Fully human single-domain antibodies against lilrb1 / lilrb2 and uses thereof

Abstract

The application discloses an anti-LILRB1 / LILRB2 fully human single-domain antibody and a preparation method and application thereof, and obtains the anti-LILRB1 / LILRB2 fully human single-domain antibody which has the function of blocking HLA-G ligand and can be combined with human LILRB1 and LILRB2 proteins on the cell surface. The antibody can be applied to the efficacy of M1 type macrophages existing in a reinforced tumor environment, weakens the inhibition capacity of M2 type macrophages, improves a tumor microenvironment, and reverses tumor immunosuppression mediated by TAM.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a fully human single-domain antibody against LILRB1 / LILRB2 and its applications. Background Technology

[0002] Nanobodies are the smallest antibodies, 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 immunoglobulin-like receptor B1 (LILRB1), also known as ILT2 or CD85j, is an immunosuppressive member of the LILR family of leukocyte immunoglobulin-like receptors. It is expressed in cells such as B cells, T cells, NK cells, dendritic cells, macrophages, and other immune cells. LILRB1 participates in signal transduction mechanisms that suppress immune cell activity by binding to both classical and non-classical MHC class I receptors. Furthermore, various cancer cells have been reported to overexpress MHC class I receptors, such as HLA-G, to evade the immune system. It is anticipated that blocking the binding of LILRB1 to HLA-G will restore suppressed immune cell activity, thereby exhibiting an anti-cancer effect.

[0004] Leukocyte immunoglobulin-like receptor 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. 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. 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 LILRB1 / LILRB2.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the amino acid sequence of the antibody is shown in SEQ ID NO:1 to 5, and the nucleotide sequence of the nucleic acid molecule encoding the antibody is shown in SEQ ID NO:6 to 10.

[0009] As a preferred embodiment of the fully human single-domain antibody against LILRB1 / LILRB2 described in this invention, the antibody is capable of targeting and binding to LILRB1 and LILRB2 molecules.

[0010] As a preferred embodiment of the fully human single-domain antibody against LILRB1 / LILRB2 described in this invention, the antibody is capable of blocking the binding of LILRB1, LILRB2 to their ligand HLA-G.

[0011] The present invention also provides a method for preparing an anti-LILRB1 / LILRB2 fully human single-domain antibody as described in claim 1, comprising,

[0012] Using recombinant human LILRB2 biotin-tagged protein and phage display antibody library as starting materials, LILRB2 was immobilized on magnetic beads through the interaction between streptavidin and biotin, and then incubated with the antibody library.

[0013] After incubation, unbound / weakly bound phages were washed away, and phages bound to LILRB2 protein were washed away. This process was repeated 3 times, with the amount of LILRB2 biotin-tagged protein and washing conditions changed each time. Weakly bound phages were eliminated one by one, while strongly bound phages were retained.

[0014] The host bacteria were infected with strongly binding bacteriophages, plated and cultured overnight to obtain single-clonal colonies, which were then monoclonalized.

[0015] Monoclonal antibody genes were obtained by ELISA screening using monoclonal culture supernatant, and the nucleic acid sequences of the monoclonal antibodies were determined.

[0016] The signal peptide, antibody gene, and coding sequence of human IgG4 Ec are linked together, read in the same frame, and a plasmid is constructed. The plasmid is then transfected to obtain the antibody.

[0017] 293F cells were transfected, and the supernatant was harvested after shaking culture. The fusion protein was then purified from the supernatant using Protein A magnetic bead affinity chromatography.

[0018] The present invention also provides a plasmid comprising the above-described nucleic acid molecules.

[0019] The present invention also provides an expression vector comprising the above-mentioned antibody gene, linked to the coding sequence of the signal peptide, the Fc of human IgG4.

[0020] The present invention also provides a fusion protein comprising the above-mentioned antibody molecule, fused with a heterologous protein, wherein the heterologous protein is the Fc of human IgG4.

[0021] The present invention also provides a conjugate comprising the above-mentioned antibody or fusion protein, conjugated to an effector molecule, wherein the effector molecule is a detectable label, including a fluorescent label, a radiolabel, avidin, biotin, or an enzyme.

[0022] The antibody described in this invention can be used to prepare drugs or drug compositions that inhibit the tumor microenvironment's resistance to immune checkpoint inhibitors.

[0023] The antibody described in this invention can be used to improve the tumor microenvironment and reverse TAM-mediated tumor immunosuppression.

[0024] To provide a more thorough understanding of this invention, the following definitions are provided, including:

[0025] In this article, "anti-LILRB1 / LILRB2 fully human single-domain antibody" refers to an antibody that can bind to both LILRB2 and LILRB1.

[0026] The term "fully human" as used in this article means that the antibodies are entirely encoded by human antibody genes.

[0027] The term "fusion protein" as used in this article refers to the expression product of a fusion gene, or two or more proteins fused together by biological or chemical methods.

[0028] As used in this article, "conjugate" refers to an antibody or protein molecule linked to another small or large molecule through biological or chemical methods.

[0029] This invention uses commercially available recombinant human LILRB2 biotinylated protein and phage display antibody library as starting materials. The LILRB2 biotinylated 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 / weakly bound phages and elute the phages bound to the LILRB2 protein.

[0030] 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 strongly binding phages, plated, and incubated overnight to obtain monoclonal colonies. The phage antibodies that could bind LILRB2 were then monocloned, and the supernatant from the monoclonal culture was used for ELISA screening. The nucleic acid sequences of the monoclonal colonies were then determined.

[0031] Furthermore, the coding sequences of the signal peptide, antibody gene, and human IgG4 Fc were linked and read in the same frame to construct a mammalian expression vector. The vector was transfected into 293F cells, cultured with shaking for 5 days, and the supernatant was harvested. The fusion protein was purified from the supernatant using protein A magnetic bead affinity chromatography.

[0032] Molecular blocking experiments were performed on the purified antibody. HLA-G tetramer was used as the coating substrate. The purified antibody and LILRB2 protein with mouse Fc tag were added together into the ELISA coated plate. Anti-mouse secondary antibody was used as the enzyme-labeled secondary antibody. Colorimetric analysis was performed after adding colorimetric solution.

[0033] The purified antibody was bound to the LILRB2 overexpressing cell line, and the binding was detected by flow cytometry. The LILRB1 protein was used as a coating substrate, the purified antibody was added to the coated plate, and the anti-human secondary antibody was used as the enzyme-labeled secondary antibody. The results were analyzed by adding chromogenic solution, which proved the effectiveness of the fully human single-domain antibody against LILRB1 / LILRB2 of the present invention.

[0034] Beneficial effects of this invention:

[0035] This invention provides a fully human single-domain antibody against LILRB1 / LILRB2. This antibody blocks HLA-G ligand function and can bind to human LILRB1 and LILRB2 proteins on the cell surface. It can be used to enhance the efficacy of M1 macrophages in the tumor environment, weaken the inhibitory ability of M2 macrophages, improve the tumor microenvironment, and reverse TAM-mediated tumor immunosuppression. Attached Figure Description

[0036] 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:

[0037] Figure 1 This is a graph showing the ELISA detection results of the binding of phage crude culture medium and human LILRB2 in Example 2 of the present invention.

[0038] Figure 2 This is a schematic diagram of the LILRB1 / 2 antibody expression vector structure in Example 3 of the present invention.

[0039] Figure 3 This is a diagram showing the SDS-PAGE electrophoresis results of the purified antibody in Example 3 of this invention.

[0040] Figure 4 This is a graph showing the results of the binding performance detection of the purified antibody and LILRB2 overexpressed on the cell surface in Example 4 of the present invention.

[0041] Figure 5 This is a graph showing the ELISA level blocking detection results of the purified antibody in Example 5 of the present invention.

[0042] Figure 6This is a graph showing the ELISA binding performance of the purified antibody with LILRA1 protein in Example 6 of the present invention. Detailed Implementation

[0043] 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.

[0044] 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.

[0045] 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.

[0046] The raw materials used in this invention have been described. Unless otherwise specified, they are all commercially available or can be obtained from the National Center for Biotechnology Information.

[0047] Example 1: Selection of Human VH Antibody Library for Phage Display

[0048] 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.

[0049] 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.

[0050] 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 with 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.

[0051] 4) Library blocking: Synchronize the binding of antigen to magnetic beads, take 10 11PFU phage virus particles (the first round of panning was from the original antibody library, and the latter two rounds of panning were from the amplified products of the panning) were incubated with 1 mL of 1.0% skim milk powder + 1.0% BSA blocking agent by rotation for 1 hour.

[0052] 5) Phage binding: Place the magnetic beads on a magnetic rack, remove the liquid, add the sealed library to the magnetic beads, resuspend and rotate for 1 hour, then remove the liquid.

[0053] 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).

[0054] 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.

[0055] 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.

[0056] 9) Phage amplification: While panning, pick TG1 single clones from mini agar plates and inoculate them into 10 mL of 2×YT medium. Incubate at 37°C with shaking at 250 rpm until mid-log phase (OD600 = 0.5). Add 200 μL of the eluted product obtained from panning and incubate at 37°C for 30 min. Add helper phage M13KO7 and incubate at 37°C for another 30 min. Incubate at 37°C with shaking at 250 rpm for 1 hour. Centrifuge to remove the supernatant and resuspend in 20 mL of 2×YT containing working concentrations of 100 μg / mL ampicillin and 50 μg / mL kanamycin. Incubate overnight at 30°C with shaking at 220 rpm.

[0057] 10) Phage precipitation: Centrifuge at 10000 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. Centrifuge at 10000 rpm for 10 min to obtain phage precipitation. Remove the residual liquid and resuspend the precipitate in 0.2 mL of 0.01M PBS (pH 7.4). Measure the titer as described above.

[0058] 11) Repeat steps 2) to 10) two or three times to obtain phage display antibodies with strong binding force. The amino acid sequences of the antibodies are shown in SEQ ID NO:1 to 5. The following examples all use these five antibodies as the research objects.

[0059] Example 2: Monoclonal ELISA Screening

[0060] 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.

[0061] 2) Single colonies picked from 2×YT-GA100 plates were cultured with shaking until mid-log phase. Helper phage M13KO7 was added, and the plates were incubated at 37°C for 30 min. The plates were then cultured at 37°C with shaking at 220 rpm for 1 hour, followed by centrifugation at 4000 rpm for 15 min. The cells were resuspended in 400 μL of 2×YT containing 100 μg / mL ampicillin and 50 μg / mL kanamycin, and cultured overnight at 30°C with shaking at 220 rpm. The cells were then centrifuged at 4000 rpm for 15 min to precipitate the bacterial cells.

[0062] 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.

[0063] 4) Remove the liquid: wash 5 times with 0.1% PBST, then wash 3 times with PBS to remove the liquid.

[0064] 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.

[0065] 6) Add 100 μL TMB chromogenic solution, incubate at 37°C for 10 min or until the blue color is fully developed, then add 100 μL 1M sulfuric acid to terminate the reaction. Read the OD450 on a microplate reader. (See attached data). Figure 1 See Table 1.

[0066] Table 1. ELISA detection of Phage crude culture medium and human LILRB2.

[0067]

[0068] From Table 1 and Figure 1 The results show that the antibodies obtained in Example 1 of this invention can all bind to LILRB2 coated on the ELISA plate at the Phage level.

[0069] Example 3: Preparation of Humanized LILRB1 / 2 Monoclonal Antibody

[0070] 1) Single colonies picked from 2×YT-GA100 plates were cultured overnight with liquid shaking, and phagemids were extracted using the plasmid extraction method.

[0071] 2) Synthesize primers and amplify the antibody gene coding region displayed by the bacteriophage using PCR.

[0072] 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.

[0073] 4) Prepare sterile, endotoxin-free plasmids. Take 23 μg and dilute with 0.75 mL of diluent (OPM-293CD05 medium). Simultaneously, add 70 μL of transfection reagent (PEI solution) to the 0.75 mL diluent (OPM-293CD05 medium) and mix gently. Add the PEI diluent to the plasmid diluent and immediately mix gently with a pipette. Let stand at room temperature for 15 min, avoiding disturbance.

[0074] 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 (OPM-293CD05), and continue incubating at 85 rpm, 37°C, and 5% CO2 for 72 hours.

[0075] 6) Centrifuge at 10,000 rpm for 10 min and collect the supernatant. Incubate with equilibrated protein A affinity beads by rotation for 1 hour, then place on a magnetic rack and remove the supernatant.

[0076] 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.

[0077] 8) SDS-PAGE detection of purified antibodies ( Figure 3 As can be seen, the antibody purification effect is very good.

[0078] Example 4: Flow cytometry detection of the cell-binding ability of purified antibodies

[0079] 1) The LILRB2 overexpressing cell line (cell binding assay, CHOK1 overexpressing LILRB2) was fully digested with 0.25% trypsin, digestion was stopped with serum, the cells were collected by centrifugation, and a single-cell suspension was prepared by gently pipetting with PBS.

[0080] 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.

[0081] 3) Take 2.5 × 10 5 Cells were collected by centrifugation in 96-well cell culture plates.

[0082] 4) Add 100 μL of the purified antibody from step 8) of Example 3 (10 μg / mL), and use IgG4 as the isotype control group and J-19 and 1E1 as the positive control group. Mix well and incubate at 4°C for 30 min to 1 hour.

[0083] 5) Centrifuge to collect cells and wash them once with 300 μL PBS.

[0084] 6) Add 100 μl of fluorescently labeled (FITC, APC) antibody diluted 200 times with PBS, and react at room temperature in the dark for 20 min.

[0085] 7) Wash the cells once with 1 mL PBS, centrifuge at 1000 rpm for 8 min, and remove the supernatant.

[0086] 8) Resuspend the cells in 100 μl PBS to form a single-cell suspension, and analyze the data using a flow cytometer. Data are as follows: Figure 4 As shown in Table 2.

[0087] Table 2. Detection of the binding performance of purified antibody to LILRB2 overexpressed on the cell surface.

[0088]

[0089] In Table 2, wild-type cells are CHOK1 cells that have not been overexpressed. It can be seen that the antibody of the present invention has excellent binding ability to LILRB2 overexpressing chok1 cells.

[0090] Example 5: Purified antibody ELISA level blocking HLA-G detection

[0091] 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.

[0092] 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.

[0093] 3) Add 100 μL of the mixed liquid to the blocked ELISA plate, bind at 30°C for 30 min, and wash with PBST.

[0094] 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.

[0095] 5) Add 100 μL TMB chromogenic solution, incubate at 37°C for 10 min or until the blue color is fully developed, then add 100 μL 1M sulfuric acid to stop the reaction. Read the OD450 on a microplate reader. Data Figure 5 As shown in Table 3.

[0096] Table 3. IC50 of purified antibody blocking HLA-G by ELISA detection

[0097]

[0098] From Table 3 and Figure 5 It can be seen that the antibody of the present invention can effectively block the binding of LILRB2 to its ligand HLA-G.

[0099] Example 6: Detection of purified antibody levels by ELISA combined with LILRB1

[0100] 1) Dilute LILRB1 protein with coating buffer and coat overnight at 4°C. Remove the coating buffer the next day and wash with PBS.

[0101] 2) Add blocking buffer and block at 37°C for 1 h, then wash with PBS. Simultaneously, dilute the antibody to 10 μg / mL and co-incubate with the coated plate at 30°C for 30 min, then wash with PBST.

[0102] 3) Add 100 μL of the mixed liquid to the blocked ELISA plate, bind at 30°C for 30 min, and wash with PBST.

[0103] 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.

[0104] 5) Add 100 μL TMB chromogenic solution, incubate at 37°C for 10 min or until the blue color is fully developed, then add 100 μL 1M sulfuric acid to stop the reaction. Read the OD450 on a microplate reader. Data as follows: Figure 6 As shown in Table 4.

[0105] Table 4. ELISA Detection of Purified Antibody Binding to LILRB1 Protein

[0106]

[0107]

[0108] from Figure 6 As can be seen from Table 4, the antibody of the present invention also has the ability to bind to LILRB1, indicating that it can effectively bind to LILRB1 and LILRB2 sites, block the binding of this site to its ligand HLA-G, and realize the antibody function.

[0109] In summary, this invention provides a fully human single-domain antibody against LILRB1 / LILRB2 that blocks HLA-G ligand function and can bind to human LILRB1 and LILRB2 proteins on the cell surface. This antibody can be used to enhance the efficacy of M1 macrophages in the tumor environment, weaken the inhibitory ability of M2 macrophages, improve the tumor microenvironment, and reverse TAM-mediated tumor immunosuppression.

[0110] Furthermore, the full-length sequences of the anti-LILRB1 / LILRB2 fully human single-domain antibodies corresponding to the antibodies No. 1 to 5 in the table of this invention are shown in SEQ ID NO: 1 to 5, and the nucleotide sequences of the nucleic acid molecules encoding the antibodies are shown in SEQ ID NO: 6 to 10.

[0111] 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 claims of the present invention.

Claims

1. A fully human single-domain antibody against LILRB1 / LILRB2, characterized in that: The amino acid sequence of the antibody is shown in SEQ ID NO:1, and the nucleotide sequence of the nucleic acid molecule encoding the antibody is shown in SEQ ID NO:6; The antibody targets and binds to LILRB1 and LILRB2 molecules and blocks the binding of LILRB1 and LILRB2 to their ligand HLA-G.

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