Monoclonal antibody for blocking invasion of mycobacterium tuberculosis and application thereof

Abstract

The invention discloses a monoclonal antibody for blocking invasion of mycobacterium tuberculosis and application of the monoclonal antibody, and belongs to the field of biological medicines. The invention provides a hybridoma cell strain Mce3A 15G4, the hybridoma cell strain Mce3A 15G4 can secrete an Mce3A monoclonal antibody, and the antibody can specifically recognize Mce3A protein on the surface of mycobacterium tuberculosis, so that the combination of Mce3A and a host cell surface receptor GPR108 is blocked. The Mce3A monoclonal antibody disclosed by the invention can effectively block the process that mycobacterium tuberculosis invades host cells, so that the mycobacterium tuberculosis is effectively inhibited from establishing infection, and the Mce3A monoclonal antibody has potential clinical application value for treating or preventing tuberculosis.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody that blocks the invasion of Mycobacterium tuberculosis and its application, belonging to the field of biomedicine. Background Art

[0002] Tuberculosis (TB) is a major chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) that seriously endangers public health for a long time. In 2021 alone, there were approximately 10.6 million new cases, and at least 1.6 million people died from the disease. The clinical treatment of tuberculosis mainly relies on the use of anti-tuberculosis drugs. However, the treatment with anti-tuberculosis drugs is prone to drug resistance, which not only affects the efficacy of the drugs but also leads to treatment failure. At present, the research and development of anti-tuberculosis drugs still face many challenges such as the emergence of drug resistance and new variants of Mycobacterium tuberculosis. Therefore, there is an urgent need to develop more new anti-tuberculosis targets and new strategies for the prevention and treatment of tuberculosis.

[0003] As an important immunotherapeutic and regulator, monoclonal antibodies have broad application prospects in the treatment of infectious diseases such as viral hepatitis, AIDS, and syphilis due to their highly specific and targeted recognition of antigens. However, the role of monoclonal antibodies in the prevention and treatment of tuberculosis is still in its early stage.

[0004] As an intracellular pathogen, Mycobacterium tuberculosis can invade a variety of cells including alveolar epithelial cells and alveolar macrophages. The research and development of drugs such as monoclonal antibodies targeting the invasion interface of Mycobacterium tuberculosis will help to discover new anti-tuberculosis drugs and provide new ideas and methods for the prevention and treatment of tuberculosis. Previous studies have found that the surface protein Mce3A of Mycobacterium tuberculosis can promote the invasion of mycobacteria into host cells in a manner dependent on the host cell surface GPR108 receptor. Summary of the Invention

[0005] In the present invention, it is found that specific monoclonal antibodies targeting the carboxyl terminus of Mce3A can block the invasion of Mycobacterium tuberculosis by blocking the interaction between Mce3A and GPR108. Such antibodies have potential effects on the prevention and treatment of tuberculosis.

[0006] The present invention first uses a polypeptide in Mce3A as an antigen, and its amino acid sequence is as shown in SEQ ID NO: 1, to obtain a hybridoma cell line that can secrete Mce3A monoclonal antibodies. The Mce3A monoclonal antibodies secreted by this hybridoma cell line can specifically recognize Mce3A on the surface of Mycobacterium tuberculosis, thereby blocking the binding of Mce3A to the cell surface receptor GPR108.

[0007] The present invention provides an Mce3A monoclonal antibody. The amino acid sequence of CDR1 in the heavy chain variable region of the Mce3A monoclonal antibody is TYWIE, the amino acid sequence of CDR2 is EILPGSGSINYFEKFKG, and the amino acid sequence of CDR3 is WDGNYVGWFAY; the amino acid sequence of CDR1 in the light chain variable region is RSSTGAVTTSNYAN, the amino acid sequence of CDR2 is GTNNRAP, and the amino acid sequence of CDR3 is ALWYSNHFV.

[0008] In one embodiment of the present invention, the heavy chain variable region of the Mce3A monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 3.

[0009] The present invention also provides a nucleic acid molecule encoding the Mce3A monoclonal antibody.

[0010] In one embodiment of the present invention, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID NO: 4, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO: 5.

[0011] The present invention also provides a recombinant plasmid expressing the nucleic acid molecule.

[0012] The present invention also provides a host cell containing the recombinant plasmid.

[0013] In one embodiment of the present invention, the host cell includes, but is not limited to, bacteria, fungi, mammalian cells or insect cells.

[0014] In one embodiment of the present invention, the fungi include yeast or mold, and the bacteria include Escherichia coli.

[0015] In one embodiment of the present invention, the mammalian cells include, but are not limited to, CHO cells and 293 cells.

[0016] The present invention provides a hybridoma cell line secreting the Mce3A monoclonal antibody, with the preservation number of CGMCC No. 45720. The hybridoma cell line was preserved on September 21, 2023 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0017] The present invention provides the application of the hybridoma cell line in the preparation of the Mce3A monoclonal antibody.

[0018] In one embodiment of the present invention, the application is to culture the hybridoma cell line.

[0019] In one embodiment of the present invention, the method is to intraperitoneally inject the hybridoma cell line into mice, collect ascites and purify it to obtain a monoclonal antibody.

[0020] The present invention also provides the use of the Mce3A monoclonal antibody, or the nucleic acid molecule, or the recombinant plasmid, or the host cell in the preparation of products for immunodiagnosis or detection of Mycobacterium tuberculosis, or in the preparation of drugs for the treatment and / or prevention of tuberculosis.

[0021] The present invention further provides a drug, which contains the Mce3A monoclonal antibody. The Mce3A monoclonal antibody includes but is not limited to murine, human-mouse chimeric and humanized monoclonal antibodies. Preferably, it includes the heavy chain variable region and the light chain variable region of the Mce3A monoclonal antibody.

[0022] In one embodiment of the present invention, the drug further contains a pharmaceutically acceptable carrier.

[0023] The present invention also provides a product for immunodiagnosis or detection of Mycobacterium tuberculosis, which contains the monoclonal antibody, or the nucleic acid molecule, or the recombinant plasmid, or the host cell.

[0024] In one embodiment of the present invention, the product contains an antigen with the amino acid sequence shown in SEQ ID NO: 1.

[0025] In one embodiment of the present invention, the product includes reagents, kits, and test strips.

[0026] The hybridoma cell line Mce3A 15G4 provided by the present invention secretes an Mce3A monoclonal antibody that can recognize Mce3A on the surface of Mycobacterium tuberculosis and block the binding of Mce3A to the cell surface receptor GPR108. The Mce3A monoclonal antibody of the present invention can effectively block the process of Mycobacterium tuberculosis invading host cells, thereby effectively inhibiting the establishment of infection by Mycobacterium tuberculosis, and thus has potential clinical application value for the treatment or prevention of tuberculosis.

[0027] Biological material preservation information:

[0028] The hybridoma cell line Mce3A 15G4 that secretes the Mce3A monoclonal antibody, classified and named as: hybridoma cell line, the preservation unit is: China General Microbiological Culture Collection Center (abbreviated as CGMCC), the address of the preservation unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation number is: CGMCC No. 45720, and the preservation date is: September 21, 2023. Brief description of the drawings

[0029] Figure 1: Purity detection of Mce3A monoclonal antibody;

[0030] Figure 2 : Titer detection of Mce3A monoclonal antibody;

[0031] Figure 3 : Blocking effect detection of Mce3A monoclonal antibody;

[0032] Figure 4 : Detection of the effect of Mce3A monoclonal antibody on inhibiting the invasion of Mycobacterium tuberculosis into host cells. Specific implementation mode

[0033] pEGFP-N1-GPR108 plasmid: Using the commercial plasmid pEGFP-N1 as the expression vector, the GPR108 gene (Gene ID: 56927) was ligated. After transferring pEGFP-N1-GPR108 into cells, the expression of GFP and GPR108 was achieved.

[0034] 7H10 plate: Weigh 1.9 g of Middlebrook 7H10, add 90 mL of double-distilled water to dissolve, autoclave at 121 °C for 30 minutes, place in an oven at 65 °C or a water bath to keep warm, add 10 mL of OADC, 1 mL of 50% glycerol, and 250 μL of 20% Tween-80 to the 7H10 medium in a laminar flow hood, mix well and pour into plates or 15 mL glass tubes to make slopes.

[0035] 7H9 liquid medium: Weigh 0.47 g of Middlebrook 7H9, add 90 mL of double-distilled water to dissolve, add 10 mL of OADC, 1 mL of 50% glycerol, and 250 μL of 20% Tween-80 to the 7H9 medium in a laminar flow hood, mix well and filter through a filter (0.22 μm) into a sterile reagent bottle. Check for contamination overnight at 37 °C and then store in a 4 °C refrigerator for later use.

[0036] Example 1: Preparation and screening of hybridoma cell lines secreting Mce3A monoclonal antibody

[0037] 1. Preparation of immunogen:

[0038] The synthesized Mce3A protein polypeptide (amino acid sequence as shown in SEQ ID NO: 1) was conjugated with bovine serum albumin (BSA) to obtain a recombinant protein. The concentration of the recombinant protein was adjusted to 1 mg / mL.

[0039] 2. Mouse immunization

[0040] Six-week-old female BALB / c mice were selected as immune animals, and recombinant protein was used as the immune antigen. Three BALB / c mice were immunized 4 times at two-week intervals. For the first 3 immunizations, 50 μL of recombinant protein was mixed with an equal volume of Freund's adjuvant and emulsified thoroughly, and then immunized by subcutaneous multi-point injection. For the 4th immunization, 100 μL of recombinant protein was directly injected intraperitoneally for boost immunization. After a 4-day interval, blood was collected from the tail vein, and the antibody titer of the antigen in the serum was measured by indirect ELISA. At the same time, the serum of non-immunized mice was used as a negative control. Mice with high tail blood titers were selected and then hybridoma cells were prepared.

[0041] 3. Cell fusion and screening:

[0042] Feeder cells (peritoneal cells of BALB / c mice) were prepared 24 h in advance, resuspended with HAT medium, and plated at 10 4 / well in a 96-well plate; SP2 / 0 mouse myeloma cells were prepared and counted for later use.

[0043] Mouse spleen cells were isolated in a laminar flow hood, ground with a cell sieve and resuspended and counted with complete medium containing 20% fetal bovine serum. SP2 / 0 cells and mouse spleen cells were added to a 50 mL centrifuge tube at a ratio of 1:5 (SP2 / 0: 2×10 6 cells, mouse spleen cells: 1×10 7 cells), centrifuged at 300 g for 10 min, and washed 3 times; pre-warmed cell fusion agent PEG1450 at 37 °C was added, allowed to stand at room temperature for 1 min, termination solution was added, centrifuged at 300 g for 10 min, and washed 3 times; the supernatant was aspirated, the cells were resuspended with 2% HAT medium, added to a pre-prepared 96-well cell culture plate containing feeder cells, 100 μL / well, and cultured in an incubator at 37 °C and 2% CO2.

[0044] 4. Selection of subclones:

[0045] After culturing for 24 h after cell fusion, the cell status was observed; after the cell mass became visible to the naked eye, the cell supernatant was aspirated, and the absorbance value was measured by indirect ELISA to determine whether it was a positive well. The positive wells were expanded and cultured, and the strongly positive wells secreting antibodies were selected for cloning. After 2 subclones, the hybridoma cell lines with higher and stable positive values were selected as the final positive hybridoma cell lines. A total of five hybridoma cell lines, namely 8E12, 10E5, 16D7, 15G4, and 4E3, were obtained.

[0046] 5. Antibody purification and purity detection

[0047] Six- to eight-week-old mice were injected intraperitoneally with 0.5 mL of mineral oil per mouse; after 9 - 12 days, the hybridoma cell lines were injected at 10×10 5 / Inject only into the abdominal cavity of mice; After 10 days, collect ascites, centrifuge at 12,000 g for 5 min, add normal saline (half of the total volume), and store at -20 °C;

[0048] Crude antibody purification: Use the ammonium sulfate precipitation method. Mix ascites, diluent, and ammonium sulfate in a ratio of 1:2:3, and incubate the mixture overnight at 4 °C. Centrifuge at 12,000 rpm / min for 10 min, discard the supernatant, wash once with purified water, and redissolve with the same volume of reconstitution solution;

[0049] Antibody fine purification: Further purify using Protein G affinity chromatography.

[0050] Use a Thermos trace element analyzer to measure the concentration of the purified monoclonal antibody. The results are shown in Table 1:

[0051] Table 1 Concentration of monoclonal antibody

[0052] Plant number Concentration Volume Mass 8E12 2 mg / mL 0.5 mL 1 mg 10E5 3 mg / mL 0.35 mL 1 mg 16D7 0.5 mg / mL 2 mL 1 mg 15G4 1.5 mg / mL 0.7 mL 1 mg 4E3 2 mg / mL 0.5 mL 1 mg

[0053] Further perform SDS-PAGE electrophoresis on the purified monoclonal antibody to analyze its purity. The results are as Figure 1 .

[0054] 6. Antibody titer detection

[0055] Coat the Mce3A protein polypeptide synthesized in step 1 onto an ELISA plate respectively, and use the indirect ELISA method to measure the antibody titer. Use an ELISA reader to measure the OD 450 absorbance and calculate the antibody titer as Figure 2 , showing that the purity of the monoclonal antibody is relatively high.

[0056] Example 2: Mce3A monoclonal antibody blocking experiment

[0057] 1. Transfect pEGFP-N1-GPR108 into 293T cells to overexpress the GFP-GPR108 fusion protein.

[0058] 2. The next day, add mouse IgG (negative control) at a final concentration of 1 μg / mL and the 5 different Mce3A monoclonal antibodies prepared in Example 1 at a final concentration of 1 μg / mL to 7 centrifuge tubes respectively. Leave one centrifuge tube untreated (blank control Blank) for the time being.

[0059] 3. Add Mce3A protein (1 μg / mL) labeled with 594 fluorescence (AAT Bioquest, 1230) to the IgG and monoclonal antibody treatment groups respectively. Add an equal volume of PBS to the other group. Incubate all samples at room temperature for 30 min without washing for the time being.

[0060] 4. After 24 hours of cell transfection, trypsinize the cells and evenly aliquot them into the above centrifuge tubes. Incubate all samples at room temperature for an additional 1 h.

[0061] 5. Wash the cells 3 times with PBS.

[0062] 6. Detect the fluorescence value of 594 on the cell surface of each sample by flow cytometry, which represents the amount of Mce3A protein bound to the surface of GFP-GPR108 overexpressing cells.

[0063] As Figure 3 shown, the monoclonal antibodies 4E3 and 10E5 could hardly play a blocking role, while the three monoclonal antibodies 8E12, 15G4 and 16D7 could all inhibit the binding of Mce3A protein to the cell surface to varying degrees, among which 15G4 had the best inhibitory effect. Compared with the IgG treatment group as the negative control, after removing the background fluorescence value shown in the Blank group, the relative fluorescence value (MFI) of Mce3A protein bound to the cell surface in the 15G4 monoclonal antibody treatment group decreased by about 50%. This result suggests that the Mce3A 15G4 monoclonal antibody can effectively block the binding between the surface protein Mce3A of Mycobacterium tuberculosis and the host cell surface receptor GPR108.

[0064] Example 3: Sequencing of the variable regions of the Mce3A 15G4 monoclonal antibody

[0065] GenScript Biotech Corporation was commissioned to sequence the variable regions of the Mce3A monoclonal antibody secreted by the hybridoma 15G4, and the nucleotide and amino acid sequences of its heavy and light chain variable regions were confirmed. That is, the nucleotide sequence of the DNA encoding the heavy chain variable region is shown in SEQ ID NO: 4, and the nucleotide sequence of the DNA encoding the light chain variable region is shown in SEQ ID NO: 5. The encoded amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3.

[0066] Analysis shows that the amino acid sequence of CDR1 of the heavy chain variable region is: TYWIE; the amino acid sequence of CDR2 is: EILPGSGSINYFEKFKG; the amino acid sequence of CDR3 is: WDGNYVGWFAY; the amino acid sequence of CDR1 of the light chain variable region is: RSSTGAVTTSNYAN; the amino acid sequence of CDR2 is: GTNNRAP; the amino acid sequence of CDR3 is: ALWYSNHFV.

[0067] Example 4: Experiment on blocking the invasion of Mycobacterium tuberculosis by Mce3A15G4 monoclonal antibody

[0068] 1. Preparation of Mycobacterium tuberculosis H37Rv:

[0069] Mycobacterium tuberculosis H37Rv is cultured on a 7H10 culture plate or slant for 3 - 4 weeks for later use.

[0070] 2. Infection of macrophages with Mycobacterium tuberculosis H37Rv:

[0071] (1) After digesting and counting RAW264.7 cells, they are seeded onto a 24-well plate and cultured overnight (1×10 5 cells per well).

[0072] (2) The next day, the H37Rv colonies on the fixed plate or slant are scraped off and added to 7H9 liquid medium, and shaken thoroughly until there are no granular bacterial masses. Take 1 mL of the H37Rv bacterial solution and measure the OD 600 value of the bacterial solution, and continuously dilute and adjust until the OD 600 of the bacterial solution = 0.6 - 0.8 (when OD 600 = 0.6, the bacterial concentration is 1×10 8 CFUs / mL).

[0073] (3) Dilute the bacterial solution with 7H9 liquid medium to 1×10 5 cells / mL.

[0074] (4) Add mouse IgG at a final concentration of 1 μg / mL (negative control) and 1 μg / mL of the 5 different Mce3A monoclonal antibodies prepared in Example 1 to the diluted bacterial solution in step (3) respectively. In addition, no treatment is added to another cell well (blank control NC), and incubate at 37 °C for 30 min.

[0075] (5) Add the bacterial solution treated in step (4) to the cells in the 24-well plate in step (1), add 100 μL of the bacterial solution to each well (MOI = 1:1), and culture at 37 °C under 5% CO2 for 2 hours.

[0076] (6) Wash the cells 3 times with 1 mL of PBS to remove extracellularly unbound Mycobacterium tuberculosis.

[0077] (7) Directly collect the cells after lysing with 0.02% SDS for 5 min.

[0078] 3. CFU counting

[0079] (1) Transfer the cells collected by lysing with 0.02% SDS in step 2 into 1.5 mL EP tubes respectively.

[0080] (2) Serial dilute the lysed cells collected in step (1) with 7H9 liquid medium and directly spread them onto 7H10 mycobacterial solid medium respectively.

[0081] (3) The 7H10 solid culture plate was inverted and placed in a 37 °C constant temperature bacterial incubator, and the number of bacterial colonies was counted after 3 - 4 weeks of culture.

[0082] The results are as Figure 4 shown. The number of bacterial colonies in the Mce3A15G4 monoclonal antibody treatment group decreased by approximately 85% compared to the IgG control treatment group, while there was no significant difference in the number of bacterial colonies between the other four Mce3A monoclonal antibody treatment groups and the IgG control treatment group. This result suggests that treatment with the Mce3A 15G4 monoclonal antibody can significantly reduce the invasion of Mycobacterium tuberculosis into macrophages to establish infection ( Figure 4 ).

[0083] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A monoclonal antibody against Mce3A, characterized in that, The amino acid sequence of CDR1 of the heavy chain variable region is: TYWIE; the amino acid sequence of CDR2 is: EILPGSGSINYFEKFKG; the amino acid sequence of CDR3 is: WDGNYVGWFAY; the amino acid sequence of CDR1 of the light chain variable region is: RSSTGAVTTSNYAN; the amino acid sequence of CDR2 is: GTNNRAP; the amino acid sequence of CDR3 is: ALWYSNHFV.

2. The Mce3A monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region of the Mce3A monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:

3.

3. A hybridoma cell line secreting the Mce3A monoclonal antibody according to any one of claims 1 to 2, characterized in that, The preservation number of the hybridoma cell line is CGMCC No. 45720. The hybridoma cell line was preserved on September 21, 2023 at the General Microbiology Center of the China Microbial Culture Collection Center, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

4. A nucleic acid molecule encoding the Mce3A monoclonal antibody according to any one of claims 1 to 2; preferably, the nucleotide sequence of the heavy chain variable region of the nucleic acid molecule is as shown in SEQ ID NO: 4, and the nucleotide sequence of the light chain variable region of the nucleic acid molecule is as shown in SEQ ID NO:

5.

5. A recombinant plasmid containing the nucleic acid molecule according to claim 4.

6. A host cell containing the recombinant plasmid according to claim 5; optionally, the host cell is selected from fungi, bacteria, mammalian cells or insect cells.

7. A human-mouse chimeric monoclonal antibody or humanized monoclonal antibody based on the Mce3A monoclonal antibody according to any one of claims 1 to 2; preferably, it includes the heavy chain variable region and the light chain variable region of the Mce3A monoclonal antibody according to any one of claims 1 to 2; Preferably, it is obtained by humanized cells such as expressing its coding DNA.

8. The application of the Mce3A monoclonal antibody according to any one of claims 1 to 2, or the nucleic acid molecule according to claim 4, or the recombinant plasmid according to claim 5, or the host cell according to claim 6 in the preparation of products for immunodiagnosis or detection of Mycobacterium tuberculosis, or in the preparation of drugs for the treatment and / or prevention of tuberculosis.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the Mce3A monoclonal antibody according to any one of claims 1 to 2, or the nucleic acid molecule according to claim 4, or the recombinant plasmid according to claim 5, or the host cell according to claim 6; preferably, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

10. A product for immunodiagnosis or detection of Mycobacterium tuberculosis, characterized in that, The product contains the monoclonal antibody according to any one of claims 1 to 2, or the nucleic acid molecule according to claim 4, or the recombinant plasmid according to claim 5, or the host cell according to claim 6.

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