Anti-human lilrb2 fully human single-domain antibodies and uses thereof

Abstract

The application discloses anti-human LILRB2 full-human single-domain antibodies and application thereof, the amino acid sequence of the antibody is shown as SEQ ID NO:1-2, the nucleic acid sequence of the antibody is shown as SEQ ID NO:3-4, the antibody can block the combination of LILRB2 and its ligand HLA-G, regulate macrophage JAK / STAT, PI3K / Akt signal pathways, reprogram M2 macrophages into M1 macrophages, and improve tumor microenvironment. The application belongs to the technical field of biotechnology, and particularly relates to the sequence of a binding unit of a single-domain antibody capable of combining with human LILRB2, a preparation method, and detection of the blocking capacity of the antibody, antibody off-target and homologous family off-target.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to fully human single-domain antibodies against human LILRB2 and their applications. Background Technology

[0002] Nanobodies are the smallest antibodies by molecular weight and have been found in camel blood. Camel-derived nanobodies are small, have strong penetrating power, and good antigen specificity; however, their immunogenicity can easily cause related safety issues. Constructing a fully human single-domain antibody library from human blood and screening for fully human single-domain antibodies can effectively reduce immunogenicity problems.

[0003] Leukocyte Ig-like receptor subfamily B2 (LILRB2), also known as ILT4, MIR-10, and CD85d, is an immunosuppressive member of the LILR family of leukocyte immunoglobulin-like receptors, regulating the activation of immune cells. LILRB2 is a typical type I transmembrane protein with four extracellular tandem Ig-like domains, a 23-amino acid transmembrane region, and a cytoplasmic tail with three immunoreceptor tyrosine inhibitory motifs (ITIMs). Physiologically, it is commonly expressed in many myeloid cells, including monocytes, macrophages, granulocytes, and dendritic cells. Tumor-associated macrophages (TAMs) in the tumor microenvironment highly express LILRB2. The most important ligand for LILRB2 is HLA-G, and related studies have shown that LILRB2 / HLA-G binding activates Akt phosphorylation and STAT6 activation, which is associated with M2 macrophage polarization. PIR-B is a mouse homolog of LILRB2. Studies have shown that PIR-B is a key target for maintaining the M2 phenotype of mouse myeloid suppressor cells (MDSCs). In PIR-B-deficient mice, TLR and IFN-γ signaling are amplified in MDSCs, IL-4 / IL-13 and IL-10 expression is suppressed, tumor burden is reduced, and Treg activation is decreased. LILRB2 is mainly expressed in myeloid cells, not in B or NK cells, and its expression is limited in other tissues. LILRB2 is a promising immune checkpoint targeting M2 macrophages, with immunomodulatory potential and therapeutic value.

[0004] Currently, there are three LILRB2-targeting drugs in the clinical stage. Existing technologies such as ScFv or all-IgG anti-LILRB2 antibody molecules have complex structures and are not easy to prepare; nanobody molecules are small and easy to manipulate, but have low humanization and low affinity. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a fully human single-domain antibody against human LILRB2.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a fully human single-domain antibody against human LILRB2, wherein the amino acid sequence of the antibody is shown in SEQ ID NO:1-2 and the nucleic acid sequence of the antibody is shown in SEQ ID NO:3-4.

[0009] As a preferred embodiment of the antibody described in this invention, the antibody targets the human LILRB2 molecule and blocks the binding of LILRB2 to its ligand HLA-G.

[0010] As a preferred embodiment of the antibody described in this invention, the antibody targets the human LILRB2 molecule and does not de-target the family or irrelevant cells.

[0011] As a preferred embodiment of the antibody described in this invention, the antibody regulates the JAK / STAT and PI3K / Akt signaling pathways of macrophages, reprograms M2 macrophages into M1 macrophages, and improves the tumor microenvironment.

[0012] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of anti-human LILRB2 fully human single-domain antibody in the preparation of immunotherapeutic drugs or pharmaceutical compositions.

[0013] In a preferred embodiment of the application described in this invention, the drug or drug composition comprises a drug or drug composition that improves the tumor microenvironment and reverses TAM-mediated tumor immunosuppression.

[0014] As a preferred embodiment of the application described in this invention, the drug or drug composition comprises a drug or drug composition that alleviates tumor resistance to immune checkpoint inhibitors.

[0015] Another object of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing a fully human single-domain antibody against human LILRB2, comprising,

[0016] LILRB2 biotin-tagged protein was co-incubated with streptavidin-coated magnetic beads. LILRB2 was immobilized on the magnetic beads through the interaction between streptavidin and biotin. Antibody library was used to incubate the beads, and unbound or weakly bound phages were washed away. Phages bound to LILRB2 protein were eluted.

[0017] This process was repeated three times, each time with changes to the amount of LILRB2 biotin-tagged protein used and the washing conditions, to gradually eliminate weakly binding phages and retain as many strongly binding phages as possible.

[0018] The host bacteria were infected with a strongly binding phage, plated, and cultured overnight to obtain monoclonal colonies, which were then monoclonalized using a phage antibody that could bind to LILRB2.

[0019] ELISA screening was performed using the culture supernatant of single clones, and the nucleic acid sequences of the single clones were determined.

[0020] The coding sequences of the signal peptide, anti-LILRB2 antibody, and human IgG4 Fc were linked and read in the same frame to construct a mammalian expression vector.

[0021] 293F cells were transfected and cultured with shaking for 5 days. The supernatant was harvested and purified from the supernatant using protein A magnetic bead affinity chromatography to obtain the fusion protein.

[0022] Beneficial effects of this invention:

[0023] Currently, therapies targeting LILRB2 mainly involve monoclonal antibodies, primarily single-chain antibodies and a small number of camel single-domain antibodies. However, these single-domain antibodies are not fully human and, compared to fully human antibodies, suffer from higher immunogenicity, poorer safety profiles, and potentially lower in vivo stability. This invention successfully prepared an antibody (fusion protein) that can bind to human LILRB2 molecules. This antibody exhibits good specificity and high affinity, effectively blocking the binding of LILRB2 to its ligand when it binds to human LILRB2 expressed on the cell surface. This anti-human LILRB2 single-domain antibody is a potential drug for immunotherapy. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0025] Figure 1 This is a graph showing the ELISA detection results of the binding of phage crude culture medium and human LILRB2 in an embodiment of the present invention.

[0026] Figure 2 This is a schematic diagram of the LILRB2 structure in an embodiment of the present invention, wherein NheⅠ is a single enzyme cleavage site, and the target gene is inserted into this position through homologous recombination.

[0027] Figure 3 This is an SDS-PAGE electrophoresis image of the purified antibody in an embodiment of the present invention; wherein, reduction electrophoresis is used, the disulfide bonds in the antibody molecule are opened, and the molecule migrates electrophoretically in an extended single peptide chain state.

[0028] Figure 4 This is a graph showing the results of the binding performance detection of purified antibody and LILRB2 overexpressed on the cell surface in an embodiment of the present invention.

[0029] Figure 5 This is a graph showing the ELISA level blocking detection results of purified antibodies in an embodiment of the present invention.

[0030] Figure 6 This is an off-target detection diagram of purified antibodies and LILRA1, LILRA2, LILRA3, and LILRB1, which have over 80% homology with the LILRA1 family, in an embodiment of the present invention.

[0031] Figure 7 This is a diagram showing the detection results of purified antibody and LILRB2 overexpressing cell line blocking in an embodiment of the present invention.

[0032] Figure 8 The image shows the FACS detection results of purified antibody and LILRB2 negative cells (MCF-10A, Jurkat, 786-0) in an embodiment of the present invention. Detailed Implementation

[0033] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0034] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0035] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0036] This invention uses commercially available recombinant human LILRB2 biotin-tagged protein and a phage display antibody library as starting materials. The LILRB2 biotin-tagged protein is co-incubated with streptavidin-coated magnetic beads. LILRB2 is immobilized on the magnetic beads through the interaction between streptavidin and biotin. The antibody library is then incubated with the beads to wash away unbound or weakly bound phages, and the phages bound to the LILRB2 protein are eluted.

[0037] This process was repeated three times, each time changing the amount of LILRB2 biotin-tagged protein and washing conditions to gradually eliminate weakly binding phages and retain as many strong-binding phages as possible. The host bacteria were then infected with the strong-binding phages, plated, and incubated overnight to obtain monoclonal colonies. This process involved monocloning the phage antibody that could bind to LILRB2.

[0038] ELISA screening was performed using the culture supernatant of single clones, and the nucleic acid sequences of the single clones were determined.

[0039] The coding sequences of the signal peptide, anti-LILRB2 antibody, and human IgG4 Fc were ligated and read in the same frame to construct a mammalian expression vector. The vector was transfected into 293F cells, and after 5 days of shaking culture, the supernatant was harvested. The fusion protein was purified from the supernatant using protein A magnetic bead affinity chromatography, and its binding characteristics to LILRB2 were identified.

[0040] The purified antibody was co-incubated with the human LILRB2 overexpression cell line for half an hour. After adding the APC secondary antibody, the antibody binding ability was detected by flow cytometry. In the ELISA experiment, HLA-G tetramer was used as a coating substrate and coated overnight. The purified antibody and LILRB2 protein with mFc tag were added to the coated plate and incubated. After adding anti-mouse HRP, TMB color development was performed until the blue reaction was complete. Then, the stop solution was added to terminate the experiment.

[0041] LILRB1, LILRA1, LILRA2, and LILRA3 were used as coating substrates and coated overnight. The binding of the purified antibody to the other homologous families was detected by ELISA.

[0042] Human LILRB2 overexpressing cell lines were co-incubated with HLA-G tetramers carrying the PE tag, followed by incubation with purified LILRB2 antibody for half an hour. The PE fluorescence intensity was detected by flow cytometry, and the blocking rate was calculated. Irrelevant cells, including MCF-10A, Jurkat, and 786-0, were co-incubated with purified antibody, and the binding was detected by flow cytometry.

[0043] Example 1

[0044] (1) Phage display antibody library selection

[0045] 1) Activation of host bacteria TG1: Prepare mini agar medium plates (1×M9 salt, 2% glucose, 2mM MgSO4, 0.1mM CaCl2, 1mM vitamin B1), and incubate TG1 overnight at 37℃ using the streak method.

[0046] 2) Magnetic bead washing and blocking: Take 50 μl of magnetic beads (purchased from Invitrogen), place them on a magnetic rack, remove the liquid after adsorption, resuspend in 1 mL PBS, wash twice, block with 1 mL of blocking agent of 1.5% skim milk powder + 1.5% BSA (the concentration of blocking agent is gradually increased in the second and third rounds of washing) for 1 hour, and remove the liquid.

[0047] 3) Antigen binding: Human LILRB2 protein (purchased from Kactus Biosystems, with the antigen concentration gradually decreasing in subsequent rounds of panning) was diluted to 1 mL in PBS (pH 7.2-7.4) at a concentration of 16 μg / mL, the magnetic beads were resuspended, and the mixture was incubated by rotation for 1 hour.

[0048] 4) Library blocking: Synchronize the binding of antigen to magnetic beads, take 10 11 PFU phage virus particles (derived from the original antibody library or panning amplification products, the phage is M13 phage) were incubated with 1 mL of 1.0% skim milk powder + 1.0% BSA blocking agent by rotation for 1 hour.

[0049] 5) Phage binding: Place the magnetic beads on a magnetic rack and remove the liquid. Add the sealed library to the magnetic beads, resuspend and incubate by rotation for 1 hour, then remove the liquid.

[0050] 6) Washing: Wash with 1 mL PBST [0.01 M PBS (pH 7.4), 0.1% Tween-20 (the concentration of Tween-20 for the second and third rounds is 0.2% and 0.3% respectively)], and then wash with 0.01 M PBS (pH 7.4).

[0051] 7) Elution: Remove the liquid, elute with 300 μL of 0.2 M glycine-hydrochloric acid (pH 2.2) for 10 min, add 20 μL of neutralization solution [1 M Tris-Cl (pH 9.0)] and mix well. Store temporarily at 4 °C.

[0052] 8) Titration: Take 2 μL, 0.2 μL (2 μL of the original solution diluted 10 times with 2×YT medium), and 0.02 μL (2 μL of the original solution diluted 100 times with 2×YT medium) of elution buffer, mix with 0.2 mL of TG1 at mid-log (OD600 = 0.5), incubate at room temperature for 30 min, and spread evenly on 2×YT-GA100 plates [containing 2% glucose and 100 μg / mL ampicillin], incubate overnight at 37°C, count the number of clones on plates with approximately 50 clones, and calculate the titer based on the dilution factor; For 2-YT medium (500 mL): Weigh 8 g of tryptone, 5 g of yeast extract, and 2.5 g of sodium chloride, add 400 mL of ultrapure water to dissolve them completely, bring the volume to 500 mL, adjust the pH to 7.4 with NaOH, autoclave at 121°C, and place at room temperature after sterilization.

[0053] 9) Phage amplification: While panning, pick TG1 single clones from mini agar plates and inoculate them into 10 mL of 2×YT culture medium. Incubate at 37°C with shaking at 250 rpm until mid-log phase (OD600 = 0.5).

[0054] Add 200 μL of the eluted product obtained by panning and incubate at 37°C for 30 min.

[0055] Add helper phage M13KO7, incubate at 37°C for 30 min, then incubate at 37°C with shaking at 250 rpm for 1 hour.

[0056] Centrifuge to remove supernatant, resuspend in 20 mL of 2×YT containing working concentrations of 100 μg / mL ampicillin and 50 μg / mL kanamycin, and incubate overnight at 30°C with shaking at 220 rpm.

[0057] 10) Phage precipitation: Centrifuge at 10,000 rpm for 15 min to remove bacterial cells, add 1 / 5 volume of 2.5M NaCl / 20% PEG8000 to the supernatant, and incubate on ice for 2 h.

[0058] Centrifuge at 10000 rpm for 10 min to obtain phage precipitate, remove the residual liquid, add 0.2 mL of 0.01 M PBS (pH 7.4) to resuspend the precipitate, and measure the titer as described above.

[0059] 11) Repeat steps 2)-10) two or three times to obtain phage display antibodies with strong binding affinity.

[0060] (2) Monoclonal ELISA

[0061] 1) Coat an ELISA plate with 0.3 μg / mL streptavidin overnight at 4°C, treat with 2% BSA / PBS blocking buffer for 2 h, and wash 3 times with PBS.

[0062] 2) Select single colonies from 2×YT-GA100 plates, shake and culture until mid-log phase, add helper phage M13KO7, and incubate at 37℃ for 30 min.

[0063] Incubate at 37℃ and 220 rpm for 1 hour with shaking, then centrifuge at 4000 rpm for 15 minutes.

[0064] Resuspend in 400 μL of 2×YT containing working concentrations of 100 μg / mL ampicillin and 50 μg / mL kanamycin, and incubate overnight at 30°C with shaking at 220 rpm.

[0065] Centrifuge at 4000 rpm for 15 min to precipitate the bacterial cells.

[0066] 3) Add 50 μL of 4% BSA / PBS to the microplate, and at the same time add 50 μL of phage supernatant. Mix well and incubate for 1 hour.

[0067] 4) Remove the liquid: wash 5 times with 0.1% PBST, then wash 3 times with PBS to remove the liquid.

[0068] 5) Dilute the HRP-labeled anti-M13 phage antibody (purchased from Beijing Yiqiao Shenzhou) 3000 times with 2% BSA, add 100 μL to the ELISA plate, incubate for 1 hour, remove the liquid, wash 3 times with 0.1% PBST, and pat dry the residual liquid.

[0069] 6) Add 100 μL TMB colorimetric solution, incubate at 37°C for 10 min or until the blue color is fully developed, add 100 μL 1M sulfuric acid to terminate the reaction, and read the OD450 on a microplate reader.

[0070] See data Figure 1 See Table 1.

[0071] Table 1

[0072] 1 0.03 1.68 56 2 0.05 2.35 47

[0073] During the phage panning process, 50 μL of 2×YT medium containing phage virus particles was used for detection. The raw data are shown in Table 1, where the values ​​are the absorbance at 450 nm and their ratios.

[0074] Note: The use of ELISA to detect the binding of Phage crude culture medium mainly demonstrates that at the Phage level, phages can bind to LILRB2 coated on the ELISA plate.

[0075] (3) Preparation and flow cytometry detection of human LILRB2 monoclonal antibody

[0076] 1) Single colonies picked from 2×YT-GA100 plates were cultured overnight with liquid shaking, and phagemids were extracted using the plasmid extraction method.

[0077] 2) Synthesize primers and amplify the antibody gene coding region displayed by the bacteriophage using PCR.

[0078] 3) The above nucleic acid fragments were digested with NheI single enzyme and then sequentially inserted into the eukaryotic expression vector Abexp-uIgG4sa using homologous recombination. Figure 2 We constructed a fusion protein encoding an N-terminal signal peptide, a mid-terminal antibody, and a C-terminal Fc tag.

[0079] 4) Prepare sterile, endotoxin-free plasmids.

[0080] Take 23 μg and dilute it with 0.75 mL of diluent (OPM-293CD05 medium). Simultaneously, add 70 μL of transfection reagent (e.g., PEI solution) to the 0.75 mL diluent (e.g., OPM-293CD05 medium) and mix gently. Add the PEI diluent to the plasmid diluent and immediately mix gently with a pipette. Let it stand at room temperature for 15 min, avoiding disturbance.

[0081] 5) Add to 25 mL of 293F cells and their culture medium, and incubate at 85 rpm, 37°C, and 5% CO2 for 24 hours. Then add 25 mL of fresh growth medium (such as OPM-293CD05), and continue incubating at 85 rpm, 37°C, and 5% CO2 for 72 hours.

[0082] 6) Centrifuge at 10,000 rpm for 10 minutes and collect the supernatant.

[0083] Incubate with equilibrated protein A affinity beads by rotation for 1 hour, then place on a magnetic rack and remove the supernatant.

[0084] 7) Wash the sample three times with 30 mL PBS, add 5 mL 0.1 M glycine (pH 3.0) and elute for 10 min. Place the sample on a magnetic rack, aspirate the supernatant and immediately neutralize with 1 M Tris-HCl buffer (pH 8.5) to obtain the purified antibody.

[0085] 8) SDS-PAGE was used to detect the purified antibody, and the concentration was determined simultaneously; the SDS-PAGE electrophoresis results are shown in [reference needed]. Figure 3 As can be seen, the purification effect is quite good.

[0086] The amino acid sequence of the antibody is shown in SEQ ID NO:1-2, and the nucleic acid sequence of the antibody is shown in SEQ ID NO:3-4, as shown in the table below:

[0087]

[0088] 9) Flow cytometry detection of the cell-binding ability of purified antibodies

[0089] ① Cells expressing LILRB2 (cell binding assay, CHOK1 cells overexpressing LILRB2) or not expressing LILRB2 (off-target assay, MCF-10A normal human breast epithelial cells, Jurkat human T lymphocyte leukemia cells, 786-O human clear cell adenocarcinoma cells) were fully digested with 0.25% trypsin. Digestion was terminated with serum. Cells were collected by centrifugation and prepared as single-cell suspensions by gentle pipetting with PBS.

[0090] ② Wash the cells once with 10 mL PBS, centrifuge at 1000 rpm for 5 min, then resuspend the cells in 1 mL PBS and count the cells.

[0091] ③ Take 2.5 × 10 5 Cells were collected by centrifugation in 96-well cell culture plates.

[0092] ④ Add 100 μL of the purified antibody from step (3) 8) with 10 μg / mL, mix well, and incubate at 4°C for 30 min to 1 hour.

[0093] ⑤ Collect cells by centrifugation and wash cells once with 300μL PBS.

[0094] ⑥ Add 100 μl of fluorescently labeled (e.g., FITC, APC) antibody diluted 200 times with PBS, and react at room temperature in the dark for 20 min.

[0095] ⑦ Wash the cells once with 1 mL PBS, centrifuge at 1000 rpm for 8 min, and remove the supernatant.

[0096] ⑧ Add 100 μl PBS to resuspend the cells in a single-cell suspension and analyze them using a flow cytometer.

[0097] Data such as Figure 4 Table 2 shows the flow cytometry used for this detection. J19 and 1E1 are positive controls, and hIgG4 is a negative control. Each antibody was detected using wild-type cells (gray curve) and LILRB2 overexpressing cells (black curve). The values ​​are median fluorescence intensity (MFI).

[0098] Table 2

[0099] hIgG4 77.5 192.6 2.48 No.ID 1 64.6 6178.1 95.64 No.ID 2 62.1 6275.3 101.05 J-19 58.8 5725.4 97.37 1E1 52 5261.4 101.18

[0100] Data such as Figure 8Table 6 shows the flow cytometry data used for this assay. Antibody concentrations of 10 μg / mL and 100 μg / mL were used, with hIgG4 as a negative control for parallel assays. The raw data are shown in Table 6, where the values ​​are median fluorescence intensities (MFI).

[0101] Table 6 Off-target detection of purified antibodies and irrelevant cells

[0102]

[0103] Example 2

[0104] Purified antibody ELISA level blocking detection:

[0105] 1) Dilute the HLA-G tetramer with coating buffer and coat overnight at 4°C. Remove the coating buffer the next day and wash with PBS.

[0106] 2) Add blocking buffer and block at 37°C for 1 h, then wash with PBS. Simultaneously, serially dilute the antibody starting from 30 μg / mL to obtain 8 antibody solutions with different concentrations. Take 50 μL of the diluted antibody and add 50 μL of LILRB2-mFc to a final concentration of 4 μg / mL. Mix well and incubate at 37°C for 30 min.

[0107] 3) Add 100 μL of the mixed liquid to the blocked ELISA plate, bind at 30°C for 30 min, and wash with PBST.

[0108] 4) Add HRP-labeled anti-mouse IgG secondary antibody (purchased from Biolegend) at a dilution ratio of 1:2000, bind at 30℃ for 30 min, and wash with PBST.

[0109] 5) Add 100 μL of TMB chromogenic solution, incubate at 37°C for 10 min or until the blue color is fully developed, then add 100 μL of 1M sulfuric acid to stop the reaction. Read the OD450 on a microplate reader. Data as follows: Figure 5 As shown in Table 3.

[0110] ELISA was used for this test, with J19 and 1E1 serving as positive controls. hIgG4 served as a negative control because it did not bind and therefore had no linear relationship and thus no IC50 value.

[0111] Table 3. IC50 detection of purified antibody blocking HLA-G tetramer on solid-phase carrier.

[0112] IC50 (μg / mL) 3.225 3.291 5.659 3.631 -

[0113] Example 3

[0114] Purified antibody ELISA levels and off-target detection of homology families:

[0115] 1) Dilute LILRA1, LILRA2, LILRA3 and LILRB1 proteins with coating buffer and coat them overnight at 4°C.

[0116] 2) Remove the coating solution the next day and wash with PBS.

[0117] 3) Add blocking buffer and block at 37°C for 1 hour, then wash with PBS. Dilute the antibody to 10 μg / mL.

[0118] 4) Add 100 μL of the 10 μg / mL antibody solution to each of the four blocked ELISA plates, incubate at 30°C for 30 min, and wash with PBST.

[0119] 4) Add HRP-labeled anti-human IgG secondary antibody (purchased from Biolegend) at a dilution ratio of 1:10000, bind at 30℃ for 30 min, and wash with PBST.

[0120] 5) Add 100 μL of TMB chromogenic solution, incubate at 37°C for 10 min or until the blue color is fully developed, then add 100 μL of 1M sulfuric acid to stop the reaction. Read the OD450 on a microplate reader. Data as follows: Figure 6 As shown in Table 4.

[0121] Table 4. Off-target detection of purified antibodies and LILRA1, LILRA2, LILRA3, and LILRB1.

[0122]

[0123]

[0124] Example 4

[0125] Purified antibody cellular level blocking assay:

[0126] 1) Prepare LILRB2-CHOK1 overexpressing cells. Resuscitate the overexpressing cell line in advance and culture it to the logarithmic growth phase. After digestion with trypsin, count the cells using a Countstar cell counter. Transfer the required cells to centrifuge tubes, centrifuge at 500×g for 5 min, and add 2E5 cells / well to a 96-well round-bottom plate.

[0127] 2) Sample incubation: Simultaneously, the antibody concentration was serially diluted from 60 μg / mL to obtain 8 gradient concentration antibody solutions. 50 μL of diluted antibody was added to 50 μL of cell suspension and mixed to obtain a co-incubation solution with 8 serially diluted concentrations of 30 μg / mL. The solution was incubated at 4℃ for 30 min.

[0128] 3) Cell washing: After sample incubation, add 150 μL of 1×PBS to each well, incubate at 600×g for 5 min, aspirate the supernatant, and gently tap the plate wall to separate the cells.

[0129] 4) Ligand incubation: Dilute PE-Labeled Human HLA-G Tetramer Protein to 1 μg / mL with 1×PBS, add 100 μL to each well of cells, and mix gently. Incubate at 4℃ for 30 min.

[0130] 5) Cell washing: After the sample incubation is complete, add 150 μL of 1×PBS to each well, 600×g, 5 min, aspirate the supernatant, gently tap the plate wall to separate the cells, and repeat the washing process once more. For the second wash, add 200 μL of PBS.

[0131] 6) Detection: Add 60 μL / well of 1×PBS to resuspend the cells and detect them using a flow cytometer.

[0132] The results of the purified antibody and LILRB2 overexpression cell line blocking assay are shown in the figure. Figure 7 Flow cytometry was used for this detection, with hIgG4 as a negative control and J19 and 1E1 as positive controls. The blocking rate is shown in the figure. The blocking rate was calculated as: median fluorescence intensity (MFI) of 1-HLA-G tetramer and antibody co-incubation / median fluorescence intensity (MFI) of HLA-G tetramer alone.

[0133] The IC50 results of purified antibody blocking HLA-G tetramer at the cellular level are shown in Table 5.

[0134] Table 5

[0135] IC50 (μg / mL) - 1.204 -

[0136] This invention provides a fully human single-domain antibody that does not bind to its homologous family, blocks HLA-G ligands, and binds to the human LILRB2 protein on the cell surface. Furthermore, this antibody was obtained through panning from a fully human phage display antibody library and is a fully human antibody with low immunogenicity. This invention successfully prepared an antibody (fusion protein) that binds to the human LILRB2 molecule. This antibody exhibits good specificity and high affinity, effectively blocking the binding of LILRB2 to its ligands when it binds to human LILRB2 expressed on the cell surface. This anti-human LILRB2 single-domain antibody is a potential drug for immunotherapy.

[0137] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A fully human single-domain antibody against human LILRB2, characterized in that: The amino acid sequence of the antibody is shown in SEQ ID NO:2, and the nucleic acid sequence encoding the antibody is shown in SEQ ID NO:

4.

2. The antibody as described in claim 1, characterized in that: The antibody targets the human LILRB2 molecule and blocks the binding of LILRB2 to its ligand HLA-G.

3. The antibody as described in claim 1, characterized in that: The antibody targets the human LILRB2 molecule and does not deviate from its family or from unrelated cells.

4. The antibody as described in claim 1, characterized in that: The antibody regulates the JAK / STAT and PI3K / Akt signaling pathways in macrophages, reprograms M2 macrophages into M1 macrophages, and improves the tumor microenvironment.

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