CN118580352A

CN118580352A - Anti-LILRB 4 antibodies and uses thereof

Info

Publication number: CN118580352A
Application number: CN202410618816.4A
Authority: CN
Inventors: 陈小凤; 杨金亮; 勾蓝图
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2023-05-20
Filing date: 2024-05-17
Publication date: 2024-09-03

Abstract

The invention relates to the technical field of antibodies, in particular to preparation of an anti-LILRB 4 antibody or a fragment thereof and application thereof. The technical problem to be solved by the present invention is to provide a series of anti-LILRB 4 antibodies or fragments thereof. The anti-LILRB 4 antibody has good affinity and functional activity, can kill tumor cells in vitro, can effectively inhibit the growth of tumors in mice, and has application potential in the aspect of cancer treatment.

Description

Anti-LILRB 4 antibodies and uses thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to preparation of an anti-LILRB 4 antibody and fragments thereof and application thereof.

Background

Antibodies are biological macromolecules composed of heavy chains and light chains, are secreted by B lymphocytes, and play an important role in humoral immunity of organisms. The heavy chain or the light chain of the antibody molecule is respectively composed of a variable region and a constant region, wherein the variable region mainly plays a role in binding a target antigen, and the constant region mainly plays an immune regulation effect. Antibodies can be classified into IgG, igM, igE, igA, igD and the like according to spatial structure and amino acid sequence characteristics, and heavy and light chains can be further subdivided into a plurality of subtypes. For example, human IgG heavy chains can be classified as IgG1, igG2, igG3, and IgG4, and light chains can be classified as kappa and lambda. Since antibodies can specifically and efficiently bind to target molecules or target cells, regulate signaling pathways downstream of target molecules, or kill target cells by immune effects, antibodies can be developed as drugs for disease treatment. Antibodies have been developed currently as an important biotechnological drug, where monoclonal antibodies take up a substantial part, which in turn are predominantly of the IgG type, so that so-called monoclonal antibodies are often referred to as IgG type antibodies.

An IgG type antibody molecule is a tetramer consisting of 2 heavy chains and 2 light chains with a molecular weight of about 150kD via interchain disulfide bonds. Antibody molecules can be divided into variable and constant regions according to structural and functional characteristics, with the variable region acting primarily for antigen binding and the constant region acting primarily for immunological effects and transport. The variable region of an antibody can be further divided into complementarity determining region CDRs and framework regions FRs, wherein each of the heavy or light chain contains 3 CDR regions (heavy chain VH-CDR1, VH-CDR2, VH-CDR3, light chain VL-CDR1, VL-CDR2, VL-CDR 3.) and 4 FR regions flanking the CDR regions (FR 1, FR2, FR3, FR 4). The loop formed by the CDR region is the main part of the binding of the antibody molecule to the antigen, and the FR region forms the supporting structure of the CDR region by spatial folding. The specific recognition of antibodies against different antigen molecules is mainly achieved by amino acid polymorphisms of the 6 CDR regions (VH-CDR 1,2,3 and VL-CDR1, 2, 3) together with conformational polymorphisms of the loop. Because of the high structural similarity of the FR regions of different antibodies, when the CDR regions of one antibody are substituted for the CDR regions of other antibody molecules, if the FR regions of different antibody molecules are appropriately matched, the conformational change of the CDR regions before and after substitution is small, so that the novel variable region formed after substitution can still retain the antigen binding ability, which is the basis of the CDR grafting (CDR GRAFTING) technique. The CDR regions of the murine antibody can be recombined with the human FR regions into humanized antibodies by CDR grafting techniques to replace the CDRs of the human antibody (humanized antibody), and if the FR regions between the human-murine antibody are properly matched, the antigen binding capacity can still be preserved.

Hybridoma technology is currently an important technology in the antibody discovery process. After the antigen is used for immunizing the mouse, the B cells of the mouse develop into germinal centers in the lymphoid tissues, and the affinity of the antibody is gradually improved through somatic cell high frequency mutation (SHM). The mouse spleen cells are isolated and fused with mouse myeloma cells in vitro to form hybridomas, and hybridomas producing the target antibodies can be selected by measuring the antibody activity in the culture supernatants of the hybridoma cells. The hybridoma cells are gradually monoclonal after subcloning, mRNA of the subcloned cells is extracted for antibody variable region gene sequencing, and the amino acid sequence of the antibody variable region can be analyzed. Currently, most antibodies on the market are obtained by hybridoma technology screening. Since hybridoma technology derived antibody molecules complete the affinity maturation process in mice, those B cells that are cross-reactive with the mouse self-protein are cleared in the mouse bone marrow by "negative selection", and thus the antibody molecules are effective in reducing non-specific binding to the self-protein or its similar proteins.

Monoclonal antibodies can exert pharmacological effects through a variety of mechanisms. The antibody variable region can bind to extracellular soluble ligand, block the binding of ligand and receptor and cut off downstream signal transmission induced by ligand, so that the antibody medicine developed by taking immune cell factor as target can improve inflammatory diseases, for example, adalimumab, belimumab, siltuximab and other antibodies are already marketed in batches. The antibody constant region can exert immune regulation effects, including Antibody Dependent Cellular Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC) and the like, so that antibody drugs developed by taking tumor cell surface molecules as targets can kill tumor cells, such as rituximab, trastuzumab, cetuximab and other antibodies on the market have been greatly successful. In addition, novel antibody technologies such as bispecific antibodies and Antibody Drug Conjugates (ADC) derived based on monoclonal antibodies have been developed rapidly in recent years, and have all achieved breakthrough of different degrees. Cancer and inflammatory diseases are currently the most used areas of disease for antibody drugs.

Cancer is a type of disease that is severely threatening to human health and life. With aging and changes in people's lifestyle, the incidence of cancer is increasing and also the trend of younger, so cancer treatment is always a research hotspot in the medical field. Cancer immunotherapy is a successful cancer treatment modality, and has gained widespread acceptance by the medical community. The immune system has the ability to recognize and kill tumor cells, however tumor cells can evade anti-tumor immunity through a variety of mechanisms. Immune checkpoint (checkpoint) molecules can regulate the degree of immune cell activation, playing an important role in the normal activation of the immune system of the body and in preventing autoimmunity. Overexpression of immune checkpoint molecules such as PD-1, PD-L1 and CTLA4 is one of important factors for tumor immune escape, and immune checkpoint inhibitors are hot spots of tumor immunotherapy. At present, although antibody drugs represented by PD-1, PD-L1, CTLA4 and other targets have achieved great success in clinic, the antibody drugs are effective only on a few tumors, but the curative effect on most tumors is still not ideal, so that development of antibody drugs aiming at other new targets is urgently needed to solve the unmet clinical demands.

Leukocyte immunoglobulin-like receptor subfamily B member 4 (Leukocyte Ig-like receptor subfamily B4, LILRB 4), also known as ILT3, is one of the LILR family members expressed in dendritic cells, monocytes, macrophages, precursor mast cells, and endothelial and osteoclasts. In addition, LILRB4 is also highly expressed on the surface of tumor cells such as Acute Myeloid Leukemia (AML), B-cell chronic lymphocytic leukemia (B-CLL), chronic myelomonocytic leukemia (CMML), non-small cell lung cancer (NSCLC), and gastric cancer. LILRB4 has a number of functionally distinct ligands, mainly including CD166, apoE, fibronectin (fibreonectin), CNTFR, and the like. APOE is an important ligand for LILRB4, which upon binding to LILRB4 recruits SHP-2 to transmit an inhibitory signal, inhibiting lymphocyte growth and cytokine release. Studies show that LILRB4 can be used as a target point of tumor immunotherapy, and the APOE/LILRB4 signal pathway inhibitor can inhibit the growth of tumors.

Disclosure of Invention

The invention aims to solve the technical problem of providing a series of anti-LILRB 4 antibodies or antibody fragments, which have good affinity and functional activity, can kill tumor cells in vitro, can effectively inhibit the growth of tumors in mice, and have application potential in the aspect of cancer treatment.

The present invention provides a series of anti-LILRB 4 antibodies or fragments thereof.

The present invention first provides an anti-LILRB 4 antibody or fragment thereof, which satisfies at least one of the following:

contains at least one of heavy chain complementarity determining regions VH-CDR1, VH-CDR2 or VH-CDR3 with amino acid sequences shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively;

Or alternatively

Contains at least one of light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 with amino acid sequences shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively.

Further, the above-mentioned anti-LILRB 4 antibody or fragment thereof contains a heavy chain variable region VH having an amino acid sequence shown in SEQ ID NO. 1.

Or the above-mentioned anti-LILRB 4 antibody or fragment thereof contains the light chain variable region VL having the amino acid sequence shown in SEQ ID NO. 5.

Further, the above-mentioned anti-LILRB 4 antibody or fragment thereof contains a heavy chain variable region VH having an amino acid sequence shown as SEQ ID NO.1 and a light chain variable region VL having an amino acid sequence shown as SEQ ID NO. 5.

An antibody comprising a heavy chain variable region VH having the amino acid sequence shown in SEQ ID NO.1 and a light chain variable region VL having the amino acid sequence shown in SEQ ID NO.5 was designated B4-A11-2.

The invention also provides an anti-LILRB 4 antibody or fragment thereof, which satisfies at least one of the following:

contains at least one of heavy chain complementarity determining regions VH-CDR1, VH-CDR2 or VH-CDR3 with amino acid sequences shown as SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 respectively;

Or alternatively

Contains at least one of light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 with amino acid sequences shown as SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16 respectively.

Further, the above-mentioned anti-LILRB 4 antibody or fragment thereof contains a heavy chain variable region VH having an amino acid sequence shown in SEQ ID NO. 9.

Or the above-mentioned anti-LILRB 4 antibody or fragment thereof contains the light chain variable region VL having the amino acid sequence shown in SEQ ID NO. 13.

Further, the above-mentioned anti-LILRB 4 antibody or fragment thereof contains a heavy chain variable region VH having an amino acid sequence shown as SEQ ID NO.9 and a light chain variable region VL having an amino acid sequence shown as SEQ ID NO. 13.

An antibody comprising a heavy chain variable region VH having the amino acid sequence shown in SEQ ID NO.9 and a light chain variable region VL having the amino acid sequence shown in SEQ ID NO.13 was designated B4-7C2.

The invention also provides another anti-LILRB 4 antibody or fragment thereof, which satisfies at least one of the following:

Contains at least one of heavy chain complementarity determining regions VH-CDR1, VH-CDR2 or VH-CDR3 with amino acid sequences shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20 respectively;

Or alternatively

Contains at least one of light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 with amino acid sequences shown as SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24 respectively.

Further, the above-mentioned anti-LILRB 4 antibody or fragment thereof contains a heavy chain variable region VH having an amino acid sequence shown as SEQ ID NO. 17.

Or the above-mentioned anti-LILRB 4 antibody or fragment thereof contains the light chain variable region VL having the amino acid sequence shown in SEQ ID NO. 21.

Further, the above-mentioned anti-LILRB 4 antibody or fragment thereof contains a heavy chain variable region VH having an amino acid sequence shown as SEQ ID NO.17 and a light chain variable region VL having an amino acid sequence shown as SEQ ID NO. 21.

An antibody comprising a heavy chain variable region VH having the amino acid sequence shown in SEQ ID NO.17 and a light chain variable region VL having the amino acid sequence shown in SEQ ID NO.21 was designated B4-12A5. Furthermore, the corresponding CDR regions of the above antibodies or fragments thereof are spliced with FRs of the framework regions of a humanized antibody to form a humanized antibody or fragment thereof.

Further, the above antibody or fragment thereof contains a heavy chain variable region VH having an amino acid sequence shown in SEQ ID NO. 25.

Or the antibody or the fragment thereof contains a light chain variable region VL of which the amino acid sequence is shown in SEQ ID NO. 26.

Further, the above antibody or fragment thereof contains a heavy chain variable region VH having an amino acid sequence shown in SEQ ID NO.25 and a light chain variable region VL having an amino acid sequence shown in SEQ ID NO. 26.

A humanized antibody comprising a heavy chain variable region VH having the amino acid sequence shown in SEQ ID NO.25 and a light chain variable region VL having the amino acid sequence shown in SEQ ID NO.26 is a humanized version of antibody B4-7C2, designated antibody hB4-7C2-14.

Further, the heavy chain constant region of the antibody may be derived from at least one of the constant regions of human immunoglobulin IgG1, igG2, igG3, igG4, igM, igE, igA, igD heavy chains: or the light chain constant region may be derived from at least one of the constant regions of a human immunoglobulin kappa or lambda light chain.

Further, the above antibody fragment may be Fab (antigen binding fragment) or scFv (single-CHAIN FRAGMENT variable).

The invention also provides nucleic acid molecules encoding the above-described anti-LILRB 4 antibodies or fragments thereof.

The present invention also provides a recombinant vector comprising a nucleic acid molecule encoding the above antibody against LILRB4, which may be a plasmid or a viral vector.

The invention also provides a cell comprising the recombinant vector, wherein the cell can be a eukaryotic cell or a prokaryotic cell.

The invention also provides the application of the anti-LILRB 4 antibody in killing tumor cells.

The antibody of the present invention can be prepared into various forms of pharmaceutical preparations according to conventional techniques of pharmacy, and liquid injections and freeze-dried injections are more preferable.

The antibodies of the invention may be formed into pharmaceutical compositions with other drugs that may be used in the treatment of diseases along with other therapeutic methods including chemotherapy, radiation therapy, biological therapy, and the like.

The invention has the beneficial effects that:

The invention provides a series of anti-LILRB 4 antibodies, which have good affinity and functional activity, can kill tumor cells in vitro, can effectively inhibit the growth of tumors in mice, and have application potential in the aspect of cancer treatment.

Drawings

FIG. 1 binding activity of hybridoma antibodies to CHO/LILRB4 cells.

FIG. 2 binding activity of hybridoma antibodies to CHO/LILRB3 cells.

FIG. 3 antitumor effect of murine antibody B4-7C2 in immunocompetent mice

FIG. 4 anti-tumor effect of chimeric antibody B4-7C2-h in a mouse model of human PBMC immune reconstitution.

FIG. 5 SPR binding kinetics analysis of humanized antibody hB4-7C 2-14.

FIG. 6 analysis of THP-1 cell binding activity of humanized antibody hB4-7C2-14 and chimeric antibody B4-A11-2-h.

FIG. 7 ADCC assay of humanized antibody hB4-7C 2-14.

FIG. 8 in vitro tumor cell killing activity of humanized antibody hB4-7C 2-14.

FIG. 9 in vitro tumor cell killing activity of chimeric antibody B4-A11-2-h.

FIG. 10 anti-tumor effects of humanized antibodies hB4-7C2-14 and chimeric antibodies B4-A11-2-h in a mouse model of human PBMC immunoreconstruction.

Detailed Description

The anti-LILRB 4 antibody is obtained by screening by adopting a hybridoma technology. The human LILRB4 protein is mixed with an adjuvant and immunized into a mouse, after the serum titer is qualified, spleen cells of the mouse are separated, and the spleen cells are fused with myeloma cells of the mouse in vitro and cultured to obtain cell culture supernatant containing the antibody. First, hybridoma antibodies having LILRB4 binding activity were screened by affinity ELISA,

Further, SPR was used to screen for hybridoma antibodies with good binding kinetics, and flow cytometry was used to screen for hybridoma antibodies with good binding activity to CHO-K1/LILRB4 cells. The hybridoma antibodies further showed binding activity to LILRB3 in addition to good binding activity to LILRB4 by flow cytometry analysis of cross-binding to LILR proteins of the same family, antibodies B4-7C2, B4-12 A5. Antibody B4-A11-2 has good binding activity only to LILRB 4.

In order to obtain the amino acid sequence of the hybridoma antibody, the invention sequences the antibody mRNA of the hybridoma to obtain the variable region sequence of the hybridoma antibody, prepares a recombinant monoclonal antibody and performs functional activity verification. The antibody variable region mRNA gene is amplified by PCR through an upstream signal peptide primer and a downstream constant region primer, and further sequenced to obtain the antibody variable region gene. The expression vector is constructed by splicing and fusing the murine antibody variable region and the antibody constant region. The HEK293 cells are transfected by expression plasmids containing antibody genes for transient expression, and the purity and the content of the antibodies are confirmed by protein G affinity purification and SDS-PAGE and spectrophotometry for further verification. The obtained recombinant monoclonal antibodies were subjected to verification of cell binding activity and binding kinetics, respectively, to ensure the activity of the recombinant monoclonal antibodies.

The invention further examines the anti-tumor effect of the antibody B4-7C2 in the mouse body, and proves that the murine antibody B4-7C2 can inhibit the tumor growth in the immune sound mouse body, and the chimeric antibody B4-7C2-h can inhibit the tumor growth in the immune reconstruction mouse body of the human PBMC.

The invention further splices the CDR region of the murine antibody B4-7C2 with the framework region FR of the humanized antibody to construct the humanized antibody. The obtained humanized antibody was subjected to a binding kinetics analysis, from which the humanized antibody hB4-7C2-14 was preferentially extracted. Flow cytometry analysis showed that the maximum binding signal was higher than that of the LILRB4 monoclonal antibody B4-A11-2-h, since the humanized antibody hB4-7C2-14 could bind to both LILRB3 and LILRB4 on the surface of THP-1 cells. Meanwhile, the luciferase reporter cell analysis system shows that the humanized antibody hB4-7C2-14 also has obvious ADCC signals.

The invention further analyzes the tumor-inhibiting effect of the antibodies in vitro and in vivo. The humanized antibody hB4-7C2-14 and the chimeric antibody B4-A11-2-h have obvious tumor cell killing activity in vitro and have good anti-tumor effect in a human PBMC immune reconstruction mouse model.

The anti-LILRB 4 antibody can be modified into Fab (antigen binding fragment), scFv (single-CHAIN FRAGMENT variable) and other antibody fragments by conventional gene recombination technology. Antibody fragments such as Fab, scFv and the like have smaller volume and strong tissue permeability, and have unique advantages in some application fields. Fab is a heterodimer consisting of a heavy chain variable region-constant region 1 (VH-CH 1) and a light chain variable region-constant region (VL-CL) with a molecular size of 1/3 of an IgG molecule. Because of the absence of the Fc segment, the Fab-induced immune effect is significantly reduced compared to IgG and cytokine release is weaker. Antibody drugs such as abciximab, ranibizumab and the like, which are currently in Fab-like structures, have been approved for the market. The scFv is formed by fusing VH and VL and a connecting peptide linker between the VH and the VL, the molecular size is only 1/6 of that of the IgG, and the scFv has the characteristics of strong tissue permeability, short half-life and the like, has unique advantages in the fields of imaging diagnosis and some treatments, and a bispecific antibody blinatumomab based on the scFv is also approved to be marketed. The antibody fragments may further be fused to other proteins, or conjugated to other small molecules, for diagnosis and treatment of diseases by targeted delivery.

The anti-LILRB 4 antibodies of the invention may be further improved in affinity by engineering techniques to mutate amino acids in the CDR regions. The antibody CDR regions play a critical role in the binding of antibodies to antigens, wherein the amino acids may interact with the amino acids of the antigen via hydrogen bonding, ionic bonding, van der waals forces, and the like. By mutating amino acids in the CDR regions of an antibody, the interaction of the CDRs with the antigen can be further enhanced, thereby increasing the affinity of the antibody. The application of the antibody library technology in the aspect of antibody affinity evolution is mature, an antibody mutation library can be established through strategies such as alanine hot spot mutation, error-prone PCR and the like, high-throughput screening of mutant antibodies is carried out, and the affinity evolution of the antibodies is realized in vitro.

The antibodies of the invention can be expressed using stable cell lines for large-scale production of large amounts of proteins. The gene encoding the antibody amino acid can be obtained by conventional gene recombination technology, and can be inserted into an expression vector after DNA sequence optimization, synthesis and PCR amplification. The vectors used may be plasmids, viruses or gene fragments which are customary for molecular biology. A protein secretion signal peptide gene is added at the front end of a DNA sequence for encoding the antibody so as to ensure that the antibody can be secreted outside cells. The vector sequence contains a promoter for gene expression, a protein translation initiation and termination signal, polyadenylation (polyA) and other elements. The vector contains antibiotic resistance genes and replication elements to facilitate replication of the vector in a host cell, such as a bacterium, for vector preparation. In addition, a selectable gene may be included in the vector to facilitate selection of stably transfected host cells for construction of stably expressed cell lines.

After construction of the vector containing the DNA sequence encoding the antibody, the vector may be used to transfect or transform a host cell for expression of the corresponding protein. There are various expression systems that can be used to express antibodies, which can be eukaryotic cells, or prokaryotic cells, including mammalian cells, insect cells, yeast, bacteria, and the like. Mammalian cells are the preferred system for expressing the protein because of the ease of inclusion bodies when prokaryotic cells express intact antibodies. There are various mammalian cells that can be used for large-scale expression of antibodies, such as CHO cells, HEK293 cells, NS0 cells, COS cells, etc., all of which are included among the cells that can be used in the present invention. Recombinant vectors containing genes encoding antibodies can be transfected into host cells by a variety of methods including electroporation, lipofection, and calcium phosphate transfection.

A preferred method of protein expression is by stably transfected host cell expression comprising a selectable gene. For example, after stably transfecting a host cell lacking Neomycin resistance with a recombinant vector containing a Neomycin (Neomycin) resistance gene, the concentration of Neomycin may be increased in the cell culture broth to select a stable cell strain with high expression; for example, after stably transfecting a host cell lacking DHFR with a recombinant vector containing the dihydrofolate reductase (DHFR) gene, the concentration of Methotrexate (MTX) may be increased in the cell culture medium to select for stable cell lines with high expression.

Other expression systems besides mammalian cells, such as insect cells, yeast, bacteria, etc., may also be used to express the antibodies or fragments thereof of the present invention, and they are also encompassed by the host cells that can be used in accordance with the present invention. The protein expression level of these expression systems is in some cases higher than that of mammalian cells, but inclusion bodies are easily formed, and thus further protein renaturation is required.

Antibodies of the invention may also be carried and expressed using viral vectors including, but not limited to, adenovirus vector (adenoviral vectors), adeno-associated virus vector (adeno-associated viral vectors), retrovirus vector (retroviral vectors), herpes simplex virus vector (herpes simplex virus-based vectors), lentivirus vector (LENTIVIRAL VECTORS), and the like.

The anti-LILRB 4 antibodies of the invention can be used for detection of LILRB4, including ELISA and flow cytometry. The anti-LILRB 4 antibody of the invention shows no binding signal with LILRB4 negative cell components of various different tissue sources through ELISA and flow cytometry analysis, which indicates that the antibody has good specificity.

The anti-LILRB 4 antibody can kill tumor cells in vitro, can effectively inhibit the growth of tumors in mice, and has application potential in the aspect of cancer treatment.

The following examples illustrate the discovery, preparation, testing and use of antibodies of the invention. The content and use of the invention is not limited to the scope of the embodiments.

Example 1 mouse immunization

Female BALB/c mice, 6-8 weeks old and weighing about 20g, were used as immunization hosts and were subjected to antigen immunization after one week of adaptive feeding. Expression of purified LILRB4-ECD-His (human ectodomain C-terminal fusion 6 XHis tag) was formulated to 1mg/mL with PBS (pH 7.2), and after filtration through a 0.22 μm filter, 50. Mu.L was mixed well with 50. Mu.L of immunoadjuvant (QuickAntibody) and injected into the calf muscle of the hind leg of mice. The immunization was boosted 1 time on day 21 in the same manner, and serum antibody titer was measured by tail vein blood sampling on day 35. The human IgG1Fc fusion protein was coated onto an ELISA plate (50 ng/well) and the mouse serum antibody titer was determined by ELISA. Mice with antibody titers greater than 32000 were given an antigen impact once and spleen cell fusion was performed after 3 days.

Example 2 spleen cell fusion

After the mice were euthanized, spleens were isolated under aseptic conditions, spleen cell suspensions were prepared using a 70 μm screen, and washed 2 times with basal medium for cell counting. SP2/0 was mixed with splenocytes in a 1:3 ratio, the supernatant was discarded after centrifugation, 1mL of PEG preheated at 37℃was added dropwise over 1min, the mixture was allowed to stand at 37℃for 90s, and then 20mL of basal medium preheated at 37℃was added over 6 min. Cells were collected by centrifugation (room temperature, 800rpm,3 min), resuspended in 20mL of HAT medium pre-warmed at 37℃and the fused cells were added to 96-well cell culture plates at a density of 1X 10 ⁵ spleen cells/well, cultured in a carbon dioxide cell incubator until the cells reached a confluence of 70% or more, and the culture supernatant was subjected to ELISA detection.

Example 3 affinity ELISA screening

LILRB4-hFc solution with concentration of 1. Mu.g/mL was prepared with PBS, and the ELISA plate (50. Mu.L/well) was added and coated overnight at 4 ℃. PBST plates were washed 3 times, 5% BSA blocking solution (200. Mu.L/well) was added and incubated for 2h at 37 ℃. PBST plates were washed 3 times, hybridoma cell culture supernatants (50. Mu.L/well) were added, and incubated at 37℃for 1h. PBST plates were washed 3 times, added with HRP-goat anti-mouse solution (50. Mu.L/well) diluted 1:5000, and incubated for 1h at 37 ℃. PBST plates were washed 3 times, added with ready-to-use TMB color development solution (100. Mu.L/well), and incubated at 37℃for 5-10min in the dark. The color development was stopped by adding 2M H ₂SO₄ (100. Mu.L/well) and the OD was measured at 450 nm. The positive original clone obtained was subjected to the next screening.

Example 4 SPR screening

The appropriate coupling amount was calculated according to the formula rl= (rmax× MWligand)/(sm× MWANALYTE), and anti-mouse anti was coupled to CM5 chip using amine coupling kit. Hybridoma cell culture supernatants were captured onto chips and the response values of the flow through LILRB4-HSA-His (fusion of human albumin and 6 xhis tag at the C-terminus of the human LILRB4 extracellular domain) in the channel were detected using Biacore 8K. Data fitting was performed by Evaluation Software to obtain binding curves and kinetic parameters. The hybridoma antibodies with good binding kinetics are preferably selected for further screening.

Example 5 hybridoma subcloning

The hybridoma cells were subcloned by limiting dilution. Hybridoma cells secreting antibody were collected and counted, diluted with complete medium, added to 96-well cell culture plates at a cell density of 0.5 cells/well for continued culture, and the remaining cells were expanded for seed retention. After 10 days of subcloning, the culture supernatants of the monoclonal wells were taken for affinity ELISA and SPR validation, respectively. And taking positive clones, continuing to perform secondary subcloning, and continuing to verify the secondary subcloning according to the screening mode of the primary subcloning. The hybridoma subclones with good binding kinetics are preferably selected for further screening.

Example 6 cell binding Activity screening

The CHO-K1 cell line (CHO-K1/LILRB 4) stably expressing LILRB4 was collected and the number of cells in each group was 1X 10 ⁶, and the cell suspension was aspirated into U-type 96-well plates. Cells were washed 1 time with PBS and centrifuged at 1200rpm for 5min, and the supernatant was discarded. The cells were resuspended in 100. Mu.L of hybridoma cell culture supernatant, PBS, isotype antibody mIgG (2. Mu.g/mL), and anti-LILRB 4 positive antibody (2. Mu.g/mL), respectively, and incubated on ice for 60min. After incubation was completed, cells were collected by centrifugation, washed with 200 μl PBS, and repeated 2 times. 100. Mu.L of Alexa Fluor-488-labeled goat anti-mouse IgG (H+L) (1:200 dilution) was added to the cell wells, resuspended, and incubated on ice for 40min in the absence of light. After incubation was completed, cells were collected by centrifugation, washed with 200 μl PBS, and repeated 2 times. Finally, the cells were resuspended in 130. Mu.L PBS and analyzed by flow cytometry. Hybridoma antibodies with good cell binding activity are preferentially selected for further screening.

Example 7, LILR Cross-Linked Screen for proteins of the same family

CHO-K1 cell lines stably expressing LILR family proteins, respectively, were collected, at CHO-K1/LILRA1、CHO-K1/LILRA2、CHO-K1/LILRA3、CHO-K1/LILRA4、CHO-K1/LILRA5、CHO-K1/LILRA6、CHO-K1/LILRB1、CHO-K1/LILRB2、CHO-K1/LILRB3、CHO-K1/LILRB4、CHO-K1/LILRB5, cells per group at 1×10 ⁶, and cell suspensions were pipetted into U-shaped 96 well plates. Cells were washed 1 time with PBS and centrifuged at 1200rpm for 5min. The cell pellet was resuspended in 100. Mu.L of hybridoma cell culture supernatant, PBS, isotype antibody mIgG (2. Mu.g/mL), anti-LILRB 4 positive antibody (2. Mu.g/mL), and incubated on ice for 60min. After incubation was completed, cells were collected by centrifugation, washed with 200 μl PBS, and repeated 2 times. 100. Mu.L of Alexa Fluor-488-labeled goat anti-mouse IgG (H+L) (1:200 dilution) was added to the cell pellet, resuspended, and incubated on ice for 40min in the absence of light. After incubation was completed, cells were collected by centrifugation, washed with 200 μl PBS, and repeated 2 times. Finally, the cells were resuspended in 130. Mu.L PBS and analyzed by flow cytometry. The results showed that antibodies B4-A11-2, B4-7C2, B4-12A5 had good binding activity to CHO-K1/LILRB4 (see FIG. 1). In addition, antibodies B4-7C2, B4-12A5 and CHO-K1/LILRB3 also showed good binding activity (see FIG. 2).

Example 8 acquisition of antibody variable region sequences

Hybridoma subclones were collected and RNA was extracted using Trizol method. Reverse transcription is carried out by taking the extracted RNA as a template to obtain cDNA. The heavy and light chain variable regions of the antibodies were PCR amplified using degenerate primers (Novagen Ig-PRIMER SETS), respectively, and the PCR amplified products were detected by agarose gel electrophoresis. And obtaining a target DNA fragment by adopting a gel recovery kit, and then performing TA cloning to construct a recombinant plasmid. And (3) transforming the recombinant plasmid into competent cells by adopting a heat shock method, and plating to perform blue and white spot screening. White single colony is picked up to 0.5mL LB liquid culture medium, shake culture is carried out for 3h at 37 ℃ and 220rpm, and bacterial liquid is taken and sent for sequencing. The amino acid sequences of the variable regions of the anti-LILRB 4 antibodies B4-A11-2, B4-7C2 and B4-12A5 are shown in Table 1.

TABLE 1 amino acid sequences of variable regions of antibodies

EXAMPLE 9 construction and preparation of recombinant monoclonal antibodies

And splicing the heavy chain variable region gene fragment and the light chain variable region gene fragment with the signal peptide, the mouse heavy chain (IgG 2 a) constant region gene fragment and the light chain (kappa) constant region gene fragment by adopting overlap PCR, and sequencing and identifying. And (3) inserting the correctly spliced heavy chain genes and light chain genes of the antibody into pTT5 plasmid respectively, transfecting the recombinant plasmid into HEK293 cells by adopting a PEI method, performing serum-free suspension culture, and transiently expressing the antibody. Cell supernatants cultured for 7 days were collected, filtered through a 0.22 μm filter, and the antibodies were purified by protein G affinity chromatography. The antibody is ultrafiltered and replaced to PBS solution, reduced SDS-PAGE and NanoDrop 2000 are adopted to identify the purity and concentration of the antibody, and the antibody is subpackaged and stored at-80 ℃ for standby. The obtained recombinant monoclonal antibodies were subjected to verification of cell binding activity and binding kinetics, respectively, to ensure the activity of the recombinant monoclonal antibodies.

Example 10 specificity detection

Culturing and collecting various cell lines, taking different anti-LILRB 4 antibodies as primary antibodies and FITC-labeled goat anti-mouse IgG (H+L) as secondary antibodies, and detecting the binding condition of the antibodies and cell surface proteins by adopting Flow Cytometry (FCM). The results showed that none of the antibodies B4-A11-2, B4-7C2 showed binding signals to HT-29, LO2, PANC-1, hela, MGC-803, hepG2, jurkat cells, whereas it showed a clear binding signal to THP-1 cells. On the other hand, a variety of cell lines were cultured, collected, cell-lysed samples were prepared using RIPA lysate and an ultrasonic cytobreaker, coated onto an elisa plate (500 ng/well), and coated with lilrb4+thp-1 cell lysate as a positive control. Different anti-LILRB 4 antibodies are used as primary antibodies, HRP-goat anti-mouse antibodies are used as secondary antibodies, and ELISA is used for detecting the binding condition of the antibodies and cell lysis components. The results showed that none of the antibodies B4-A11-2 and B4-7C2 showed binding signals to LO2, hela, MGC-803, hepG2, jurkat, K562, raji cells, while showing significant binding signals to LILRB4+THP-1 cells.

EXAMPLE 11 antitumor Effect of murine antibody B4-7C2 in immunocompetent mice

Mice colon cancer cells (CT 26-hLILRB 4) from stable human LILRB4 were inoculated subcutaneously on the backs (3.5X10 ⁶/mouse) of 6 week old female BALB/c mice and randomly grouped when tumor volumes reached 50-100mm ³. The drug was administered to the abdominal cavity (10 mg/kg) on days 0,3, 6, 9, and 12, respectively. The murine antibody B4-7C2 was prepared by purification from mouse ascites, and murine isotype antibody (mIgG) was used as a control. Tumor volume size (calculated as v=1/2×long×short×short) was measured every 3 days, and the body weight and state of the mice were monitored. The results showed that murine antibody B4-7C2 was effective in inhibiting tumor growth (see fig. 3).

EXAMPLE 12 anti-tumor Effect of chimeric antibody B4-7C2-h in mouse model of human PBMC immune reconstitution

Human PBMC (8X 10 ⁶/mouse) were subcutaneously inoculated in combination with human acute myelogenous leukemia cells THP-1 (4X 10 ⁶/mouse) on the backs of NOD mice, and when tumor volumes reached 50-100mm ³, the mice were randomly grouped. The chimeric antibodies B4-7C2-h were administered intraperitoneally (10 mg/kg) on days 0, 3, 6, 9, and 12, respectively, with human (murine-human chimeric antibody, human hIgG1 kappa type constant region) as a control. Tumor volume size (calculated as v=1/2×long×short×short) was measured every 3 days, and the body weight and state of the mice were monitored. The results showed that chimeric antibody B4-7C2-h was effective in inhibiting tumor growth (see FIG. 4).

EXAMPLE 13 humanization of murine antibody B4-7C2

The variable region of antibody B4-7C2 was homologously modeled according to the antibody structure in the PDB database, and the CDR regions were determined based on the amino acid primary sequence characteristics and the spatial conformation of the variable region. The antibody variable region is compared with amino acid sequences encoded by human antibody germ line genes V and J, and 5V genes and 1J gene in an IGHV library are preferably transplanted and spliced with heavy chain CDR according to the factors of the consistency, the similarity, the conservation and the like of the frame region FR to form 5 different humanized heavy chain variable regions. Similarly, it is preferred that 4V genes and 1J gene in the IGKV library be graft spliced with the light chain CDRs to form 4 different humanized light chain variable regions. Humanized heavy and light chain variable regions were fused to human IgG1 kappa heavy and light chain constant regions, respectively, to form complete heavy and light chains, and 5 heavy chains were combined with 4 light chains to construct 20 different humanized antibodies. By Biacore binding kinetics analysis, humanized antibodies hB4-7C2-14 showed good binding kinetics with both LILRB4 and LILRB3 (see fig. 5), with binding kinetics parameters as shown in table 2, and amino acid sequences of the variable regions as shown in table 3.

TABLE 2 binding kinetics parameters

TABLE 3 amino acid sequences of variable regions of antibodies

Example 14 analysis of THP-1 cell binding Activity of humanized antibody hB4-7C2-14 and chimeric antibody B4-A11-2-h.

THP-1 cells were collected, 1X 10 ⁶ cells per group, and the cell suspension was pipetted into a U-shaped 96-well plate. Cells were washed 1 time with PBS and centrifuged at 1200rpm for 5min. Humanized antibody hB4-7C2-14, chimeric antibody B4-A11-2-h, isotype antibody hIgG solution concentration 80000ng/mL、40000ng/mL、20000ng/mL、10000ng/mL、5000ng/mL、2500ng/mL、1250ng/mL、625ng/mL、312.5ng/mL、156.25ng/mL、78.125ng/mL、39.0625ng/mL、19.53125ng/mL, were prepared separately, 100. Mu.L of antibody solution was added to 96-well plates containing THP-1 cells and incubated on ice for 60min. After the incubation was completed, the cells were collected by centrifugation and washed 2 times with 200 μl PBS. 100. Mu.L of Alexa Fluor-488-labeled goat anti-mouse IgG (H+L) (1:200 dilution) was added to the cell wells, resuspended, and incubated on ice for 40min in the absence of light. After the incubation was completed, the cells were collected by centrifugation and washed 2 times with 200 μl PBS. Finally, the cells were resuspended with 130. Mu.L PBS and analyzed by flow cytometry. Flow cytometry analysis showed that humanized antibody hB4-7C2-14 and antibody B4-A11-2-h (murine-human chimeric antibody, constant region of human hIgG1 kappa type) had good cell binding activity to THP-1 cells and exhibited concentration-dependent characteristics. Since the humanized antibody hB4-7C2-14 can bind to both LILRB3 and LILRB4 on the surface of THP-1 cells, its maximum binding signal is higher than that of the LILRB4 monoclonal antibody B4-A11-2-h (see FIG. 6).

EXAMPLE 15 ADCC Effect assay of humanized antibody hB4-7C2-14

THP-1 cells were collected as target cells and the density was adjusted to 1.1X10 ⁶/mL. The cell suspension was added to 96-well plates at 90 μl per well, and 3 multiplex wells were placed per group. Preparation of humanized antibody hB4-7C2-14 and isotype antibody hIgG solution at concentration 20000ng/mL,4000ng/mL,800ng/mL,160ng/mL,32ng/mL,6.4ng/mL,1.28ng/mL,0.256ng/mL,0.0512ng/mL,0.01024ng/mL,. Mu.L of antibody solution was added to 96-well plates while medium negative control was set. The cell density was adjusted to 2.2X10 ⁶ cells/mL using Jurkat-Lucia-NFAT-CD16a luciferase reporter cell strain as effector cells, and 90. Mu.L of the cell suspension was added to a 96-well plate. After mixing the 96-well plates, placing the mixture in a cell culture box for 6 hours. After the incubation, the 96-well plate was removed and left at room temperature for 30min to equilibrate to room temperature. 20 mu L of cell culture supernatant is sucked, quanti-LUC detection reagent (50 mu L/hole) is added, the reaction is allowed to stand for 20s, and a multifunctional enzyme-labeled instrument is adopted for luminous signal detection. The results showed that the humanized antibody hB4-7C2-14 showed a significant ADCC signal (see fig. 7).

Example 16 in vitro tumor cell killing Activity of humanized antibody hB4-7C2-14 and chimeric antibody B4-A11-2-h

THP-1 cells were collected as target cells, and the cell suspension was added to a 96-well plate at 50. Mu.L per well, 4.5X10 ⁴ cells/well. Antibody solution concentrations (0.2. Mu.g/mL, 2. Mu.g/mL, 20. Mu.g/mL) and isotype antibody hIgG (20. Mu.g/mL) were prepared and gently mixed with the target cell suspension, and incubated at 37℃in a 5% CO2 incubator for 30min. Effector cells PBMC to target cells THP-1 ratio E: t=10:1, 100uL of PBMC cell suspension (4.5×10 ⁵/well) was added per well and incubated at 37 ℃ in a 5% CO2 incubator for 20h. After incubation was completed, cells were collected by centrifugation, washed with 1mL of PBS, 400g, and centrifuged at 4℃for 5min. To the cell pellet, 100. Mu.L PBS was added to resuspend the cells while 0.3. Mu.L dead living cell dye (Fixable Viability Stain 450,450) was added. Incubate at room temperature in the dark for 10min. After incubation was completed, cells were collected by centrifugation, washed with 1mL PBS, and repeated 2 times. Finally, 200. Mu.L PBS was used to resuspend the cells for flow cytometry analysis. The results show that humanized antibodies hB4-7C2-14 (hIgG 1 kappa type) (see FIG. 8) and B4-A11-2-h (mouse-human chimeric antibody, constant region is human hIgG1 kappa type) (see FIG. 9) all have good in vitro killing effect.

EXAMPLE 17 anti-tumor Effect of humanized antibodies hB4-7C2-14 and chimeric antibodies B4-A11-2-h in human PBMC immunoreconstruction mouse model

Human PBMC (2.7X10 ⁶/mouse) were tail-injected into NSG mice 5-6 weeks old and 7 days later were subcutaneously inoculated with human acute myelogenous leukemia cells THP-1 (8X 10 ⁶/mouse) on the backs of the mice. When the tumor volume reached 50-100mm ³, mice were randomly grouped and given intraperitoneally (10 mg/kg) on days 0, 3, 7, 10, 13, respectively. Humanized antibody hB4-7C2-14 (capable of recognizing LILRB3/4 simultaneously) was human IgG1 kappa type (hIgG 1 kappa), the constant region of antibody B4-A11-2-h (recognizing only LILRB 4) was human (mouse-human chimeric antibody, constant region was human hIgG1 kappa type), and human isotype antibody (hIgG) was used as a control. Tumor volume size (calculated as v=1/2×long×short×short) was measured every 3 days, and the body weight and state of the mice were monitored. The results showed that the humanized antibody hB4-7C2-14 and the chimeric antibody B4-A11-2-h can effectively inhibit the growth of THP-1 tumor, and that the growth inhibition effect of hB4-7C2-14 on THP-1 tumor is stronger than that of B4-A11-2-h (see FIG. 10).

Sequence listing

SEQ ID NO.1

Antibody B4-A11-2 heavy chain variable region VH amino acid sequence

QVQLQQPGAELVKPGASVKLSCKASGYTFISYWMHWVKQRPGQGLEWIGEINPSNGRTNYNEKFKSKAT

LTVDKSSSTAYMQLSSLTSEDSAVYYCARPTYGNYWYLDVWGVGTTVTVSS

SEQ ID NO.2

Antibody B4-A11-2 heavy chain variable region VH-CDR1 amino acid sequence

GYTFISYWMH

SEQ ID NO.3

Antibody B4-A11-2 heavy chain variable region VH-CDR2 amino acid sequence

EINPSNGRTNYNEKFKS

SEQ ID NO.4

Antibody B4-A11-2 heavy chain variable region VH-CDR3 amino acid sequence

PTYGNYWYLDV

SEQ ID NO.5

Antibody B4-A11-2 light chain variable region VL amino acid sequence

DVQMTQTTSSLSASLGDRVTISCRASQDIGNYLNWYQQKPDGTFKLLIFYTSRLHSGVPSRFSGSGSGTDY

SLTISNLEHEDVATYFCQQGDTLPWTFGGGTKLEIK

SEQ ID NO.6

Antibody B4-A11-2 light chain variable region VL-CDR1 amino acid sequence

RASQDIGNYLN

SEQ ID NO.7

Antibody B4-A11-2 light chain variable region VL-CDR2 amino acid sequence

YTSRLHS

SEQ ID NO.8

Antibody B4-A11-2 light chain variable region VL-CDR3 amino acid sequence

QQGDTLPWT

SEQ ID NO.9

Antibody B4-7C2 heavy chain variable region VH amino acid sequence

EVQLQQSGPELVKPGVSMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLIDPYNGVTNYNQKFKGKA

TLTVDKSSSTAYMELLSLTSEDSAVYYCARNYGNYGGYGMDYWGQGTSVTVSS

SEQ ID NO.10

Antibody B4-7C2 heavy chain variable region VH-CDR1 amino acid sequence

GYSFTGYTMN

SEQ ID NO.11

Antibody B4-7C2 heavy chain variable region VH-CDR2 amino acid sequence

LIDPYNGVTNYNQKFKG

SEQ ID NO.12

Antibody B4-7C2 heavy chain variable region VH-CDR3 amino acid sequence

NYGNYGGYGMDY

SEQ ID NO.13

Antibody B4-7C2 light chain variable region VL amino acid sequence

DIVMTQSPSSLAVTAGEKVTMSCKSSQSLLWSVTQKNYLSWYQQKQRQPPKLLIYGASIRESWVPDRFTG

SGSGTDFTLTISSVHAEDLAVYYCQHNHGSFLPLTFGAGTKLELK

SEQ ID NO.14

Antibody B4-7C2 light chain variable region VL-CDR1 amino acid sequence

KSSQSLLWSVTQKNYLS

SEQ ID NO.15

Antibody B4-7C2 light chain variable region VL-CDR2 amino acid sequence

GASIRES

SEQ ID NO.16

Antibody B4-7C2 light chain variable region VL-CDR3 amino acid sequence

QHNHGSFLPLT

SEQ ID NO.17

Antibody B4-12A5 heavy chain variable region VH amino acid sequence

QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVNQRPGQGLEWIGEINPSNGRSNYNEKFKNKA

TLTVDKSSSTAYMQLSSLTSEDSAVYYCARSVYYGFDWYFDVWGAGTTVTVSS

SEQ ID NO.18

Antibody B4-12A5 heavy chain variable region VH-CDR1 amino acid sequence

GYTFTSYWMH

SEQ ID NO.19

Antibody B4-12A5 heavy chain variable region VH-CDR2 amino acid sequence

EINPSNGRSNYNEKFKN

SEQ ID NO.20

Antibody B4-12A5 heavy chain variable region VH-CDR3 amino acid sequence

SVYYGFDWYFDV

SEQ ID NO.21

Antibody B4-12A5 light chain variable region VL amino acid sequence

DIVMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDY

SLTINSLEYEDMGIYYCLQSDEFLTFGSGTKLEIK

SEQ ID NO.22

Antibody B4-12A5 light chain variable region VL-CDR1 amino acid sequence

KASQDINSYLS

SEQ ID NO.23

Antibody B4-12A5 light chain variable region VL-CDR2 amino acid sequence

RANRLVD

SEQ ID NO.24

Antibody B4-12A5 light chain variable region VL-CDR3 amino acid sequence

LQSDEFLT

SEQ ID NO.25

Antibody hB4-7C2-14 heavy chain variable region VH amino acid sequence

QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLIDPYNGVTNYNQKFKGR

VTMTRDTSTSTVYMELSSLRSEDTAVYYCARNYGNYGGYGMDYWGQGTLVTVSS

SEQ ID NO.26

Antibody hB4-7C2-14 light chain variable region VL amino acid sequence

DIVMTQSPLSLPVTPGEPASISCKSSQSLLWSVTQKNYLSWYLQKPGQSPQLLIYGASIRESGVPDRFSGSG

SGTDFTLKISRVEAEDVGVYYCQHNHGSFLPLTFGGGTKVEIK

Claims

1. An anti-LILRB 4 antibody or fragment thereof, characterized in that: at least one of the following is satisfied:

1) Contains at least one of heavy chain complementarity determining regions VH-CDR1, VH-CDR2 or VH-CDR3 with amino acid sequences shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively;

2) Or at least one of light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively.

2. The anti-LILRB 4 antibody or fragment thereof according to claim 1, characterized in that at least one of the following is satisfied:

1) Contains a heavy chain variable region VH with an amino acid sequence shown as SEQ ID NO. 1;

2) Contains a light chain variable region VL with an amino acid sequence shown in SEQ ID NO. 5.

3. The anti-LILRB 4 antibody or fragment thereof according to any one of claims 1 or 2, characterized in that: comprises a heavy chain variable region VH with an amino acid sequence shown as SEQ ID NO.1 and a light chain variable region VL with an amino acid sequence shown as SEQ ID NO. 5.

4. An anti-LILRB 4 antibody or fragment thereof, characterized by at least one of:

1) Contains at least one of heavy chain complementarity determining regions VH-CDR1, VH-CDR2 or VH-CDR3 with amino acid sequences shown as SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 respectively;

2) Or at least one of the light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID No.14, SEQ ID No.15 and SEQ ID No.16 respectively.

5. The anti-LILRB 4 antibody or fragment thereof according to claim 4, characterized in that at least one of the following is satisfied:

1) Comprises a heavy chain variable region VH with an amino acid sequence shown as SEQ ID NO. 9;

2) Contains a light chain variable region VL with an amino acid sequence shown in SEQ ID NO. 13.

6. The anti-LILRB 4 antibody or fragment thereof according to any one of claims 4 or 5, characterized in that: comprising a heavy chain variable region VH with an amino acid sequence shown as SEQ ID NO.9 and a light chain variable region VL with an amino acid sequence shown as SEQ ID NO. 13.

7. An anti-LILRB 4 antibody or fragment thereof, characterized in that: at least one of the following is satisfied:

1) Contains at least one of heavy chain complementarity determining regions VH-CDR1, VH-CDR2 or VH-CDR3 with amino acid sequences shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20 respectively;

2) Or at least one of the light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 shown by SEQ ID No.22, SEQ ID No.23 and SEQ ID No.24 respectively.

8. The anti-LILRB 4 antibody or fragment thereof according to claim 7, characterized in that at least one of the following is satisfied:

1) Comprises a heavy chain variable region VH with an amino acid sequence shown as SEQ ID NO. 17;

2) Contains a light chain variable region VL with an amino acid sequence shown in SEQ ID NO. 21.

9. The anti-LILRB 4 antibody or fragment thereof according to any one of claims 7 or 8, characterized in that: comprising a heavy chain variable region VH with an amino acid sequence shown as SEQ ID NO.17 and a light chain variable region VL with an amino acid sequence shown as SEQ ID NO. 21.

10. The anti-LILRB 4 antibody or fragment thereof according to any one of claims 1 to 9, characterized in that: the variable region of the antibody is formed by splicing the CDR of the complementarity determining region of the corresponding antibody and the FR of the framework region of the human antibody.

11. The anti-LILRB 4 antibody or fragment thereof according to any one of claims 1 to 10, characterized in that: the antibody is a humanized antibody and satisfies at least one of the following:

1) The amino acid sequence of the heavy chain variable region VH is shown as SEQ ID NO. 25;

2) Or the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO. 26.

12. The anti-LILRB 4 antibody or fragment thereof according to any one of claims 1 to 11, characterized in that at least one of the following is satisfied:

1) The heavy chain constant region of the antibody is selected from at least one of the constant regions of human immunoglobulins IgG1, igG2, igG3, igG4, igM, igE, igA, or IgD heavy chains;

2) The light chain constant region of the antibody is selected from at least one of the constant regions of human immunoglobulin kappa or lambda light chains.

13. The anti-LILRB 4 antibody or fragment thereof according to any one of claims 1 to 12, characterized in that: the antibody fragment is at least one of Fab (antigen binding fragment) or scFv (single-CHAIN FRAGMENT variable).

14. A nucleic acid molecule encoding the anti-LILRB 4 antibody or fragment thereof of any one of claims 1 to 13.

15. A recombinant vector comprising the nucleic acid molecule of claim 14.

16. A cell comprising the recombinant vector of claim 15.

17. Use of an anti-LILRB 4 antibody or fragment thereof as claimed in any one of claims 1 to 13 for killing acute myelogenous leukemia cells.

18. Use of an anti-LILRB 4 antibody or fragment thereof as claimed in any one of claims 1 to 13 in the manufacture of a medicament for the treatment or prophylaxis of tumors.

19. Use according to claim 18, characterized in that: the tumor is acute myelogenous leukemia.