It can be other antibodies that target the Yersinia pestis F1 protein or antibodies that target any antigen other than the Yersinia pestis F1 protein. Those skilled in the art can make conventional selections according to actual needs.

[0019] A third aspect of the present invention provides a polynucleotide molecule or a carrier comprising the polynucleotide molecule, said polynucleotide molecule encoding the plague 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.

[0020] In a specific embodiment of the present invention, the nucleotide sequences encoding the heavy chain variable regions HCDR1-3 of the antibody or its antigen-binding fragment described in 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 LCDR1-3 of the antibody or its antigen-binding fragment described in the first aspect of the present invention are shown in SEQ ID NO:13-15.

[0021] 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.

[0022] In this invention, the polynucleotide molecule may comprise natural, non-natural, or modified nucleotides; and it may comprise natural, non-natural, or modified internucleotide linkages, such as aminophosphate linkages or thiophosphate linkages, instead of phosphodiester linkages present between unmodified oligonucleotide nucleotides. In some embodiments, the nucleotides do not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, it may be suitable for a nucleotide to contain one or more insertions, deletions, inversions, and / or substitutions, and therefore, nucleotides formed by these insertions, deletions, inversions, and / or substitutions are also within the scope of this invention.

[0023] Those skilled in the art can readily mutate the nucleotide sequence corresponding to the antibody described in this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 75% or more identity with the nucleotide sequence corresponding to the plague antibody or its antigen-binding fragment described in this invention, as long as they encode the aforementioned plague antibody or its antigen-binding fragment, are all derived from and equivalent to the nucleotide sequence of this invention, and are also included within the scope of protection of this invention.

[0024] When applied to polynucleotide molecules, the term "encoding" refers to a polynucleotide that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce an mRNA containing a polypeptide and / or fragments thereof, is called "encoding" the polypeptide. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.

[0025] In some embodiments, the vector may contain expression regulatory sequences, such as transcription and translation start and stop codons, which 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 whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0026] 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.); and prototrophic complementation in auxotrophic hosts. Suitable marker genes for the expression vector of this invention include neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, and puromycin resistance genes.

[0027] Various vectors known in the art can be used, such as commercially available vectors, and then a polynucleotide encoding the antibody or its antigen-binding fragment can be operatively linked to the expression regulatory sequence to form an expression vector. In some embodiments, the vector includes, but is not limited to, plasmids, phage particles, granules, artificial chromosomes, and virus-derived vectors.

[0028] A fourth aspect of the present invention provides a modified host cell or a population of host cells comprising thereto, said modified host cell comprising the polynucleotide molecule described in the third aspect of the present invention or a vector comprising thereto.

[0029] Furthermore, the host cell population also includes host cells other than the modified host cells.

[0030] Furthermore, the modified host cells include prokaryotic cells and eukaryotic cells.

[0031] In some implementations, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsia.

[0032] In some implementations, the bacteria include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Staphylococcus.

[0033] In some implementations, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0034] In some embodiments, the modified host cells are prepared by introducing the polynucleotide molecules or vectors containing them as described above into the host cells. The methods of introduction include, but are not limited to, physical, chemical, and biological methods. The physical methods include, but are not limited to, microinjection, electroporation, calcium phosphate precipitation, lipid transfection, and particle bombardment. The chemical methods include, but are not limited to, colloidal dispersion systems and lipid-based systems. The colloidal dispersion systems include, but are not limited to, macromolecular complexes, nanocapsules, microspheres, and beads. The lipid-based systems include, but are not limited to, oil-in-water emulsions, micelles, mixed micelles, and liposomes. The biological methods include, but are not limited to, DNA vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors.

[0035] The fifth aspect of the present invention provides an antibody derivative of a plague antibody or its antigen-binding fragment thereof, the antibody derivative comprising a complex formed by direct or indirect coupling of the plague 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 or therapeutic agent.

[0036] Furthermore, the detectable markers include fluorescent dyes, enzymes, chemiluminescent markers, radioactive isotopes, electron-dense reagents, colored particles, biotin, or digoxin.

[0037] In some implementations, the detectable marker often generates 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, and phycoerythrin; examples of suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; chemiluminescent markers include, but are not limited to, luminol and its derivatives, isoluminol and its derivatives, acridine esters and their derivatives, adamantane, rare earth elements, and ruthenium bipyridine complexes; radioactive isotopes include, but are not limited to, 68Ga, 86Y, 110In, 111In, 177Lu, 18F, 52Fe, 62Cu, 64Cu, 11C, 67Cu, 94Tc, 99mTc, 120I, 123I, 124I, and 15O. The aforementioned detectable markers can be directly linked or conjugated with antibodies or indirectly via intermediates such as known linkers in the art using techniques known in the art.

[0038] Furthermore, the therapeutic agents include cytotoxic agents, hormonal preparations, targeted small molecule preparations, proteasome inhibitors, chemotherapeutic agents, oncolytic drugs, cytokines, activators of co-stimulatory molecules, or inhibitors of inhibitory molecules.

[0039] The sixth aspect of the present invention provides a detection reagent or detection product, the detection reagent or detection product comprising the plague 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, or the antibody derivative as described in the fifth aspect of the present invention.

[0040] Furthermore, the product includes reagent kits, chips, and test strips.

[0041] Furthermore, the kits include, but are not limited to, ELISA kits, immunofluorescence kits, flow cytometry kits, and immunohistochemistry kits.

[0042] In some embodiments, the kit may include a container, instructions, buffers, etc. In other embodiments, the kit may also include 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 test kit can be an in vitro diagnostic device.

[0043] The seventh aspect of the present invention provides a pharmaceutical composition for treating plague, the pharmaceutical composition comprising the plague 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, or the antibody derivative described in the fifth aspect of the present invention.

[0044] Furthermore, the plague is a disease caused by infection with Yersinia pestis.

[0045] Furthermore, the plague bacterium is the Yersinia pestis strain, and also includes its highly virulent strains and / or drug-resistant strains.

[0046] Furthermore, the highly virulent strain is 141 strains, and the drug-resistant strain is S19960127 strain.

[0047] In this invention, the treatment refers to the suppression, containment, relief, improvement, slowing, cessation, delay, or reversal of the course of a disease in order to prevent and reduce the occurrence or development of the disease. Various indicators of maintaining and / or using the medication during the course of the disease, disorder, or pathological state include the reduction or elimination of symptoms or complications, or the cure or elimination of the disease, disorder, or condition.

[0048] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. Excipients refer to additives in a pharmaceutical preparation other than the active pharmaceutical ingredient (API), also known as excipients. General requirements for excipients include: stability, no incompatibility with the API, no side effects, no impact on efficacy, resistance to deformation, cracking, mold, and insect infestation at room temperature, harmlessness to humans, no physiological effects, no chemical or physical reactions with the API, and no interference with the determination of the API's content.

[0049] In some embodiments, the excipients include binders, fillers, disintegrants, lubricants, ointments, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solvents, osmotic pressure regulators, and colorants.

[0050] In some embodiments, the above-described pharmaceutical composition may be administered via 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 fluid administration.

[0051] The eighth aspect of the present invention provides any of the following methods: 1) A method for preparing the plague 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, the method comprising the following steps: culturing the modified host cells or host cell populations containing the modified host cells as described in the fourth aspect of the present invention, and isolating the plague antibody or its antigen-binding fragment or bispecific antibody from the culture.

[0052] 2) A method for detecting Yersinia pestis F1 protein in a test sample, the method comprising the following steps: contacting the test sample with the plague 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 derivative as described in the fifth aspect of the present invention, or the detection reagent or detection product as described in the sixth aspect of the present invention, and detecting the formation of the corresponding antibody-antigen complex.

[0053] 3) A method for preparing the modified host cell or host cell population containing the present invention according to the fourth aspect of the present invention, the method comprising the following steps: introducing the polynucleotide molecule or vector containing the present invention according to the third aspect of the present invention into the host cell.

[0054] In some implementations, the sample or test sample may be selected from blood, serum, plasma, urine, saliva, ascites, brain tissue, cerebrospinal fluid, non-tissue-associated cells, tissues, histological preparations, etc., from the test subject. The present invention does not have any particular limitation on the specific type of the sample or test sample, and any sample that may contain Yersinia pestis F1 protein may be used as the sample or test sample.

[0055] The ninth aspect of the present invention provides any of the following applications: 1) The use of the plague 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 polynucleotide molecule or a carrier containing the same as described in the third aspect of the present invention, the modified host cell or a host cell population containing the same as described in the fourth aspect of the present invention, or the antibody derivative as described in the fifth aspect of the present invention in the preparation of a detection reagent or detection product for detecting the F1 protein of Yersinia pestis.

[0056] 2) The use of the plague 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 polynucleotide molecule or a carrier containing the same as described in the third aspect of the present invention, the modified host cell or a host cell population containing the same as described in the fourth aspect of the present invention, the antibody derivative as described in the fifth aspect of the present invention, or the detection reagent or detection product as described in the sixth aspect of the present invention in the preparation of diagnostic products for the diagnosis or auxiliary diagnosis of plague.

[0057] 3) The use of the plague 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 polynucleotide molecule or a carrier containing the thereof as described in the third aspect of the present invention, or the modified host cell or a population of host cells containing the thereof as described in the fourth aspect of the present invention in the preparation of a medicament for the treatment and / or prevention of plague.

[0058] 4) The application of the plague 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 polynucleotide molecule or a carrier containing the same as described in the third aspect of the present invention, the modified host cell or a host cell population containing the same as described in the fourth aspect of the present invention, the antibody derivative as described in the fifth aspect of the present invention, or the detection reagent or detection product as described in the sixth aspect of the present invention in the detection of plague F1 protein for non-diagnostic and non-therapeutic purposes.

[0059] Advantages and beneficial effects of the present invention: This invention discloses the plague antibody Fm25 and its applications, wherein the plague antibody Fm25 is an antibody targeting the F1 protein of Yersinia pestis. The plague antibody or its antigen-binding fragment of this invention can bind to the Yersinia pestis F1 protein with high activity and specificity. This invention also relates to polynucleotide molecules encoding the plague antibody or its antigen-binding fragment, vectors, host cells, and antibody derivatives or other products derived from the plague antibody or antigen-binding fragment according to this invention. Attached Figure Description

[0060] Figure 1 The titer of F1 antibody in the serum of immunized mice is given.

[0061] Figure 2To screen for target antibodies in a library for phage ELISA.

[0062] Figure 3 The results show the specificity of Fm25 antibody binding to F1; where A is the ELISA result of Fm25 antibody binding to F1 protein; B is the ELISA result of Fm25 antibody not binding to irrelevant antigen (SARS-CoV-2 NP); and C is the Western Blot result of Fm25 antibody binding to F1 protein.

[0063] Figure 4 The affinity of Fm25 antibody to F1 was determined; where A is the dissociation curve of Fm25 antibody to F1; and B is the relevant kinetic parameters of Fm25 antibody to F1.

[0064] 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

[0065] 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.

[0066] 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.

[0067] Example 1: Preparation of Plague Antibody Fm25 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.

[0068] 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... 7 Six 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.

[0069] 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.

[0070] Table 1 Primer Sequences

[0071] 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.

[0072] 5. Select single clones that specifically bind to F1. 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.

[0073] 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.

[0074] 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 Mice of the m6 genotype, after immunization 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.

[0075] 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 Fm25. The sequence information is shown in Tables 2 and 3.

[0076] Table 2. Fm25 antibody amino acid sequence information

[0077] Table 3. Nucleotide sequence information of Fm25 antibody

[0078] Example 2: Detection of Fm25 antibody specificity 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. 450 The absorbance value was calculated by taking the average value of three replicates for each sample.

[0079] 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.

[0080] II. Experimental Results ELISA results as follows Figure 3 A. After Fm25 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 Fm25... 50 The value was 34 ng / µL. Fm25 does not cross-react with irrelevant antigens, namely the SARS-CoV-2 NP antigen, such as... Figure 3 B. In Western blotting experiments, the Fm25 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 B.

[0081] Example 3: Detection of the affinity between Fm25 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.

[0082] II. Experimental Results The results showed that Fm25 antibody concentrations from 200 nM to 3.13 nM could produce obvious binding and dissociation curves for the F1 antigen, and the curves exhibited a clear dose-response relationship. Figure 4 A). Figure 4 B represents the relevant kinetic parameters of the reaction between Fm25 and F1. The affinity constant KD value of the Fm25 antibody is 1.3 nM, indicating high affinity.

[0083] Example 4: In vivo challenge experiment of Fm25 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.

[0084] 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.

[0085] Table 4 Animal Experiment Grouping Information

[0086] II. Experimental Results 100 µg of Fm25 provided 100% protection against challenge with strain 141; all animals in the 20 µg and 4 µg groups died around day 10, indicating that the 20 µg and 4 µg groups had no protective effect. Figure 5 A). In the control MLD group, only one animal in the 0.5MLD 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.

[0087] like Figure 5 Similar results were observed in mice challenged with the drug-resistant strain S19960127. No animals died in the 100 µg Fm25 group. All animals in the 20 µg and 4 µg groups died within approximately 10 days, indicating that the two low-dose groups offered no protective effect. In the control MLD groups, no clear dose-response relationship was observed; all animals in the four control groups died within 10 days. These results suggest 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.

[0088] 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.

[0089] 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 plague antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the light chain variable region includes LCDR1, LCDR2 and LCDR3. The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:1, 2 and 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:5, 6 and 7, respectively.

2. The plague antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region of the plague 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, and 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 plague 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 polynucleotide molecule or a carrier comprising the thereof, characterized in that, The polynucleotide molecule encodes the plague antibody or its antigen-binding fragment as described in claim 1 or 2, or the bispecific antibody as described in claim 3.

5. A modified host cell or a population of host cells containing the same, characterized in that, The modified host cell comprises the polynucleotide molecule of claim 4 or a vector containing thereto; Preferably, the host cell population further includes host cells other than the modified host cells; Preferably, the modified host cells include prokaryotic cells and eukaryotic cells.

6. An antibody derivative thereof, comprising a plague antibody or an antigen-binding fragment thereof, characterized in that, The antibody derivative comprises the plague 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, directly or indirectly coupled to a detectable marker or therapeutic agent to form a complex. Preferably, the detectable markers include fluorescent dyes, enzymes, chemiluminescent markers, radioactive isotopes, electron-dense reagents, colored particles, biotin, or digoxin; Preferably, the therapeutic agent includes cytotoxic agents, hormonal preparations, targeted small molecule preparations, proteasome inhibitors, chemotherapeutic agents, oncolytic drugs, cytokines, activators of co-stimulatory molecules, or inhibitors of inhibitory molecules.

7. A testing reagent or testing product, characterized in that, The detection reagent or detection product includes the plague antibody or its antigen-binding fragment as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, or the antibody derivative as described in claim 6; Preferably, the product includes a reagent kit, a chip, and a test strip.

8. A pharmaceutical composition for treating plague, characterized in that, The pharmaceutical composition comprises the plague antibody or its antigen-binding fragment as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, or the antibody derivative as described in claim 6; 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.

9. Any of the following methods: 1) A method for preparing the plague 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, the method comprising the following steps: culturing the modified host cell or host cell population containing the modified host cell as described in claim 5, and isolating the plague antibody or its antigen-binding fragment or bispecific antibody from the culture; 2) A method for detecting Yersinia pestis F1 protein in a test sample, the method comprising the following steps: contacting the test sample with the plague antibody or its antigen-binding fragment as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the antibody derivative as described in claim 6, or the detection reagent or detection product as described in claim 7, and detecting the formation of the corresponding antibody-antigen complex; 3) A method for preparing the modified host cell or a host cell population containing the present invention as described in claim 5, the method comprising the step of introducing the polynucleotide molecule or a vector containing the present invention as described in claim 4 into the host cell.

10. Any of the following applications: 1) The use of the plague 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 polynucleotide molecule or a carrier containing the thereof as described in claim 4, the modified host cell or a population of host cells containing the thereof as described in claim 5, or the antibody derivative as described in claim 6 in the preparation of a detection reagent or detection product for detecting the F1 protein of Yersinia pestis. 2) The use of the plague 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 polynucleotide molecule or a carrier containing the thereof as described in claim 4, the modified host cell or a host cell population containing the thereof as described in claim 5, the antibody derivative as described in claim 6, or the detection reagent or detection product as described in claim 7 in the preparation of diagnostic products for the diagnosis or auxiliary diagnosis of plague. 3) The use of the plague 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 polynucleotide molecule or a carrier containing the thereof as described in claim 4, or the modified host cell or a population of host cells containing the thereof as described in claim 5 in the preparation of a medicament for the treatment and / or prevention of plague. 4) The use of the plague antibody or its antigen-binding fragment as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the polynucleotide molecule or a carrier containing the same as described in claim 4, the modified host cell or a host cell population containing the same as described in claim 5, the antibody derivative as described in claim 6, or the detection reagent or detection product as described in claim 7 in the detection of plague F1 protein for non-diagnostic, non-therapeutic purposes.

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