Protective monoclonal antibody targeting Mycobacterium tuberculosis OmpA and its preparation method and application

Abstract

The present invention provides a protective monoclonal antibody targeting Mycobacterium tuberculosis OmpA, as well as a preparation method and application. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The sequences of HCDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 1, 2, and 3, and the sequences of LCDR1-3 of the light chain variable region are shown in SEQ ID NOs: 4, 5, and 6. The provided antibody can be used to treat tuberculosis, especially drug-resistant tuberculosis, and can also be used to prevent tuberculosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a protective monoclonal antibody targeting Mycobacterium tuberculosis OmpA, a preparation method and an application thereof. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease transmitted through the respiratory tract, resulting in millions of deaths annually. Currently, the only commercially approved TB vaccine is Bacillus Calmette-Guérin (BCG), which is highly effective in preventing TB infection in infants and young children. However, BCG's limitations in preventing TB infection in adults and the development of meningitis-causing TB have led to an urgent need for new TB vaccines. Traditionally, vaccine development for this intracellular parasite has focused primarily on T cell-mediated cellular immunity, such as MVA85A. However, recent research has prompted researchers to reevaluate the importance of humoral immunity in TB vaccine design. Recent reports have shown that antibodies that recognize TB surface antigens can confer protective immunity against TB. Mycobacterium tuberculosis (Mtb) displays numerous proteins and polysaccharides on its surface, and antibodies targeting a few of these surface proteins and polysaccharides have been shown to exert anti-TB activity. However, bioinformatics predictions indicate that Mtb contains 144 surface proteins. The role of antibodies induced by these surface proteins in the anti-tuberculosis process is not very clear. Establishing and characterizing the role of antibodies produced by these antigens in the anti-tuberculosis process will greatly promote the design of anti-tuberculosis vaccines.

[0003] The overuse of antibiotics has led to the emergence of drug-resistant Mycobacterium tuberculosis, particularly multidrug-resistant tuberculosis, posing a significant challenge to global public health. With the advancement of monoclonal antibody production technology, their applications in pathogenic medicine have become increasingly widespread, including immunotherapy, diagnostics, and cancer treatment. Because antibodies are less likely to induce bacterial tolerance and produce the toxicity associated with small-molecule drugs, antibody therapy offers a promising alternative to antibiotics. For complex bacterial typing, antibodies that recognize conserved antigens are more effective therapeutics. An antibody targeting the Salmonella type III secretion system antigen SipD elicits broad-spectrum activity against different Salmonella serotypes, providing important insights for the development of broad-spectrum therapeutic antibodies targeting different subspecies. The OmpA protein, encoded by the Mycobacterium tuberculosis gene Rv0899, is located on the surface of M. tuberculosis and shares high homology among known M. tuberculosis subspecies. Therefore, establishing and characterizing antibodies induced by OmpA is crucial for anti-tuberculosis therapy. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. The present invention provides a protective monoclonal antibody that specifically targets the Mycobacterium tuberculosis OmpA antigen and a preparation method thereof, and the application of the monoclonal antibody can exert an anti-tuberculosis effect.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] In a first aspect of the present invention, there is provided a protective monoclonal antibody or an antigen-binding fragment thereof targeting Mycobacterium tuberculosis OmpA, comprising a heavy chain variable region and a light chain variable region;

[0007] The HCDR1-3 sequences of the heavy chain variable region are as shown in SEQ ID NOs: 1, 2 and 3, respectively, or are sequences having an amino acid substitution, deletion or addition compared to the sequences shown in SEQ ID NOs: 1, 2 or 3;

[0008] The sequences of LCDR1 to LCDR3 of the light chain variable region are as shown in SEQ ID NOs: 4, 5, and 6, respectively, or are sequences having an amino acid substitution, deletion, or addition compared to the sequences shown in SEQ ID NOs: 4, 5, or 6;

[0009] The sequences represented by the HCDR1 to 3 and the sequences represented by the LCDR1 to 3 are obtained based on the IMGT definition scheme.

[0010] According to an embodiment of the present invention, the monoclonal antibody or antigen-binding fragment thereof has:

[0011] (a) the heavy chain variable region set forth in SEQ ID NO:7, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the heavy chain variable region sequence set forth in SEQ ID NO:7; and

[0012] (b) the light chain variable region set forth in SEQ ID NO:8, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the light chain variable region sequence set forth in SEQ ID NO:8.

[0013] According to an embodiment of the present invention, the monoclonal antibody or antigen-binding fragment thereof has a heavy chain variable region shown in SEQ ID NO: 7 and a light chain variable region shown in SEQ ID NO: 8.

[0014] In a second aspect, the present invention provides an isolated polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to the first aspect.

[0015] According to an embodiment of the present invention, the polynucleotide includes a sequence encoding a heavy chain variable region as shown in SEQ ID NO: 9 and a sequence encoding a light chain variable region as shown in SEQ ID NO: 10.

[0016] The third aspect of the present invention provides a construct comprising the isolated polynucleotide according to the second aspect.

[0017] In a fourth aspect, the present invention provides a host cell, wherein the host cell contains the isolated polynucleotide according to the second aspect or the construct according to the third aspect.

[0018] In a fifth aspect of the present invention, a method for producing a monoclonal antibody or an antigen-binding fragment thereof is provided, comprising:

[0019] Cultivating a host cell, wherein the host cell is the host cell described in the fourth aspect, and

[0020] The monoclonal antibody or antigen-binding fragment thereof is collected from the culture.

[0021] In a sixth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0022] The monoclonal antibody or antigen-binding fragment thereof according to any embodiment of the first aspect; and

[0023] Pharmaceutically acceptable carrier.

[0024] In a seventh aspect, the present invention provides a kit comprising: the monoclonal antibody or antigen-binding fragment thereof according to any embodiment of the first aspect.

[0025] In an eighth aspect, the present invention provides use of the monoclonal antibody or antigen-binding fragment thereof according to the first aspect in preparing a reagent for detecting Mycobacterium tuberculosis OmpA, or in preparing a drug for preventing or treating a disease.

[0026] According to an embodiment of the present invention, the disease is tuberculosis caused by Mycobacterium tuberculosis.

[0027] According to an embodiment of the present invention, the drug is a preventive vaccine or a therapeutic drug.

[0028] In a ninth aspect, the present invention provides an antigenic protein or polypeptide having the sequence set forth in SEQ ID NO: 11. The provided antigenic protein or polypeptide can be used to immunize animals, thereby enabling the development of monoclonal antibodies or antigen-binding fragments that specifically target Mycobacterium tuberculosis OmpA, thereby treating or preventing tuberculosis or drug-resistant tuberculosis. The provided antigenic protein or polypeptide is obtained by truncating the N-terminal 50 amino acids (located in the transmembrane region) of the OmpA protein.

[0029] According to a specific embodiment, the antigenic protein or polypeptide is encoded by the nucleic acid sequence shown in SEQ ID NO:12. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 1 is a flow chart of antibody preparation and functional identification according to an embodiment of the present invention.

[0031] Figure 2 The preparation of the OmpA antigen and its antibody according to Example 1 of the present invention is shown. A shows the results of agarose gel electrophoresis of the amplified OmpA gene; B shows the results of SDS-PAGE identification of the OmpA protein; C shows the results of Western blot identification of the OmpA protein; D shows the serum titer of mice immunized with the OmpA antigen; E shows the SDS-PAGE results of the purified and identified antibody 1E1; F shows the subtype identification of antibody 1E1; and G shows the recognition of antibody 1E1 against BCG and whole Mycobacterium bovis.

[0032] Figure 3 These are the antibody functional identification results provided in Example 2 of the present invention. A shows the flow cytometric analysis of the effect of antibody 1E1 on macrophage phagocytosis; B shows the statistical flow cytometric analysis of macrophage phagocytosis; C shows the fluorescence confocal microscopy analysis of phagolysosome formation; D shows the percentage of phagolysosome formation as determined by the statistical confocal microscopy; E shows the antibody's inhibition of BCG growth in mouse whole blood; and F shows the reduction of bacterial load in mouse lungs by antibody 1E1.

[0033] Figure 4 This is the antibody homology modeling result provided in Example 3 of the present invention.

[0034] Figure 5 This is the result of predicting the structure of the antigen OmpA using Alphafold3 provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0035] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention, but should not be construed as limiting the present invention. To facilitate understanding by those skilled in the art, some terms in the text are explained and illustrated. It should be noted that these explanations and illustrations are for ease of understanding only and should not be regarded as limiting the scope of protection of the present invention.

[0036] As used herein, the term "antibody" is used in the broadest sense to refer to a protein or polypeptide comprising an antigen-binding site, antigen-binding fragment, or antigen-binding portion, encompassing natural and artificial antibodies of various structures, including but not limited to intact antibodies or antigen-binding fragments of antibodies. According to specific embodiments, the antibodies provided are monoclonal antibodies.

[0037] Monoclonal antibodies or their antigen-binding fragments mentioned herein are generally separable or recombinant. "Separable" refers to being able to identify and separate and / or recycle from cells or cell cultures expressing polypeptides or proteins. Typically, separated polypeptides (e.g., separated antibodies) can be prepared by at least one purification step. "Separated antibodies" refer to being substantially free of other antibodies or antigen-binding fragments (e.g., antibodies that specifically bind to Mycobacterium tuberculosis OmpA antigens that specifically bind to antigens other than the OmpA antigen) that are not substantially free of different antigenic specificities. "Recombinant" means that antibodies can be produced in exogenous host cells using genetic recombination techniques.

[0038] As used herein, "comprising" or "including" means including the elements or steps mentioned, but does not exclude other elements or steps. Of course, unless otherwise indicated, "comprising" or "including" also encompasses situations where the elements or steps are composed of the elements or steps mentioned. For example, when a reference is made to comprising a specific sequence, it is also intended to encompass an antibody consisting of that specific sequence.

[0039] The "affinity" or "binding affinity" mentioned herein is understood according to the common meaning in the art, and is used to reflect the strength and / or stability of the binding sites between an antigen and an antibody or antigen-binding fragment.

[0040] "Specific binding" or "specifically binds to" a specific antigen or epitope, or "having specificity" for a specific antigen or epitope means distinguishing from non-specific interactions, and such specific binding can be measured by methods commonly used in the art. The ability of an antibody or antigen to bind can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art.

[0041] As used herein, the names of amino acids are represented by standard single-letter or three-letter codes commonly used in the art. As used herein, "homology" of a sequence refers to the degree of sequence identity when comparing a protein, polypeptide, or nucleic acid sequence. To determine homology, various methods known in the art can be used. For example, publicly available software such as BLAST, ALIGN, BLAST-2, etc. can be used. Amino acid substitutions as used herein may be conservative amino acid substitutions. As used herein, "conservative amino acid substitutions" of a sequence refer to the replacement of an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative amino acid substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative amino acid substitutions generally do not cause significant changes in the conformational structure of the protein, and therefore, the biological activity of the protein can be retained.

[0042] The antibody CDR sequences shown herein were obtained by combining existing database definition schemes, such as those of the IMGT database (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38: D301-D307 (2010); Ehrenmann, F., Lefranc, M.-P. Cold Spring Harbor Protoc., 6: 737-749 (2011)). Of course, they can also be obtained by combining the definition schemes of Kabat (e.g., see US Pat. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)), Chothia (e.g., see J. Mol. Biol. 196: 901-917 (1987)), etc. It will be appreciated by those skilled in the art that differences in CDR sequences due to differences in database definition schemes are also encompassed within the scope of protection of the present invention.

[0043] The monoclonal antibody provided can also be a humanized antibody. "Humanized antibody" refers to an antibody based on a molecule of an antigen binding site derived from a non-human species and based on the partial structure and sequence of a human immunoglobulin molecule. Compared to non-humanized antibodies, humanized antibodies retain the binding activity to the antigen while reducing the immunogenicity. The immunogenicity of humanized antibodies is lower while retaining the binding activity of the antibody to the OmpA antigen. In at least some embodiments, the humanized antibody provided retains the CDR region amino acid sequence and is replaced with a humanized framework region sequence in the FR framework region. This replacement of the framework region sequence can be a partial replacement or a complete replacement. According to a specific embodiment, the partially humanized framework region sequence mentioned can be obtained by replacing the framework region sequence with a humanized framework region sequence, and then performing a back mutation on the key amino acids that affect the structure and function of the antibody. Humanized antibodies can be obtained by methods commonly used in the art (such as complementary determining region transplantation). Humanized framework region sequences can be obtained by published human sequences. For example, through database comparison and computer homology modeling, human FR region templates with the greatest homology are identified. Comprehensive consideration is then used to determine key residues in the FR region that require backmutation, thereby generating high-affinity humanized antibodies. These sequences are typically documented in common databases such as the PDB protein structure database, IMGT, and Genebank. Humanized antibodies can be further backmutated to retain certain properties of the original sequence, such as affinity and physiological activity.

[0044] The monoclonal antibodies or antigen-binding fragments thereof referred to herein are substantially pure. Substantially pure means that the purity of the produced monoclonal antibodies or antigen-binding fragments thereof can reach 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, etc.

[0045] The present invention provides a monoclonal antibody or an antigen-binding fragment thereof targeting Mycobacterium tuberculosis OmpA, comprising a heavy chain variable region and a light chain variable region;

[0046] The HCDR1 to 3 sequences of the heavy chain variable region are as shown in SEQ ID NOs: 1, 2, and 3, respectively, or are sequences having an amino acid substitution, deletion, or addition compared to the sequences shown in SEQ ID NOs: 1, 2, or 3;

[0047] The sequences of LCDR1 to LCDR3 of the light chain variable region are as shown in SEQ ID NOs: 4, 5, and 6, respectively, or are sequences having an amino acid substitution, deletion, or addition compared to the sequences shown in SEQ ID NOs: 4, 5, or 6;

[0048] The sequences represented by the HCDR1 to 3 and the sequences represented by the LCDR1 to 3 are obtained based on the IMGT definition scheme.

[0049] According to a specific embodiment, the monoclonal antibody or antigen-binding fragment thereof has:

[0050] (a) the heavy chain variable region of SEQ ID NO:7, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the heavy chain variable region of SEQ ID NO:7; and

[0051] (b) the light chain variable region set forth in SEQ ID NO:8, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the light chain variable region sequence set forth in SEQ ID NO:8.

[0052] According to a specific embodiment, the monoclonal antibody or antigen-binding fragment thereof has a heavy chain variable region shown in SEQ ID NO: 7 and a light chain variable region shown in SEQ ID NO: 8.

[0053] The provided monoclonal antibody or antigen-binding fragment thereof may further include an Fc region, which may be humanized or non-humanized.

[0054]

[0055]

[0056] Isolated polynucleotides

[0057] The present invention also provides an isolated polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof described above. The isolated polynucleotide may be DNA, RNA, or cDNA, etc. The polynucleotide sequence encoding the monoclonal antibody can be obtained according to conventional methods of those skilled in the art. According to a specific embodiment, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:9, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:10.

[0058] Construct

[0059] The present invention also provides a construct comprising the isolated polynucleotide described herein. The construct can be obtained using methods commonly used in the art, such as in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. For example, the polynucleotide can be inserted into the multiple cloning site of an expression vector to form the construct. The construct can contain various operators, such as promoters, terminators, and marker genes, as needed, operably linked to the polynucleotide. Promoters are typically used to provide a signal to initiate transcription. Promoters can include the lactose promoter (Lac), Trp promoter, Tac promoter, or phage PL and PR promoters. Terminators provide a signal to terminate transcription during the transcription process, and marker genes in the construct are often used for screening. Enhancers can also be included as needed to enhance protein expression. The expression vector is not particularly limited and can be any commercially available expression vector or an artificially modified expression vector, such as a plasmid, phage, or virus. Viruses can include plant cell viruses, mammalian cell viruses, and the like. The construct can express antibodies or proteins in vitro or be transfected into cells for expression.

[0060] host cells

[0061] The present invention also provides a host cell containing the isolated polynucleotide or construct described above. Any cell suitable for expressing an antibody or protein using a polynucleotide or construct can be used as a host cell. The host cell can be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a yeast cell or a mammalian cell. Commonly used host cells include yeast cells, CHO cells, HEK-293 cells, COS cells, and Drosophila S2 or Sf9 insect cells. Host cells containing the polynucleotide or construct can be obtained using methods commonly used in the art, such as microinjection, electroporation, chemical transfection, and virus-mediated transformation.

[0062] Method for producing monoclonal antibodies or antigen-binding fragments thereof

[0063] The present invention further provides a method for producing a monoclonal antibody or an antigen-binding fragment thereof, comprising: culturing the host cell described herein; and collecting the monoclonal antibody or antigen-binding fragment thereof. The collected antibody is purified to obtain a substantially pure antibody. Substantially pure means that the purity of the produced antibody can reach greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or the like.

[0064] Pharmaceutical composition

[0065] The present invention also provides a pharmaceutical composition comprising the monoclonal antibody or the antigen-binding fragment thereof; and a pharmaceutically acceptable carrier.

[0066] The pharmaceutically acceptable carriers mentioned are acceptable to the subject at the dosage or concentration used. Pharmaceutically acceptable carriers include, but are not limited to: buffers or salts, such as Tris, phosphates, citrates, sodium acetate, citric acid, sodium citrate and other organic acids; antioxidants, such as ascorbic acid; preservatives, such as benzalkonium chloride; amino acids, such as histidine, histidine hydrochloride, glycine, glutamine, asparagine, arginine or lysine. Pharmaceutical preparations for use in subjects are generally sterile, and sterile pharmaceutical preparations can be obtained by methods commonly used in the art, such as by filtration with a sterile filtration membrane. Those skilled in the art can select suitable pharmaceutically acceptable carriers according to the dosage form prepared by the pharmaceutical composition to prepare different dosage forms, such as injections, lyophilized agents, tablets and other dosage forms.

[0067] Reagent test kit

[0068] The present invention also provides a kit comprising the monoclonal antibody or antigen-binding fragment thereof described above. The kit may further comprise a container, a buffer reagent, and controls such as a positive control and a negative control, as needed. Those skilled in the art can make appropriate selections as needed. Accordingly, the kit may further comprise instructions for use to facilitate operation and use by those skilled in the art. Of course, the kit may also be prepared in the form of a test strip for specific detection of the Mycobacterium tuberculosis OmpA antigen.

[0069] The provided monoclonal antibody or antigen-binding fragment thereof can be used for the development of preventive vaccines or therapeutic drugs, thereby being used to treat or prevent tuberculosis or drug-resistant tuberculosis with high specificity.

[0070] The present invention further provides a method for preventing or treating a disease, comprising administering to a subject a preventively or therapeutically effective amount of the aforementioned monoclonal antibody or antigen-binding fragment thereof, wherein the disease is tuberculosis. The provided monoclonal antibody or antigen-binding fragment thereof can be used to treat or prevent tuberculosis or drug-resistant tuberculosis.

[0071] The provided monoclonal antibodies or antigen-binding fragments thereof can be used to prepare drugs or test kits. Drugs prepared as such can be used to prevent tuberculosis. They can also be used as reagent components in test kits for the diagnosis of specific antigens.

[0072] The technical solution of the present invention is described below by means of specific examples. It should be noted that these examples are only used to facilitate the understanding of those skilled in the art and should not be regarded as limiting the scope of protection of the present invention. Unless otherwise specified, the reagents used in the examples can be obtained commercially. Figure 1 As shown, the OmpA protein was first expressed using a prokaryotic expression system. The expressed OmpA protein was mixed with an equal volume of aluminum adjuvant and injected subcutaneously three times, two weeks apart, for immunization. Mouse spleen cells with high serum titers were selected and fused with SP2 / 0 cells, and cells secreting specific antibodies were screened using HAT / HT medium. Sequences of the corresponding specific antibodies were obtained by sequencing. The function, subtype, and related structure of the specific antibodies were simultaneously identified using various methods, including but not limited to: identification of antibody subtypes using an antibody subtype identification kit; whole-bacterium ELISA to identify antibodies against Mycobacterium tuberculosis; verification of Mycobacterium tuberculosis growth inhibition using a mouse PBMC model; immune protection experiments against bovine Mycobacterium tuberculosis following passive antibody injection; and bioinformatics prediction of antigen-antibody binding sites.

[0073] Example 1

[0074] Example 1 An antibody targeting the surface protein OmpA of Mycobacterium tuberculosis was obtained by the following method, comprising:

[0075] 1. Prepare and screen antibodies induced by the Mycobacterium tuberculosis surface protein OmpA. This includes the following steps:

[0076] First, upstream primers (OmpA-F) and downstream primers (OmpA-R) were designed to amplify the OmpA gene fragment. The primers used are shown below. The amplified OmpA gene fragment is shown in SEQ ID NO: 12 (the agarose gel electrophoresis results are shown in Figure 2 The sequence is shown in A in the figure, which encodes the protein sequence shown in SEQ ID NO: 11. The plasmid pET28a(+) was digested with EcoRI and HindIII and then homologous recombination was used to construct the vector. After sequencing, the vector was transformed into BL21 competent cells. The protein was then expressed using IPTG induction and Ni was used. 2+ The protein was affinity purified and identified by SDS-PAGE and Western blot.

[0077] OmpA-F: 5'-CCGGAATTCGAGCGGCCCCAGTCCGTTAC-3' (SEQ ID NO: 13);

[0078] OmpA-R: 5'-CCCAAGCTTGTTGACCACGATCTCGACGCGAC-3' (SEQ ID NO: 14)

[0079] SDS-PAGE results are as follows Figure 2 As shown in B, label M represents protein marker, label 1 represents protein after IPTG induction, label 2 represents protein after Ni induction, 2+ The protein was purified by column affinity. The results showed that after IPTG induction, the protein showed obvious expression. 2+ After column affinity purification, all impurity proteins were removed and the target protein was prepared.

[0080] The results of Western-blot are as follows Figure 2 As shown in middle C. The results showed that the Mycobacterium tuberculosis surface protein OmpA was successfully obtained.

[0081] 2. Mix 100 μg (1 mg / ml) of the OmpA protein prepared in Step 1 with an aluminum adjuvant (v / v = 3:1) and immunize mice subcutaneously in the nape of the neck, once every two weeks for a total of three immunizations. Blood was collected from the eye sockets of the mice, allowed to stand at room temperature for 1 hour, and then centrifuged to obtain serum. Serum titers were determined using ELISA.

[0082] The results are as follows Figure 2 As shown in D, Balb / C-1, Balb / C-2, and Balb / C-3 in C represent three immunized mice, and NC represents the blank control, which means that the three immunized mice were not immunized. The serum titer of the mice was determined by ELISA to be 1.0935×10 6 above.

[0083] 3. Select the mouse with the highest titer and intraperitoneally inject 100 μg (1 mg / ml) of OmpA protein. Three days later, remove the spleen and fuse SP2 / 0 and spleen cells with PEG1450. HAT / HT medium is used to screen for cell lines with specific antibodies to the OmpA antigen. The corresponding cell lines and antibodies are both represented by 1E1 (the expressed antibody 1E1 is purified and the identification results are as follows Figure 2 (As shown in E, the heavy and light chains are shown in the figure).

[0084] The subtype of antibody 1E1 was identified as IgG2b using a mouse antibody subtype kit (manufacturer: Frdbio). Figure 2 (as shown in F in the figure).

[0085] In addition, the recognition of antibody 1E1 against whole Mycobacterium bovis (M. bovis) and Bacillus Calmette-Guérin (BCG) was investigated. Bacterial ELISA was used to identify M. bovis and BCG cultured in 7H9 medium. The cultured bacteria were first inactivated by heating at 80°C for 20 minutes and then inoculated with 1×10 7 The bacteria were plated in a 96-well ELISA plate at a concentration of CFU / well and dried at 60°C. Antibody 1E1 (500 μg / ml) was then serially diluted and added to the 96-well plate at 100 μl / well. The plate was reacted with the bacteria at 37°C for 1 hour, followed by incubation with 100 μl of horseradish peroxidase (HRP)-labeled secondary antibody (1:10,000) at 37°C for 1 hour. After TMB color development, the absorbance at OD450 nm was measured. The results are shown in Figure 2. Figure 2 As shown in G.

[0086] Example 2

[0087] Example 2 further identified the function of antibody 1E1.

[0088] After incubation with BCG at different concentrations, macrophages J774a.1 were infected (MOI = 10). Flow cytometry analysis of BCG phagocytosis by mouse macrophages J774a.1 was performed. The results showed that antibody 1E1 promoted the phagocytosis of BCG (Mycoabcterium bovis BCG Pasteur) by macrophages in a concentration-dependent manner (the results are shown in Figure 2). Figure 3 MGO53 monoclonal antibody does not recognize Mycobacterium tuberculosis and was used as a negative control in this study.

[0089] To determine whether the antibody mediates the formation of phagolysosomes, the antibody was incubated with BCG and then infected with macrophages J774a.1. Fluorescence confocal microscopy analysis showed that antibody 1E1 could significantly promote the formation of phagolysosomes (the results are shown in Figure 2). Figure 3 C and D in the figure).

[0090] BCG was cultured in mouse whole blood mixed with different concentrations of antibody 1E1 at 37°C for 96 hours. The surviving BCG in the system was counted by plating the culture on 7H10 plates. Significant BCG growth inhibition by antibody 1E1 was observed (the results are shown in Figure 2). Figure 3 (as shown in E).

[0091] Mice were intraperitoneally injected with 500 μg of antibody 1E1 and then infected with 200 CFU of M. bovis. Two weeks later, the mice were killed and their lungs were ground and plated to count the bacterial load in the lungs. Figure 3 As shown in Figure F, the results indicate that antibody 1E1 can reduce M. bovis colonization in the lungs.

[0092] Example 3

[0093] In Example 3, antibody 1E1 was sequenced. The sequencing results showed that the protein sequence encoded by the heavy chain variable region was shown in SEQ ID NO: 7, and the protein sequence encoded by the light chain variable region was shown in SEQ ID NO: 8.

[0094] The results of homology modeling of antibody 1E1 using Discovery Studio (DS) are as follows: Figure 4 As shown in the figure, it can be seen from the results that the complementarity determining region 3 (CDR3) of the H and L chains were annotated after homology modeling to predict the amino acid residues on the antigen-antibody contact surface.

[0095] Alphafold 3 predicted the structure of OmpA antigen, and the results are as follows Figure 5 As shown, the results show that the OmpA protein presents a protein structure with a long tail, which is anchored to the N-terminus of the bacterial outer membrane, while most of the C-terminal structure is exposed on the bacterial surface, which will be beneficial to antibody recognition.

[0096] In the description of this specification, the reference terms "one embodiment", "some embodiments", "specific implementation methods", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0097] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A protective monoclonal antibody or antigen-binding fragment thereof targeting Mycobacterium tuberculosis OmpA, characterized in that: including heavy chain variable regions and light chain variable regions; The HCDR1-3 sequences of the heavy chain variable region are shown in SEQ ID NOs: 1, 2, and 3, respectively; The LCDR1-3 sequences of the light chain variable region are shown in SEQ ID NOs: 4, 5 and 6, respectively.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein have: (a) the heavy chain variable region set forth in SEQ ID NO: 7, or a sequence having at least 85% sequence homology to the heavy chain variable region sequence set forth in SEQ ID NO: 7; and (b) the light chain variable region set forth in SEQ ID NO: 8, or a sequence having at least 85% sequence homology to the light chain variable region sequence set forth in SEQ ID NO:

8.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein It has a heavy chain variable region shown in SEQ ID NO: 7 and a light chain variable region shown in SEQ ID NO:

8.

4. An isolated polynucleotide, characterized in that The isolated polynucleotide encodes the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. The isolated polynucleotide according to claim 4, characterized in that The polynucleotide comprises a sequence encoding a heavy chain variable region as shown in SEQ ID NO: 9 and a sequence encoding a light chain variable region as shown in SEQ ID NO:

10.

6. A construct, characterized in that Contains the isolated polynucleotide according to claim 4 or 5.

7. A host cell, characterized in that Containing the isolated polynucleotide of claim 4 or 5 or the construct of claim 6.

8. A method for producing a monoclonal antibody or an antigen-binding fragment thereof, characterized in that: include: Cultivating a host cell, wherein the host cell is the host cell according to claim 7, and The monoclonal antibody or antigen-binding fragment thereof is collected from the culture.

9. A pharmaceutical composition, characterized in that include: The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; as well as Pharmaceutically acceptable carrier.

10. A kit, characterized in that include: The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

11. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a reagent for detecting Mycobacterium tuberculosis OmpA, or in the preparation of a medicament for treating a disease; The disease is tuberculosis caused by Mycobacterium tuberculosis.

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