Lilrb1 monoclonal antibody and application thereof

Abstract

The application belongs to the field of biological medicine, and specifically discloses a leukocyte immunoglobulin-like receptor B1 monoclonal antibody and application thereof. The LILRB1 monoclonal antibody is obtained by taking the extracellular segment of human LILRB1 protein expressed by Escherichia coli as an antigen, can specifically recognize the cell surface LILRB1 receptor, and thus blocks the combination of LILRB1 and its ligand, human leukocyte antigen-G. The LILRB1 monoclonal antibody disclosed by the application can effectively reverse the immune exhaustion state of natural killer cells (NK), improve the killing function of NK cells on target cells, and promote the clearance of Mycobacterium tuberculosis, and thus has the application value in clinical treatment or prevention of tuberculosis.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody and its applications, belonging to the field of biomedicine. Background Technology

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). Over the past two millennia, TB has caused more than one billion deaths; in recent years, approximately 1.5 million people worldwide still die from TB annually. In 2014, the World Health Organization and the United Nations set the goal of "ending the TB epidemic by 2035." However, current TB prevention and control methods and technologies are insufficient to achieve this ambitious vision, a major reason being that existing TB treatments are inadequate for current clinical needs. Isoniazid and rifampin, which have been available for nearly 70 years, are still widely used but suffer from severe drug resistance, and the long treatment cycle for TB leads to unavoidable toxic side effects. Furthermore, co-infection with TB and human immunodeficiency virus (HIV) further complicates clinical TB treatment. Currently, global TB drug development is slow, and antibiotics are rapidly facing the threat of drug resistance. Therefore, we urgently need to develop new anti-tuberculosis drugs or new anti-tuberculosis strategies to address the current severe TB epidemic (especially drug-resistant TB). In recent years, novel host immunomodulators have become an important direction for future TB drug development. The concept is to eliminate immune escape and immune exhaustion caused by pathogens, enhance the host's protective immune response, and reduce tissue damage caused by excessive inflammatory response. Combining these drugs with traditional TB drugs can also shorten the treatment course and alleviate the development of drug resistance.

[0003] Natural killer (NK) cells are important effector cells in innate immunity. They can directly kill pathogen-infected cells without prior antigen stimulation and have no cytotoxicity to autologous tissue cells. We found that an inhibitory receptor on the surface of NK cells in tuberculosis (TB) patients—leukocyte immunoglobulin-like receptor B1 (LILRB1)—is specifically highly expressed compared to healthy controls and individuals with latent TB infection. This high expression is a key reason for NK cell immune function depletion in TB patients, making it difficult for Mtb to be cleared by immune cells. We further discovered that using a specific monoclonal antibody against LILRB1 to block the interaction between LILRB1 and its ligand—human leukocyte antigen-G (HLA-G, class I histocompatibility antigen)—can effectively alleviate NK cell depletion in TB patients, thereby enhancing their killing activity against infecting pathogens. This type of antibody has potential therapeutic effects in tuberculosis prevention and treatment. Summary of the Invention

[0004] To address the aforementioned issues, this invention first uses the extracellular domain (amino acid positions 24-461) of the human LILRB1 protein expressed in *E. coli* as an antigen, the amino acid sequence of which is shown in SEQ ID NO: 1, to obtain a hybridoma cell line capable of secreting a LILRB1 monoclonal antibody. The LILRB1 monoclonal antibody secreted by this hybridoma cell line specifically recognizes the LILRB1 receptor on the cell surface, thereby blocking the binding of LILRB1 to the ligand HLA-G.

[0005] where SEQ ID The specific sequence of NO:1 is as follows: GHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYI KPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIV APEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQ GWMQTFLLTKEGAADDPWLRRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV.

[0006] This invention provides a murine LILIRB1 monoclonal antibody. The amino acid sequence of the heavy chain variable region CDR1 is: SYWMN; the amino acid sequence of CDR2 is: QIYPGDGDTNYNGEFKD; the amino acid sequence of CDR3 is: SGDYYGYSPFY; the amino acid sequence of the light chain variable region CDR1 is: LASQTIGTWLA; the amino acid sequence of CDR2 is: AATSLAD; the amino acid sequence of CDR3 is: QQLYSTPYT.

[0007] Preferably, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO: 2, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO: 3.

[0008] The specific sequence of SEQ ID NO: 2 is as follows: MEWPLIFLFLLSGTAGVQSQIQLQQSGAELVKPGASVKISCKASGYAFSSYWMNWVKQRPGKGLEWIGQIYPGDGDTNYNGEFKDKATLTADKSSSTAFMQLSSLTSEDSAVYFCARSGDYYGYSPFYWGQGTTLTVSS;

[0009] The specific sequence of SEQ ID NO: 3 is as follows: MLTQLLGLLLLWFAGGKCDIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIYAATSLADGVPSRFSGSGSGTKFSFKISSLQAEDFVSYYCQQLYSTPYTFGGGTKLEIK.

[0010] More specifically, the antibody, named 16G10, was produced by a hybridoma cell line with accession number CGMCC No. 45305.

[0011] The present invention also provides the encoding DNA of the LILRB1 monoclonal antibody, preferably, the nucleotide sequence of the DNA encoded by the heavy chain variable region is shown in SEQ ID NO: 4, and the nucleotide sequence of the DNA encoded by the light chain variable region is shown in SEQ ID NO: 5.

[0012] The nucleotide coding sequence of the heavy chain is as follows (SEQ ID NO: 4):ATGGAATGGCCTTTGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCAATCCCAAATTCAGCTGCAGCATCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATTTCCTGCAAAGCTTCTGGCTACGCATTCAGTAGCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGAAAGGGTCTTGAGTGGATTGGACA GATTTATCCTGGAGATGGTGATACTAACTACAACGGAGAGTTCAAGGACAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTTCATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCGGGGGATTACTACGGTTATAGCCCCTTCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0013] The nucleotide coding sequence of the light chain is as follows (SEQ ID NO: 5): ATGCTCACTCAGCTCCTGGGATTACTGCTGCTCTGGTTTGCAGGTGGTAAATGTGACATTCAGATGACCCAGTCTCCTGCCTCCCAGTCTGCATCTCTGGGAGAAAGTGTCACCATCACATGCCTGGAAGTCAGACCATTGGTACATGGTTAGCATGGTATCAGCAGAAACCAGGGAAATCTCC TCAGCTCCTGATTTATGCTGCAACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGTGGTAGTGGATCTGCACAAAATTTTCTTTCAAGATCAGCAGCCTACAGGCTGAAGATTTTGTAAGTTATTACTGTCAACAACTTTACAGTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA.

[0014] The present invention also provides a LILRB1 human-mouse chimeric monoclonal antibody, which is based on a human IgG1 total antibody of the LILRB1 monoclonal antibody as described in any one of claims 1 to 2; preferably, its heavy chain H includes a signal peptide, a heavy chain variable region of the LILRB1 monoclonal antibody as described in any one of claims 1 to 2, and a human IgG1 heavy chain constant region, and its light chain κ includes a signal peptide, a light chain variable region of the LILRB1 monoclonal antibody as described in any one of claims 1 to 2, and a human IgG1 light chain constant region.

[0015] Therefore, the present invention provides DNA encoding the LILRB1 human-mouse chimeric monoclonal antibody, wherein the nucleotide sequence encoding the heavy chain of the LILRB1 monoclonal antibody includes a CMV promoter sequence, a signal peptide sequence, a sequence encoding the variable region of the heavy chain, and a sequence encoding the constant region of the heavy chain; the sequence encoding the light chain of the antibody includes a CMV promoter sequence, a signal peptide sequence, a sequence encoding the variable region of the light chain, and a sequence encoding the constant region of the light chain.

[0016] More preferably, the nucleotide sequence encoding the heavy chain of the LILRB1 monoclonal antibody further includes a CMV promoter sequence, a leader sequence, a sequence encoding the variable region of the heavy chain, and a sequence encoding the constant region of the heavy chain; the sequence encoding the light chain of the antibody includes a CMV promoter sequence, a leader sequence, a sequence encoding the variable region of the light chain, and a sequence encoding the constant region of the light chain.

[0017] The present invention also provides a vector containing the DNA and recombinant cells containing the vector.

[0018] Accordingly, the present invention also provides a hybridoma cell line that secretes LILRB1 monoclonal antibody. The hybridoma cell line was deposited on September 2, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 45305.

[0019] This invention provides the application of the hybridoma cell line that secretes LILRB1 monoclonal antibody in the preparation of LILRB1 monoclonal antibody.

[0020] In one embodiment of the present invention, the method involves taking 8-10 week old BALB / c mice, injecting each mouse intraperitoneally with 1 mL of berberine, and then, 7 days later, injecting each mouse intraperitoneally with 1 × 10 6 Ascites fluid was collected from the hybridoma cell line with accession number CGMCC No.45305 starting from day 7. The ascites fluid was purified, and the obtained monoclonal antibody was stored at -20°C.

[0021] This invention provides the application of the above-mentioned LILRB1 antigen in the preparation of hybridoma cell lines that secrete LILRB1 monoclonal antibodies and LILRB1 monoclonal antibodies.

[0022] The present invention also provides the application of the monoclonal antibody 16G10 in the preparation of drugs.

[0023] In one embodiment of the present invention, the drug is a drug for treating and / or preventing tuberculosis.

[0024] The present invention further provides a pharmaceutical composition comprising the monoclonal antibody 16G10 described above.

[0025] In one embodiment of the invention, the pharmaceutical composition further comprises a medically acceptable carrier.

[0026] Finally, the present invention also provides a reagent, kit, test strip, etc. for immunodiagnosis or detection, wherein the kit contains the monoclonal antibody 16G10, or an antigen of the monoclonal antibody 16G10, or a DNA molecule encoding the antigen, or a recombinant vector / expression cassette / transgenic cell line / recombinant bacteria expressing the antigen.

[0027] The hybridoma cell line LILRB1 16G10 provided by this invention secretes a LILRB1 monoclonal antibody that recognizes the extracellular domain of the LILRB1 receptor on the cell surface and blocks the binding of LILRB1 to the ligand HLA-G. The LILRB1 monoclonal antibody of this invention can effectively reverse the immune exhaustion of NK cells, enhance the killing function of NK cells against target cells, and promote the clearance of Mycobacterium tuberculosis. The LILRB1 monoclonal antibody produced by the hybridoma cell line of this invention has clinical application value for the treatment or prevention of tuberculosis. Attached Figure Description

[0028] Figure 1 Detection of LILRB1 protein purification.

[0029] Figure 2 LILRB1 monoclonal antibody blocking assay.

[0030] Figure 3 LILRB1 human-mouse chimeric 16G10 monoclonal antibody (LILRB1 hu-16G10 mAb) blocking assay

[0031] Figure 4 Effects of LILRB1 monoclonal antibody on NK cell killing activity.

[0032] Information on the preservation of biological materials:

[0033] The hybridoma cell line LILRB1 16G10, which secretes the LILRB1 monoclonal antibody, is classified as a hybridoma cell line. Its depository is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 45305, and the deposit date is September 2, 2022. Detailed Implementation

[0034] Example 1: LILRB1 protein expression and purification

[0035] 1. The extracellular region DNA fragment of human LILRB1 protein (amino acid sequence as shown in SEQ ID NO: 1) was digested with BglⅡ and XhoI and then ligated into the pET30a (BamHI / XhoI) vector.

[0036] 2. The ligation product was transformed into BL21 Escherichia coli competent cells, and the ligation product was plated on LB plates containing kanamycin resistance and incubated overnight at 37°C.

[0037] 3. Select single-clone colonies and extract plasmids for enzyme digestion and sequencing identification.

[0038] 4. Transform the successfully ligated plasmid into DH5α E. coli competent cells again, plate them on LB plates containing kanamycin resistance, and incubate overnight at 37°C.

[0039] 5. Select a single positive bacterial clone and inoculate it into 40 mL of LB medium, and incubate for 6-8 hours.

[0040] 6. Inoculate the bacterial culture again into 4L of LB medium and incubate at 37°C until OD500. 600 =0.6-0.8, add protein expression inducer IPTG to a final concentration of 0.1mM, and continue culturing at 37℃ for 4-6 hours.

[0041] 7. Harvest inclusion bodies and refold them using the dilution method.

[0042] 8. After concentrating the refolding buffer, replace it with a 20mM Tris-HCl, 150mM NaCl, pH 8.0 buffer. Purify the concentrated protein solution sequentially using a Ni-NTA affinity column and a HiLoad 16 / 600 Superdex 200pg gel column, monitoring the OD value simultaneously. 280 The ultraviolet absorbance value was measured to collect the target protein.

[0043] 9. High-purity monomeric protein, approximately 55 kDa in size, was obtained by polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining (including LILRB1 with a molecular weight of approximately 49 kDa and the tag sequence with a molecular weight of approximately 6 kDa). Figure 1 ).

[0044] Example 2: Preparation and screening of hybridoma cell lines secreting LILRB1 monoclonal antibodies

[0045] 1. Animal immunization:

[0046] Five BALB / c mice and five C57BL / 6 mice were immunized with purified LILRB1 protein. The immunization regimen is shown in Table 1.

[0047] Table 1 Immunization regimens

[0048]

[0049] 2. Blood collection and testing:

[0050] Seven days after each immunization, the serum of immunized animals was measured using an indirect enzyme-linked immunosorbent assay (ELISA) to determine the level of immune response. After the first phase of routine immunization, if the immunized animals achieve an immune response level (OD) against the immunogen... 450If the valence is >1.0 and the valence reaches 1:8,000, then it is selected to proceed to the next stage.

[0051] 3. Cell fusion and screening:

[0052] ① Cell fusion and plating: Two rounds of cell fusion were performed using an electrofusion method. The average fusion efficiency was approximately 2,500 spleen cells to produce one hybridoma cell; therefore, the expected hybridoma cell count could reach approximately 2 × 10⁻⁶. 4 Each round of fusion will result in all cells being seeded into 96-well plates, with approximately 25 plates seeded per round, for a total of two rounds of fusion.

[0053] ② Initial screening: The supernatant of fused cells was screened using an indirect ELISA method to select clones that were positive for LILRB1 protein. This step yielded the 100 positive clones with the highest antibody titers.

[0054] ③ Confirmation screening: For all 100 positive clones obtained in the initial screening stage, the cell supernatant of all positive maternal clones was screened using the indirect ELISA method. At the same time, the supernatant was reverse screened with His tag protein to obtain 5 maternal clones that were positive for the immunogen and negative for the reverse His tag.

[0055] ④ Clonal Expansion Culture and Cryopreservation: Transfer positive mother clone cells to 24-well plates for expansion culture. Collect 2 mL of supernatant from each expanded clone for indirect ELISA detection. Cryopreserve all specific positive clone cells to avoid clone loss.

[0056] Example 3: Culture and cryopreservation of hybridoma cell lines

[0057] 1. Culture of hybridoma cells:

[0058] ① Hybridoma cells were cultured in IMDM medium containing 20% ​​fetal bovine serum (FBS) under a non-toxic, sterile culture environment of 37°C and 5% CO2.

[0059] ② Hybridoma cells are transparent and round, and are in a semi-suspended state.

[0060] ③ When passaged hybridoma cells, gently pipette the adherent cells off the culture vessel and centrifuge at 1,000 rpm for 5 minutes. Resuspend the precipitated cells in the above culture medium and passage them at a ratio of 1:4.

[0061] 2. Cryopreservation of hybridoma cells:

[0062] ① Hybridoma cell lines were collected in centrifuge tubes, centrifuged at 1,000 rpm for 5 minutes, and the supernatant was discarded.

[0063] ② Add an appropriate amount of cell cryopreservation solution (FBS + 10% DMSO) to a centrifuge tube, mix slowly and thoroughly to prepare a cell suspension, with a cell cryopreservation concentration of 5 × 10⁻⁶. 6 / mL.

[0064] ③ Dispense the hybridoma cell mixture into fully labeled cryovials and place them in a cell cryopreservation box. Then place them in a -80°C freezer and transfer them to a liquid nitrogen tank for long-term cryopreservation after 24 hours.

[0065] The five hybridoma cell lines obtained are shown in Table 2.

[0066] Table 2 List of five hybridoma cell lines

[0067]

[0068] Example 4: Preparation of LILRB1 monoclonal antibody

[0069] 1. Monoclonal antibodies were obtained from cell culture supernatant:

[0070] ① Hybridoma cells are cultured normally in vitro. The culture supernatant is collected during the passage process, and the cells and their debris are removed by centrifugation to obtain the desired monoclonal cells.

[0071] ② Once the cells have expanded to a sufficient number, culture them in medium containing 5% FBS to secrete antibodies. Collect the supernatant after 3-5 days. At this point, the cells have aged and are not suitable for further culture. Centrifuge the supernatant to remove cells and debris, and then purify the monoclonal antibodies.

[0072] 2. Monoclonal antibodies obtained from mouse ascites:

[0073] ① Administer 0.5 mL of the immunoadjuvant norperidine to mice via intraperitoneal injection 10-14 days in advance.

[0074] ② Culture hybridoma cells in IMDM medium in a culture dish until they reach the logarithmic growth phase. Resuspend the cells in 50 mL of sterile PBS and wash. Centrifuge at 1,000 rpm for 5 minutes at room temperature and discard the supernatant. Then resuspend the cells in 5 mL of PBS.

[0075] ③ Each mouse was intraperitoneally injected with 0.5 mL of 0.5 × 10⁻⁶ solution. 7 Hybridoma cells in the logarithmic growth phase.

[0076] ④ After 7-15 days, the mouse abdomen will gradually swell (the weight gain before harvesting ascites should not exceed 20% of the baseline weight). At this time, use a 10mL syringe needle to aspirate ascites through abdominal puncture. Disinfect the abdomen with sterile alcohol gauze pads. Collect 4-5mL each time. Gently move the needle appropriately to ensure unobstructed drainage of the ascites, and keep the ascites fluid sterile. Aspiration can be performed again after about 2 days. If the mouse's health does not significantly decline, puncture and aspiration can be performed again. The ascites fluid should accumulate for approximately 48 hours after the second aspiration. 24-48 hours after the second aspiration, euthanize the mouse and perform a third aspiration. All harvested ascites fluid can be centrifuged and combined into the same centrifuge tube.

[0077] ⑤ The extracted ascites fluid contains a large amount of cell debris, fat, and oily Freund's adjuvant. The ascites fluid needs to be centrifuged promptly at 2,000 rpm for 5 minutes, and the intermediate aqueous phase should be collected. Aliquot 1 mL into each tube and store at -80°C.

[0078] Example 5: LILRB1 Monoclonal Antibody Titer Detection

[0079] 1. Thaw LILRB1 protein on ice beforehand, dilute appropriately (100 ng / 100 μL), add 100 μL to each well of a 96-well plate, and incubate overnight at 4°C.

[0080] 2. Discard the LILRB1 protein solution in the 96-well plate, add 200 μL of PBST (10 mL of PBS solution with 10 mL of L-80) buffer, and wash the plate on a horizontal shaker at room temperature for 5 minutes. Repeat 3 times.

[0081] 3. Dissolve 5g of skim milk powder in 100mL of PBST buffer to obtain a 5% (w / v) skim milk powder solution, and add it sequentially to a 96-well plate, 200μL per well, and incubate at 37℃ for 1 hour.

[0082] 4. Discard the skim milk powder solution in the 96-well plate, add 200 μL of PBST buffer, and wash the plate on a horizontal shaker at room temperature for 5 minutes. Repeat 3 times.

[0083] 5. Serially dilute the monoclonal antibody, add 100 μL of antibody of different dilutions to each well, and incubate at 37°C for 1 hour.

[0084] 6. Discard the antibody in the 96-well plate, add 200 μL of PBST buffer, and wash the plate on a horizontal shaker at room temperature for 5 minutes. Repeat 3 times.

[0085] 7. Dilute the Goat anti-mouse secondary antibody in skim milk powder at a ratio of 1:5,000 and add 100 μL to each well of a 96-well plate. Incubate at 37°C for 1 hour.

[0086] 8. Discard the secondary antibody in the 96-well plate, add 200 μL of PBST buffer, and wash the plate on a horizontal shaker at room temperature for 5 minutes. Repeat 3 times.

[0087] 9. Add 50 μL of TMB colorimetric solution to each well and develop at 37°C for 15 minutes.

[0088] 10. Add 50 μL of 2M H2SO4 to each well to stop the reaction, and measure the OD using a microplate reader. 450 Record the absorbance.

[0089] We performed titer tests on the culture supernatants of five hybridoma cell lines. The results showed that all five hybridoma cell lines produced effective monoclonal antibodies with titers greater than 1:2430, as shown in Table 3.

[0090] Table 3. Detection of monoclonal antibody titers secreted by five hybridoma cell lines.

[0091]

[0092] Example 6: Purification of LILRB1 monoclonal antibody

[0093] 1. Column packing: Fill an empty PD-10 column with 2 mL of binding / washing buffer (NaCl, 0.5 M; Na2HPO4, 20 mM; pH 8.0), and then fill the column with 5 mL of well-mixed Protein A / G gel resin.

[0094] 2. Column washing: Drain the liquid from the column and wash the column again with 10 times the volume of binding / washing buffer.

[0095] 3. Sample loading: Add the prepared sample to be purified (ascites or cell culture supernatant) into the column. The sample can be added in batches according to the column's capacity.

[0096] 4. Elute contaminating proteins: Rinse the column with binding / washing buffer until the buffer is free of proteins.

[0097] 5. Antibody Collection: Add elution buffer (7.507g glycine dissolved in double-distilled water, pH adjusted to 3.0 with hydrochloric acid, and finally brought to a final volume of 500mL) to the column, and collect the eluent (approximately 0.3-0.4mL / tube) until the eluent is free of protein. Measure the protein content in each collection tube and combine the protein collection tubes. (Note: Approximately 150μL of 1M Tris-HCl, pH 9.0 buffer should be added to the collection tubes beforehand to prevent antibody inactivation in an overly acidic environment).

[0098] 6. The eluted antibody was collected by PBS dialysis. The obtained antibody protein was quantified and used to process cells for functional studies.

[0099] Example 7: LILRB1 Monoclonal Antibody Blockade Assay

[0100] 1. Transfect pEGFP-N1-LILRB1 into 293T cells to overexpress the GFP-LILRB1 fusion protein.

[0101] 2. After transfecting the cells for 24 hours, digest them with trypsin and aliquot them evenly into 10 1.5 mL centrifuge tubes.

[0102] 3. Eight centrifuge tubes were treated with mouse IgG (negative control), LILRB1 monoclonal antibody (Biolegend), LILRB1 polyclonal antibody (self-made), 16G10 monoclonal antibody (hybridoma secretion), 19H12 monoclonal antibody (hybridoma secretion), 20H9 monoclonal antibody (hybridoma secretion), 22A10 monoclonal antibody (hybridoma secretion), and 23H10 monoclonal antibody (hybridoma secretion) at a final concentration of 1 μg / mL, respectively. The other two centrifuge tubes were left untreated.

[0103] 4. Wash the cells three times with PBS.

[0104] 5. Add 1 μg / mL of HLA-G protein labeled with 647 fluorescence (AAT Bioquest, 1235) to one of the eight antibody-treated centrifuge tubes. Add 1 μg / mL of 647-labeled HLA-G protein to one of the other two centrifuge tubes, and add an equal volume of PBS to the other. Incubate all samples at room temperature for 1 hour.

[0105] 6. Flow cytometry was used to detect the APC fluorescence value on the cell surface of each sample, which represents the amount of HLA-G protein bound to the surface of GFP-LILRB1 overexpressing cells.

[0106] like Figure 2 As shown, all LILRB1 antibodies inhibited HLA-G protein binding to the cell surface to varying degrees, with 16G10 showing the best inhibitory effect. Compared with the IgG treatment group (as a negative control), the amount of HLA-G protein bound to the cell surface decreased by approximately 85% in the 16G10 monoclonal antibody treatment group. Figure 2 ).

[0107] Example 8: Sequencing of the variable region of LILRB116G10 monoclonal antibody

[0108] Genscript Biotech Inc. was commissioned to sequence the variable region of the LILRB1 clonal antibody secreted by hybridoma 16G10, confirming the nucleotide and amino acid sequences of its heavy and light chain variable regions. Specifically, the nucleotide sequence encoding DNA in the heavy chain variable region is shown in SEQ ID NO: 4, and the nucleotide sequence encoding DNA in the light chain variable region is shown in SEQ ID NO: 5. The amino acid sequences of the encoded heavy chain variable region are shown in SEQ ID NO: 2, and the amino acid sequences of the light chain variable region are shown in SEQ ID NO: 3.

[0109] Example 9: Construction, expression, and purification of LILRB1 human-mouse chimeric 16G10 monoclonal antibody (LILRB1 hu-16G10 mAb)

[0110] To obtain human antibodies for further evaluation, a human IgG1 total antibody against LILRB1 was designed and constructed.

[0111] 1. The strategy is as follows:

[0112] Heavy chain H: CMV promoter-EcoRI-signal peptide (SP)-mouse 16G10 monoclonal antibody heavy chain variable region (VH)-human IgG1 heavy chain constant region (CH)-Xhol;

[0113] Light chain κ: CMV promoter-EcoRI-signal peptide (SP)-mouse 16G10 monoclonal antibody light chain variable region (VK)-human IgG1 light chain constant region (CLκ)-Xhol;

[0114] 2. The heavy chain H and light chain κ were cloned into the expression vector pCAGGS to obtain recombinant plasmids containing the light and heavy chain encoding genes of specific antibodies.

[0115] 3. Expression and purification of LILRB1 human-mouse chimeric 16G10 monoclonal antibody

[0116] ① The plasmids containing the heavy chain H and light chain κ encoding genes constructed above were co-transfected at a density of 3 x 102 at a heavy chain:light chain ratio of 1:1.5. 6 In 293T cells: In a 2mL sterile centrifuge tube, dilute the plasmid with 1mL of OPTI-MEM (total plasmid amount not exceeding 20μg); in a 2mL sterile centrifuge tube, dilute 60μL of Lipo2000 transfection reagent with 1mL of OPTI-MEM and let stand for 5min; after thoroughly mixing the two, let stand for 20min and add dropwise to 293T cells.

[0117] ② After 24 hours of transfection, the cell culture medium was collected, and the cells were centrifuged at 6500 rpm for 30 minutes to remove the cell pellet. The supernatant was filtered through a 0.22 μm filter and then thoroughly mixed with Protein A / GPlus Agarose (1 mL, Santa Cruz) at 4°C for 4 hours. The bound antibody protein was eluted with 10 mM glycine (pH 3.0). The protein was collected, concentrated, and quantified, and the antibody titer was detected by ELISA.

[0118] The results of ELISA showed that the titer of the LILRB1 human-mouse chimeric monoclonal antibody was greater than 1:2430 (as shown in Table 4).

[0119] Table 4. Titer Detection of LILRB1 Human-Mouse Chimeric Monoclonal Antibody

[0120]

[0121] Example 10: Blocking assay of LILRB1 human-mouse chimeric 16G10 monoclonal antibody (LILRB1 hu-16G10 mAb)

[0122] 1. Transfect pEGFP-N1-LILRB1 into 293T cells to overexpress the GFP-LILRB1 fusion protein;

[0123] 2. After transfecting cells for 24 hours, digest them with trypsin and aliquot them evenly into 10 1.5 mL centrifuge tubes;

[0124] 3.5 centrifuge tubes were treated with mouse IgG (negative control), LILRB1 16G10 monoclonal antibody (hybridoma secretion) and LILRB1 human-mouse chimeric 16G10 monoclonal antibody (LILRB1 hu-16G10 mAb) at a final concentration of 1 μg / mL, respectively. The other 2 centrifuge tubes were left untreated.

[0125] 4. Wash the cells three times with PBS;

[0126] 5. Add 1 μg / mL of HLA-G protein labeled with 647 fluorescence (AAT Bioquest, 1235) to three centrifuge tubes treated with the antibody. Add 1 μg / mL of 647-labeled HLA-G protein to one of the other two centrifuge tubes, and add an equal volume of PBS to the other. Incubate all samples at room temperature for 1 hour.

[0127] 6. Flow cytometry was used to detect the APC fluorescence value on the cell surface of each sample, which represents the amount of HLA-G protein bound to the surface of GFP-LILRB1 overexpressing cells;

[0128] like Figure 3As shown, the LILRB1 human-mouse chimeric 16G10 monoclonal antibody (LILRB1 hu--16G10 mAb) can effectively inhibit the binding of HLA-G protein to LILRB1 on the cell surface, and its inhibitory effect is comparable to that of the murine 16G10 monoclonal antibody (LILRB1 16G10 mAb) secreted by hybridoma. Compared with the IgG treatment group as a negative control, the amount of HLA-G protein bound to the cell surface in the 16G10 monoclonal antibody treatment group decreased by approximately 85%. Figure 3 ).

[0129] Example 11: Experiment on the regulation of NK cell killing activity by LILRB1 monoclonal antibody

[0130] 1. Target cell preparation:

[0131] ① Take 10 mL of peripheral blood from a healthy person and dilute it with an equal volume of sterile PBS. At the same time, take a centrifuge tube and add an equal volume of lymphocyte separation medium to the peripheral blood.

[0132] ② Carefully aspirate the blood sample with a pipette and slowly add it to the surface of the lymphocyte separation solution. Centrifuge at 600g for 30 minutes (Note: The larger the blood sample, the greater the centrifugal force and the longer the centrifugation time, but not exceeding 1,200g).

[0133] ③ Carefully aspirate the central ring of milky white lymphocytes into a 15mL centrifuge tube using a pipette. Add 5 times the volume of PBS to wash the cells, centrifuge at 350g for 5 minutes, and wash twice.

[0134] ④ Resuspend the precipitated cells in RPMI 1640 cell culture medium containing 10% fetal bovine serum.

[0135] ⑤ After incubating at 37℃ and 5% CO2 for 1.5-2 hours (to allow monocytes to adhere to the culture dish), remove non-adhesive lymphocytes (they must be completely removed, otherwise residual NK cells will affect the induction of monocyte-macrophage differentiation). Add fresh culture medium and continue culturing the adherent cells overnight.

[0136] ⑥ On the second day, replace the culture medium with fresh medium and add 30 mg / L GM-CSF inducer to the medium. Replace the culture medium with fresh medium and add 30 mg / L GM-CSF every three days thereafter to induce culture.

[0137] ⑦ One week after induction of differentiation, observe whether the macrophages have adhered and differentiated, and use them as target cells.

[0138] 2. Preparation of Mycobacterium tuberculosis H37Rv:

[0139] Mycobacterium tuberculosis H37Rv can be cultured on 7H10 plates or slant for 3-4 weeks before use.

[0140] The preparation method for 7H10 plates is as follows: Weigh 1.9g Middlebrook 7H10, add 90mL of double-distilled water to dissolve, autoclave at 121℃ for 30 minutes, and keep warm in a 65℃ oven or water bath. In a clean bench, add 10mL OADC, 1mL 50% glycerol and 250μL 20% Tween-80 to the 7H10 medium, mix thoroughly, and pour into a plate or 15mL glass tube to make a slant.

[0141] 3. Mycobacterium tuberculosis H37Rv infects target cells:

[0142] ① After digesting and counting the induced target cells, they were transferred to 24-well plates and cultured overnight (1×10⁶ cells per well). 5 (cells).

[0143] ② Scrape off H37Rv colonies from the fixed plate or slant and add them to 7H9 liquid medium. Shake thoroughly until no granular bacterial clumps remain. Take 1 mL of H37Rv and measure the OD of the bacterial solution. 600 The value was continuously diluted and adjusted until the bacterial culture OD 600 =0.6-0.8 (OD) 600 When the concentration is 0.6, the bacterial concentration is 1×10⁶. 8 (CFUs / mL).

[0144] The 7H9 culture medium formula is as follows: Weigh 0.47g Middlebrook 7H9, dissolve it in 90mL double-distilled water, and in a clean bench, add 10mL OADC, 1mL 50% glycerol, and 250μL 20% Tween-80 to the 7H9 medium. Mix thoroughly and filter through a 0.22μm filter into a sterile reagent bottle. Incubate overnight at 37℃ to check for contamination, then store at 4℃ until use.

[0145] ③ Dilute the bacterial culture to 1×10⁻⁶ using 7H9 medium. 5 per mL.

[0146] ④ Add 100 μL of bacterial culture (MOI=1:1) to each well of a 24-well plate and incubate at 37°C and 5% CO2 for 2 hours.

[0147] ⑤ Wash the cells three times with 1 mL PBS to remove extracellular Mycobacterium tuberculosis.

[0148] 4. NK cell preparation:

[0149] ① Take 10 mL of peripheral blood from a tuberculosis patient and dilute it with an equal volume of sterile PBS. At the same time, take a centrifuge tube and add an equal volume of lymphocyte separation medium to the peripheral blood.

[0150] ② Carefully aspirate the blood sample with a pipette and slowly add it to the surface of the lymphocyte separation medium. Centrifuge at 600g for 30 minutes (Note: The larger the blood sample volume, the greater the centrifugal force and the longer the centrifugation time, but not exceeding 1,200g). After centrifugation, transfer the uppermost plasma layer to a new sterile centrifuge tube, heat-inactivate at 55℃ for 30 minutes, centrifuge at 500g for 25 minutes, collect the supernatant in a laminar flow hood, and filter it through a 0.22μm filter membrane for later use.

[0151] ③ Carefully aspirate the central ring of milky white lymphocytes into a 15mL centrifuge tube using a pipette. Add 5 times the volume of PBS to wash the cells, centrifuge at 350g for 5 minutes, and wash twice.

[0152] ④ Resuspend the precipitated cells in PBS and separate NK cells using NK cell negative selection magnetic beads.

[0153] ⑤ NK cells were resuspended in RPMI 1640 complete culture medium containing 200 IU / mL recombinant human interleukin-2 (rhIL2) and 10% autologous serum (cell concentration 5 × 10⁶ cells / mL medium). 5 (cells).

[0154] 5. Incubation of NK cells with target cells:

[0155] ① Add 1 mL of NK cells (5 × 10⁶) to each well of target cells infected with Mycobacterium tuberculosis. 5 (100 NK cells), while the cell culture medium was treated with mouse IgG (negative control) at a final concentration of 1 μg / mL, commercial LILRB1 monoclonal antibody, LILRB1 polyclonal antibody, 16G10 monoclonal antibody, 19H12 monoclonal antibody, 20H9 monoclonal antibody, 22A10 monoclonal antibody, and 23H10 monoclonal antibody.

[0156] ② After co-culturing at 37℃ and 5% CO2 for 24 hours, suspension NK cells were collected for flow cytometry analysis. The main steps included: resuspending cells in Cell Staining Buffer, adding flow cytometry antibodies (including PE-CD3, FITC-CD56, and APC-CD107a), and incubating on ice in the dark for 20 minutes. After washing cells twice with Cell Staining Buffer, cells were fixed with 4% paraformaldehyde and then analyzed.

[0157] ③ Regarding CD3 - CD56 + The fluorescence intensity of CD107a in the NK cell population was quantitatively analyzed.

[0158] The results are as follows Figure 4As shown: After NK cells were treated with LILRB1 antibody, the secretion of granzyme and perforin by NK cells from tuberculosis patients was increased by approximately 1-2 times compared to NK cells that had not been treated with LILRB1 antibody (IgG treatment group), suggesting that LILRB1 antibody treatment can enhance the killing function of NK cells. Among them, the 16G10 monoclonal antibody showed the best effect. Figure 4 ).

[0159] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A LILRB1 monoclonal antibody, characterized in that, The amino acid sequence of CDR1 in the heavy chain variable region is: SYWMN; the amino acid sequence of CDR2 is: QIYPGDGDTNYNGEFKD; and the amino acid sequence of CDR3 is: SGDYYGYSPFY. The amino acid sequence of CDR1 in the light chain variable region is: LASQTIGTWLA; the amino acid sequence of CDR2 is: AATSLAD; and the amino acid sequence of CDR3 is: QQLYSTPYT.

2. The LILRB1 monoclonal antibody as described in claim 1, characterized in that, The 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.

3. The LILRB1 monoclonal antibody as described in claim 2, characterized in that, It was produced by a hybridoma cell line with accession number CGMCCNo.45305.

4. A hybridoma cell line secreting a LILRB1 monoclonal antibody, characterized in that, The hybridoma cell line has the accession number CGMCC No. 45305.

5. A LILRB1 human-mouse chimeric monoclonal antibody based on the LILRB1 monoclonal antibody as described in any one of claims 1 to 3.

6. The LILRB1 human-mouse chimeric monoclonal antibody as described in claim 5, characterized in that, It is a human IgG1 total antibody.

7. The LILRB1 human-mouse chimeric monoclonal antibody as described in claim 6, characterized in that, Its heavy chain H includes a signal peptide, a heavy chain variable region and a heavy chain constant region of the LILRB1 monoclonal antibody as described in any one of claims 1 to 3, and its light chain κ includes a signal peptide, a light chain variable region and a light chain constant region of the LILRB1 monoclonal antibody as described in any one of claims 1 to 3.

8. The LILRB1 human-mouse chimeric monoclonal antibody as described in claim 7, characterized in that, It is obtained by expressing its encoding DNA through humanized cells.

9. A nucleic acid molecule encoding the LILRB1 human-mouse chimeric monoclonal antibody as described in claim 5, 6, or 7, characterized in that, The nucleotide sequence encoding the heavy chain of the LILRB1 monoclonal antibody as described in any one of claims 1 to 3 includes a CMV promoter sequence, a signal peptide sequence, a sequence encoding the variable region of the heavy chain, and a sequence encoding the constant region of the heavy chain; the sequence encoding the light chain of the LILRB1 monoclonal antibody as described in any one of claims 1 to 3 includes a CMV promoter sequence, a signal peptide sequence, a sequence encoding the variable region of the light chain, and a sequence encoding the constant region of the light chain.

10. The use of the LILRB1 monoclonal antibody according to any one of claims 1 to 3 in the preparation of a medicament for the treatment and / or prevention of tuberculosis.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the LILRB1 monoclonal antibody as described in any one of claims 1 to 3.

12. The pharmaceutical composition of claim 11, characterized in that, It also contains medically acceptable carriers.

13. A kit for immunodiagnosis or detection, said kit containing the monoclonal antibody according to any one of claims 1 to 3.

14. The kit according to claim 13, characterized in that, The kit is an enzyme-linked immunosorbent assay (ELISA) kit.

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