Anti-LILRB4 antibodies and their uses

Abstract

This invention relates to the field of antibody technology, specifically to the preparation of anti-LILRB4 antibodies or fragments thereof and their uses. The technical problem this invention aims to solve is to provide a series of anti-LILRB4 antibodies or fragments thereof. The anti-LILRB4 antibodies of this invention possess good affinity and functional activity, can kill tumor cells in vitro, and can effectively inhibit tumor growth in mice, showing potential for application in cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically to the preparation of antibodies against LILRB4 and their fragments, as well as their uses. Background Technology

[0002] Antibodies are biological macromolecules composed of heavy and light chains, secreted by B lymphocytes, and play a crucial role in humoral immunity. The heavy or light chain of an antibody molecule consists of variable and constant regions, respectively. The variable region primarily binds to target antigens, while the constant region primarily exerts immunomodulatory effects. Based on their spatial structure and amino acid sequence characteristics, antibodies can be classified into IgG, IgM, IgE, IgA, IgD, etc., and both the heavy and light chains can be further subdivided into multiple subtypes. For example, human IgG heavy chains can be divided into IgG1, IgG2, IgG3, and IgG4, while the light chains can be divided into κ and λ. Because antibodies can specifically and efficiently bind to target molecules or target cells, regulate downstream signaling pathways of target molecules, or kill target cells through immune effects, they can be developed into drugs for disease treatment. Currently, antibodies have become an important type of biotechnology drug, with monoclonal antibodies accounting for the majority, especially IgG type antibodies. Therefore, the term "monoclonal antibody" usually refers to IgG type antibodies.

[0003] IgG antibody molecules are tetramers composed of two heavy chains and two light chains linked by interchain disulfide bonds, with a molecular weight of approximately 150 kDa. Based on structural and functional characteristics, antibody molecules can be divided into variable regions and constant regions. The variable region primarily functions in antigen binding, while the constant region primarily performs immunological effects and transport functions. The variable region of an antibody can be further divided into complementarity-determining regions (CDRs) and framework regions (FRs). Each heavy chain or light chain contains three CDRs (heavy chain VH-CDR1, VH-CDR2, VH-CDR3; light chain VL-CDR1, VL-CDR2, VL-CDR3) and four FRs flanking the CDRs (FR1, FR2, FR3, FR4). The loop formed by the CDRs is the primary site of antibody-antigen binding, while the FRs form the supporting structure of the CDRs through spatial folding. The specific recognition of different antigen molecules by antibodies is mainly achieved through the amino acid polymorphisms of the six CDR regions (VH-CDR1, 2, 3 and VL-CDR1, 2, 3) and the conformational polymorphism of the loop. Because the structural similarity of the FR regions of different antibodies is high, when the CDR region of one antibody replaces the CDR region of another antibody molecule, if the FR regions of the different antibody molecules match appropriately, the conformational change of the CDR region before and after the replacement is small. Therefore, the new variable region formed after replacement can still retain antigen-binding ability. This characteristic is the basis of CDR grafting technology. The CDR region of a murine antibody can be replaced with the CDR of a human antibody through CDR grafting technology, thereby recombining with the human FR region to form a humanized antibody. If the FR regions of the human and murine antibodies match appropriately, then the antigen-binding ability can still be retained.

[0004] Hybridoma technology is currently a crucial technique in antibody discovery. After mice are immunized with an antigen, mouse B cells develop germinal centers in lymphoid tissue. Through somatic high-frequency mutation (SHM), antibody affinity gradually increases. Mouse spleen cells are isolated and fused in vitro with mouse myeloma cells to form hybridomas. By measuring antibody activity in the culture supernatant of hybridoma cells, hybridomas producing target antibodies can be screened. Hybridoma cells are subcloned and gradually monocloned. The mRNA of subcloned cells is extracted for antibody variable region gene sequencing, allowing analysis of the amino acid sequence of the antibody variable region. Currently, most commercially available antibodies are obtained through hybridoma screening. Because antibody molecules derived from hybridoma technology complete the affinity maturation process in the mouse body, B cells that cross-react with mouse proteins are eliminated through "negative selection" in the mouse bone marrow. Therefore, antibody molecules can effectively reduce non-specific binding to their own proteins or similar proteins.

[0005] Monoclonal antibodies exert their pharmacological effects through multiple mechanisms. The variable region of an antibody can bind to soluble extracellular ligands, blocking the binding of ligands to receptors and disrupting ligand-induced downstream signal transduction. Therefore, antibody drugs targeting immune cytokines can improve inflammatory diseases; for example, antibodies such as adalimumab, belimumab, and siltuximab have already been approved for marketing. The constant region of an antibody can exert immunomodulatory effects, including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Therefore, antibody drugs targeting tumor cell surface molecules can kill tumor cells; for example, marketed antibodies such as rituximab, trastuzumab, and cetuximab have achieved great success. Furthermore, novel antibody technologies derived from monoclonal antibodies, such as bispecific antibodies and antibody-drug conjugates (ADCs), have also developed rapidly in recent years, achieving varying degrees of breakthroughs. To date, cancer and inflammatory diseases are the disease areas where antibody drugs are most widely used.

[0006] Cancer is a serious threat to human health and life. With aging and changes in lifestyle, the incidence of cancer is constantly rising and showing a trend towards affecting younger people. Therefore, cancer treatment has always been a research hotspot in the medical field. Cancer immunotherapy is a successful cancer treatment method that has gained widespread recognition in the medical community. The immune system has the ability to recognize and kill tumor cells; however, tumor cells can evade anti-tumor immunity through various mechanisms. Immune checkpoint molecules can regulate the activation level of immune cells and play an important role in the normal activation of the body's immune system and the prevention of autoimmunity. Overexpression of immune checkpoint molecules such as PD-1, PD-L1, and CTLA4 is one of the important factors leading to tumor immune escape, and immune checkpoint inhibitors are also a hot topic in tumor immunotherapy. Currently, although antibody drugs targeting PD-1, PD-L1, and CTLA4 have achieved great success in clinical practice, they are only effective against a few tumors, and their efficacy against most tumors remains unsatisfactory. Therefore, there is an urgent need to develop antibody drugs targeting other new targets to address unmet clinical needs.

[0007] Leukocyte Ig-like receptor subfamily B4 (LILRB4), also known as ILT3, is a member of the LILR family and is expressed on dendritic cells, monocytes, macrophages, precursor mast cells, endothelial cells, and osteoclasts. Furthermore, LILRB4 is 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 several ligands with different functions, mainly including CD166, ApoE, fibronectin, and CNTFR. APOE is an important ligand for LILRB4; after binding to LILRB4, it recruits SHP-2 to transmit inhibitory signals, inhibiting lymphocyte growth and cytokine release. Studies have shown that LILRB4 can serve as a target for tumor immunotherapy, and inhibitors of the APOE / LILRB4 signaling pathway can inhibit tumor growth. Summary of the Invention

[0008] The technical problem to be solved by this invention is to provide a series of anti-LILRB4 antibodies or antibody fragments with good affinity and functional activity, which can kill tumor cells in vitro and effectively inhibit tumor growth in mice, and have application potential in cancer treatment.

[0009] This invention provides a series of antibodies or fragments thereof against LILRB4.

[0010] This invention first provides an antibody or fragment thereof against LILRB4, which satisfies at least one of the following:

[0011] Contains at least one of the heavy chain complementarity-determining regions VH-CDR1, VH-CDR2, or VH-CDR3 shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively;

[0012] or,

[0013] It contains at least one of the 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.

[0014] Furthermore, the above-mentioned anti-LILRB4 antibody or fragment thereof contains the heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO.1.

[0015] Alternatively, the antibody against LILRB4 or its fragments may contain a light chain variable region VL with the amino acid sequence shown in SEQ ID NO.5.

[0016] Furthermore, the aforementioned anti-LILRB4 antibody or its fragment contains a heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO.1 and a light chain variable region VL with the amino acid sequence shown in SEQ ID NO.5.

[0017] The antibody containing the heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO.1 and the light chain variable region VL with the amino acid sequence shown in SEQ ID NO.5 is named B4-A11-2.

[0018] The present invention also provides an antibody or fragment thereof against LILRB4, which satisfies at least one of the following:

[0019] Contains at least one of the heavy chain complementarity-determining regions VH-CDR1, VH-CDR2, or VH-CDR3 shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively;

[0020] or,

[0021] It contains 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.

[0022] Furthermore, the aforementioned anti-LILRB4 antibody or fragment thereof contains the heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO.9.

[0023] Alternatively, the antibody against LILRB4 or its fragments may contain a light chain variable region VL with the amino acid sequence shown in SEQ ID NO.13.

[0024] Furthermore, the aforementioned anti-LILRB4 antibody or its fragment contains a heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO.9 and a light chain variable region VL with the amino acid sequence shown in SEQ ID NO.13.

[0025] The antibody containing the heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO.9 and the light chain variable region VL with the amino acid sequence shown in SEQ ID NO.13 is named B4-7C2.

[0026] This invention also provides another antibody or fragment thereof against LILRB4, which satisfies at least one of the following:

[0027] Contains at least one of the heavy chain complementarity-determining regions VH-CDR1, VH-CDR2, or VH-CDR3 shown in SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.20, respectively;

[0028] or,

[0029] It contains at least one of the light chain complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3 shown in SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24, respectively.

[0030] Furthermore, the above-mentioned anti-LILRB4 antibody or fragment thereof contains the heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO.17.

[0031] Alternatively, the antibody against LILRB4 or its fragments may contain a light chain variable region VL with the amino acid sequence shown in SEQ ID NO.21.

[0032] Furthermore, the aforementioned anti-LILRB4 antibody or its fragment contains a heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO.17 and a light chain variable region VL with the amino acid sequence shown in SEQ ID NO.21.

[0033] An antibody containing the heavy chain variable region VH (amino acid sequence shown in SEQ ID NO. 17) and the light chain variable region VL (amino acid sequence shown in SEQ ID NO. 21) is named B4-12A5. Furthermore, splicing the corresponding CDR region of the above antibody or its fragment with the human antibody frame region FR can form a humanized antibody or its fragment.

[0034] Furthermore, the antibody or its fragment contains the heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO.25.

[0035] Alternatively, the antibody or its fragment may contain a light chain variable region VL with the amino acid sequence shown in SEQ ID NO.26.

[0036] Furthermore, the antibody or its fragment contains a heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO.25 and a light chain variable region VL with the amino acid sequence shown in SEQ ID NO.26.

[0037] The humanized antibody containing the heavy chain variable region VH with the amino acid sequence shown in SEQ ID NO.25 and the light chain variable region VL with the amino acid sequence shown in SEQ ID NO.26 is the humanized form of antibody B4-7C2, named antibody hB4-7C2-14.

[0038] Furthermore, the heavy chain constant region of the aforementioned antibody may be derived from at least one of the constant regions of the heavy chains of human immunoglobulins IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, and IgD; or the light chain constant region may be derived from at least one of the constant regions of the light chains of human immunoglobulins κ or λ.

[0039] Furthermore, the antibody fragments mentioned above can be Fab (antigen binding fragment) or scFv (single-chain fragment variable).

[0040] The present invention also provides nucleic acid molecules encoding the above-mentioned anti-LILRB4 antibody or fragments thereof.

[0041] The present invention also provides a recombinant vector comprising a nucleic acid molecule encoding the above-mentioned anti-LILRB4 antibody, wherein the recombinant vector may be a plasmid or a viral vector.

[0042] The present invention also provides cells comprising the above-described recombinant vector, wherein the cells may be eukaryotic cells or prokaryotic cells.

[0043] The present invention also provides the use of the above-mentioned anti-LILRB4 antibody in killing tumor cells.

[0044] The antibodies of the present invention can be prepared into various forms of pharmaceutical formulations according to conventional pharmaceutical techniques, with liquid injections and freeze-dried injections being more preferred.

[0045] The antibodies of the present invention can form pharmaceutical compositions with other drugs, and the compositions can be used in conjunction with other treatment methods to treat diseases, including chemotherapy, radiotherapy, biological therapy, etc.

[0046] The beneficial effects of this invention are as follows:

[0047] This invention provides a series of anti-LILRB4 antibodies with good affinity and functional activity. They can kill tumor cells in vitro and effectively inhibit tumor growth in mice, showing potential for application in cancer treatment. Attached Figure Description

[0048] Figure 1 The binding activity of hybridoma antibodies to CHO / LILRB4 cells.

[0049] Figure 2 The binding activity of hybridoma antibodies to CHO / LILRB3 cells.

[0050] Figure 3 Antitumor activity of murine antibody B4-7C2 in immunocompetent mice

[0051] Figure 4 Antitumor effect of chimeric antibody B4-7C2-h in a human PBMC immune reconstitution mouse model.

[0052] Figure 5 SPR binding kinetics analysis of humanized antibody hB4-7C2-14.

[0053] Figure 6 Analysis of the binding activity of humanized antibody hB4-7C2-14 and chimeric antibody B4-A11-2-h to THP-1 cells.

[0054] Figure 7 Analysis of ADCC activity of humanized antibody hB4-7C2-14.

[0055] Figure 8 In vitro tumor cell killing activity of humanized antibody hB4-7C2-14.

[0056] Figure 9 In vitro tumor cell killing activity of chimeric antibody B4-A11-2-h.

[0057] Figure 10 Antitumor effects of humanized antibody hB4-7C2-14 and chimeric antibody B4-A11-2-h in a human PBMC immune reconstitution mouse model. Detailed Implementation

[0058] This invention provides a series of anti-LILRB4 antibodies with good affinity and functional activity. They can kill tumor cells in vitro and effectively inhibit tumor growth in mice, showing potential for application in cancer treatment.

[0059] The anti-LILRB4 antibody of this invention was obtained through hybridoma screening. Human LILRB4 protein was mixed with an adjuvant and used to immunize mice. After the serum titer reached a suitable level, mouse spleen cells were isolated, fused with mouse myeloma cells in vitro, and cultured to obtain cell culture supernatant containing the antibody. First, affinity ELISA was used to screen for hybridoma antibodies with LILRB4 binding activity.

[0060] Hybridoma antibodies with good binding kinetics were further screened using SPR (Self-Range Phosphate) and flow cytometry was used to screen for hybridoma antibodies with good binding activity to CHO-K1 / LILRB4 cells. The cross-binding of hybridoma antibodies with LILR family proteins was further analyzed by flow cytometry. Antibodies B4-7C2 and B4-12A5 showed good binding activity with LILRB4 and also with LILRB3. Antibody B4-A11-2 showed good binding activity only with LILRB4.

[0061] To obtain the amino acid sequence of the hybridoma antibody, this invention sequenced the hybridoma antibody mRNA to obtain the variable region sequence, and prepared a recombinant monoclonal antibody for functional activity verification. The variable region mRNA gene of the antibody was amplified by PCR using an upstream signal peptide primer and a downstream constant region primer, and further sequenced to obtain the antibody variable region gene. An expression vector was constructed by splicing and fusing the murine antibody variable region with the antibody constant region. The expression plasmid containing the antibody gene was transfected into HEK293 cells for transient expression. The antibody was purified by protein G affinity, and its purity and content were confirmed by SDS-PAGE and spectrophotometry for further verification. The obtained recombinant monoclonal antibody was then subjected to cell binding activity and binding kinetics verification to ensure its activity.

[0062] This invention further investigated the antitumor effect of antibody B4-7C2 in mice, confirming that murine antibody B4-7C2 can inhibit tumor growth in immunocompetent mice, and that chimeric antibody B4-7C2-h can inhibit tumor growth in human PBMC-reconstituted mice.

[0063] This invention further allows the CDR region of the murine antibody B4-7C2 to be spliced ​​with the FR region of the human antibody to construct a humanized antibody. Binding kinetics analysis of the obtained humanized antibody preferentially identified the humanized antibody hB4-7C2-14. Flow cytometry analysis showed that, because the humanized antibody hB4-7C2-14 can simultaneously bind to 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. Simultaneously, luciferase reporter cell analysis showed that the humanized antibody hB4-7C2-14 also exhibited a significant ADCC signal.

[0064] This invention further analyzed the tumor-suppressive effects of the antibodies in vitro and in vivo. The humanized antibody hB4-7C2-14 and the chimeric antibody B4-A11-2-h exhibited significant tumor cell-killing activity in vitro and good anti-tumor effects in a human PBMC immune reconstitution mouse model.

[0065] The anti-LILRB4 antibody of this invention can be modified into antibody fragments such as Fab (antigen-binding fragment) and scFv (single-chain fragment variable) using conventional gene recombination technology. Fab and scFv antibody fragments are small in size, have strong tissue penetration, and possess unique advantages in certain application areas. Fab is a heterodimer composed of a heavy chain variable region-constant region 1 (VH-CH1) and a light chain variable region-constant region (VL-CL), with a molecular size one-third that of IgG. Due to the absence of an Fc fragment, the immune effect induced by Fab is significantly lower than that of IgG, and its cytokine release is weaker. Currently, antibody drugs with Fab structures, such as abciximab and ranibizumab, have been approved for marketing. scFv is formed by the fusion of VH and VL and the linker peptide between them. Its molecular size is only one-sixth that of IgG, exhibiting strong tissue penetration and a short half-life, giving it unique advantages in imaging diagnostics and some therapeutic areas. The scFv-based bispecific antibody blinatumomab has also been approved for marketing. Antibody fragments can also be further fused with other proteins or conjugated with other small molecules for targeted delivery in the diagnosis and treatment of diseases.

[0066] The anti-LILRB4 antibody of this invention can further enhance affinity by mutating amino acids in the CDR region using genetic engineering techniques. The antibody CDR region plays a crucial role in the binding of the antibody to the antigen, and the amino acids within it can interact with the antigen's amino acids through hydrogen bonds, ionic bonds, van der Waals forces, and other mechanisms. By mutating the amino acids in the antibody's CDR region, the interaction between the CDR and the antigen can be further enhanced, thereby increasing the antibody's affinity. Antibody library technology is well-established for antibody affinity evolution. Antibody mutation libraries can be established using strategies such as alanine hotspot mutation and error-prone PCR, enabling high-throughput screening of mutant antibodies and achieving antibody affinity evolution in vitro.

[0067] The antibodies of this invention can be expressed in stable cell lines for large-scale protein production. The gene encoding the antibody amino acids can be obtained using conventional gene recombination techniques. After DNA sequence optimization, synthesis, and PCR amplification, it can be inserted into an expression vector. The vector used can be a plasmid, virus, or gene fragment commonly used in molecular biology. A protein secretion signal peptide gene is added to the front end of the antibody-encoding DNA sequence to ensure that the antibody can be secreted extracellularly. The vector sequence contains elements such as a promoter for gene expression, protein translation initiation and termination signals, and polyadenylated amino acids (PolyA). The vector contains antibiotic resistance genes and replication elements to facilitate vector replication in host cells such as bacteria, used for vector preparation. Additionally, the vector may contain selection genes to facilitate the selection of stable transfection host cells for constructing stable expression cell lines.

[0068] After constructing the vector containing the antibody-encoding DNA sequence, the vector can be used to transfect or transform host cells to express the corresponding protein. Various expression systems can be used to express antibodies, including eukaryotic and prokaryotic cells, such as mammalian cells, insect cells, yeast, and bacteria. Because prokaryotic cells readily form inclusion bodies when expressing complete antibodies, mammalian cells are the preferred system for expressing this protein. Several mammalian cells can be used for large-scale antibody expression, such as CHO cells, HEK293 cells, NSO cells, and COS cells, all of which are included in the cell types applicable to this invention. The recombinant vector containing the antibody-encoding gene can be transfected into host cells. Various transfection methods exist, including electroporation, liposome transfection, and calcium phosphate transfection.

[0069] A preferred method for protein expression is to utilize stable transfection of host cells containing selective genes. For example, after stably transfecting host cells lacking neomycin resistance with a recombinant vector containing a neomycin resistance gene, the concentration of neomycin in the cell culture medium can be increased to screen for stable cell lines with high expression. Similarly, after stably transfecting host cells lacking DHFR with a recombinant vector containing a dihydrofolate reductase (DHFR) gene, the concentration of methotrexate (MTX) in the cell culture medium can be increased to screen for stable cell lines with high expression.

[0070] Other expression systems besides mammalian cells, such as insect cells, yeast, and bacteria, can also be used to express the antibodies or fragments thereof of the present invention, and they are also included in the category of host cells that can be used by the present invention. The protein expression levels of these expression systems are sometimes higher than those of mammalian cells, but they are prone to forming inclusion bodies, thus requiring further protein refolding.

[0071] The antibodies of the present invention can also be delivered and expressed using viral vectors, including but not limited to adenovirus vectors, adeno-associated viral vectors, retroviral vectors, herpes simplex virus-based vectors, and lentiviral vectors.

[0072] The anti-LILRB4 antibody of the present invention can be used for the detection of LILRB4, including ELISA and flow cytometry. ELISA and flow cytometry analyses showed that the anti-LILRB4 antibody of the present invention did not show binding signals with LILRB4-negative cell components from various tissue sources, indicating that the antibody has good specificity.

[0073] The anti-LILRB4 antibody of the present invention can kill tumor cells in vitro and effectively inhibit tumor growth in mice, and has potential applications in cancer treatment.

[0074] The antibodies of the present invention can be prepared into various forms of pharmaceutical formulations according to conventional pharmaceutical techniques, with liquid injections and freeze-dried injections being more preferred.

[0075] The antibodies of the present invention can form pharmaceutical compositions with other drugs, and the compositions can be used in conjunction with other treatment methods to treat diseases, including chemotherapy, radiotherapy, biological therapy, etc.

[0076] The following examples illustrate in detail the discovery, preparation, testing, and application of the antibodies involved in this invention. However, the content and uses of this invention are not limited to the scope of these examples.

[0077] Example 1: Mouse Immunization

[0078] Female BALB / c mice aged 6-8 weeks and weighing approximately 20g were used as immunization hosts. After one week of acclimatization, antigen immunization was performed. The purified LILRB4-ECD-His (human extracellular domain C-terminus fused with a 6×His tag) was prepared to 1 mg / mL in PBS (pH 7.2), filtered through a 0.22 μm filter, and 50 μL of the solution was thoroughly mixed with 50 μL of QuickAntibody and injected into the calf muscle of the mouse's hind leg. A booster immunization was performed on day 21, and serum antibody titers were measured by tail vein blood collection on day 35. LILRB4-hFc (human LILRB4 extracellular domain C-terminus fused with human IgG1Fc) protein was coated onto ELISA plates (50 ng / well), and mouse serum antibody titers were measured by ELISA. Mice with antibody titers greater than 32000 were given a single antigen shock, and spleen cell fusion was performed 3 days later.

[0079] Example 2: Spleen cell fusion

[0080] After mouse euthanasia, spleens were isolated under aseptic conditions. Spleen cell suspensions were prepared using a 70 μm mesh and washed twice with basal culture medium for cell counting. SP2 / 0 was mixed with spleen cells at a 1:3 ratio. After centrifugation, the supernatant was discarded, and 1 mL of pre-warmed PEG (at 37°C) was added dropwise over 1 min. The mixture was incubated at 37°C for 90 s, followed by the addition of 20 mL of pre-warmed basal culture medium (at 37°C) over 6 min. Cells were collected by centrifugation (room temperature, 800 rpm, 3 min), resuspended in 20 mL of pre-warmed HAT medium (at 37°C), and cultured at a ratio of 1 × 10⁻⁶ cells / mL. 5 The density of spleen cells per well was determined by adding the fused cells to a 96-well cell culture plate and incubating them in a CO2 cell culture incubator. Once the cells reached a confluence of more than 70%, the culture supernatant was collected for ELISA detection.

[0081] Example 3: Affinity ELISA Screening

[0082] A 1 μg / mL LILRB4-hFc solution was prepared using PBS and added to an ELISA plate (50 μL / well), incubated overnight at 4°C. The plate was washed three times with PBST, and 200 μL of 5% BSA blocking buffer was added, incubated at 37°C for 2 h. The plate was washed three times with PBST, and 50 μL of hybridoma cell culture supernatant was added, incubated at 37°C for 1 h. The plate was washed three times with PBST, and 50 μL of 1:5000 diluted HRP-goat anti-mouse solution was added, incubated at 37°C for 1 h. The plate was washed three times with PBST, and 100 μL of ready-to-use TMB chromogenic solution was added, incubated at 37°C in the dark for 5–10 min. The chromogenic process was stopped by adding 2 M H2SO4 (100 μL / well), and the OD value was measured at 450 nm. The obtained positive primitive clones were then used for further screening.

[0083] Example 4: SPR Screening

[0084] The appropriate coupling amount was calculated using the formula RL = (Rmax × MWligand) / (Sm × MWanalyte), and the anti-mouse antibody was coupled to the CM5 chip using an amine coupling kit. Hybridoma cell culture supernatant was captured onto the chip, and the response value of LILRB4-HSA-His (human LILRB4 extracellular domain C-terminus fused with human albumin and a 6×His tag) flowing through the channel was detected using a Biacore 8K detector. Data fitting was performed using evaluation software to obtain binding curves and kinetic parameters. Hybridoma antibodies with good binding kinetics were selected for further screening.

[0085] Example 5: Hybridoma Subcloning

[0086] Hybridoma cells were subcloned using a limiting dilution method. Antibody-secreting hybridoma cells were collected and counted. The cells were diluted with complete culture medium and added to 96-well cell culture plates at a density of 0.5 cells / well for further culture. The remaining cells were expanded and seeded. After 10 days of subcloning culture, the culture supernatant from each subcloning well was used for affinity ELISA and SPR verification. Positive clones were used for a second subcloning test, and the second subcloning was verified using the same screening method as the first subcloning. Hybridoma subclones with good binding kinetics were selected for further screening.

[0087] Example 6: Screening for cell binding activity

[0088] Collect CHO-K1 cell lines stably expressing LILRB4 (CHO-K1 / LILRB4), with 1 × 10⁶ cells per group. 6 Cell suspension was aspirated into U-shaped 96-well plates. Cells were washed once with PBS, centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. Cells were resuspended in 100 μL of hybridoma cell culture supernatant, PBS, isotype antibody mIgG (2 μg / mL), and anti-LILRB4 positive antibody (2 μg / mL), and incubated on ice for 60 min. After incubation, cells were collected by centrifugation, washed with 200 μL of PBS, and repeated twice. 100 μL of Alexa Fluor-488-labeled goat anti-mouse IgG (H+L) (1:200 dilution) was added to the cell wells, resuspended and mixed, and incubated on ice in the dark for 40 min. After incubation, cells were collected by centrifugation, washed with 200 μL of PBS, and repeated twice. Finally, cells were resuspended in 130 μL of PBS for flow cytometry analysis. Hybridoma antibodies with good cell-binding activity were prioritized for further screening.

[0089] Example 7: Screening for cross-binding of LILR family proteins

[0090] CHO-K1 cell lines stably expressing LILR family proteins were collected, namely 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, and CHO-K1 / LILRB5, with 1 × 10⁻⁶ cells per group. 6 Cell suspension was aspirated into U-shaped 96-well plates. Cells were washed once with PBS and centrifuged at 1200 rpm for 5 min. The cell pellet was resuspended in 100 μL of hybridoma cell culture supernatant, PBS, isotype antibody mIgG (2 μg / mL), and anti-LILRB4 positive antibody (2 μg / mL), and incubated on ice for 60 min. After incubation, cells were collected by centrifugation and washed twice with 200 μL of PBS. 100 μL of Alexa Fluor-488-labeled goat anti-mouse IgG (H+L) (1:200 dilution) was added to the cell pellet, resuspended and mixed, and incubated on ice in the dark for 40 min. After incubation, cells were collected by centrifugation and washed twice with 200 μL of PBS. Finally, cells were resuspended in 130 μL of PBS for flow cytometry analysis. The results showed that antibodies B4-A11-2, B4-7C2, and B4-12A5 exhibited good binding activity with CHO-K1 / LILRB4 (see [link to results]). Figure 1 In addition, antibodies B4-7C2 and B4-12A5 also showed good binding activity with CHO-K1 / LILRB3 (see [link to article]). Figure 2 ).

[0091] Example 8: Obtaining the antibody variable region sequence

[0092] Hybridoma cell subclones were collected, and RNA was extracted using the Trizol method. Using the extracted RNA as a template, cDNA was obtained through reverse transcription. PCR amplification of the variable regions of the heavy and light chains of the antibodies was performed using degenerate primers (Novagen Ig-Primer Sets), and the PCR products were detected by agarose gel electrophoresis. The target DNA fragment was obtained using a gel extraction kit, and then TA cloning was performed to construct recombinant plasmids. The recombinant plasmids were transformed into competent cells using the heat shock method, and the cells were plated for blue-white screening. Single white colonies were picked and cultured in 0.5 mL of LB liquid medium at 37°C and 220 rpm for 3 h with shaking. The bacterial culture was then sent for sequencing. The amino acid sequences of the variable regions of anti-LILRB4 antibodies B4-A11-2, B4-7C2, and B4-12A5 are shown in Table 1.

[0093] Table 1. Amino acid sequence of the variable region of the antibody

[0094]

[0095] Example 9: Construction and preparation of recombinant monoclonal antibodies

[0096] Overlap PCR was used to splice the variable region gene fragments of the heavy and light chains with the signal peptide and the constant region gene fragments of the mouse heavy chain (IgG2a) and light chain (κ), respectively, and the sequences were used for identification. The correctly spliced ​​antibody heavy and light chain genes were inserted into the pTT5 plasmid, and the recombinant plasmids were transfected into HEK293 cells using the PEI method. Cells were then cultured in serum-free suspension for transient antibody expression. Cell supernatant was collected after 7 days of culture, filtered through a 0.22 μm filter, and purified by protein G affinity chromatography. The antibody was ultrafiltered and replaced with PBS solution. The purity and concentration of the antibody were determined using reducing SDS-PAGE and NanoDrop 2000. The aliquots were then stored at -80℃ for later use. The obtained recombinant monoclonal antibodies were validated for cell binding activity and binding kinetics to ensure their activity.

[0097] Example 10, Specificity Detection

[0098] Multiple cell lines were cultured and collected, using different anti-LILRB4 antibodies as primary antibodies and FITC-labeled goat anti-mouse IgG (H+L) as secondary antibodies. Flow cytometry (FCM) was used to detect the binding of antibodies to cell surface proteins. Results showed that antibodies B4-A11-2 and B4-7C2 did not show binding signals with HT-29, LO2, PANC-1, HeLa, MGC-803, HepG2, or Jurkat cells, but showed a significant binding signal with THP-1 cells. On the other hand, multiple cell lines were cultured and collected, and cell lysate samples were prepared using RIPA lysis buffer and an ultrasonic cell disruptor. These samples were then coated onto ELISA plates (500 ng / well), with LILRB4+THP-1 cell lysis buffer used as a positive control. ELISA was used to detect the binding of antibodies to cell lysate components using different anti-LILRB4 antibodies as primary antibodies and HRP-goat anti-mouse antibody as secondary antibodies. The results showed that antibodies B4-A11-2 and B4-7C2 did not show binding signals with LO2, Hela, MGC-803, HepG2, Jurkat, K562, or Raji cells, but showed significant binding signals with LILRB4+THP-1 cells.

[0099] Example 11: Antitumor effect of murine antibody B4-7C2 in immunocompetent mice

[0100] Mouse colon cancer cells (CT26-hLILRB4) stably transformed with human LILRB4 were subcutaneously inoculated into the backs of 6-week-old female BALB / c mice (3.5 × 10⁻⁶ cells). 6 (each tumor), until the tumor volume reaches 50-100 mm. 3 Mice were randomly assigned to groups. They were administered the drug intraperitoneally (10 mg / kg) on ​​days 0, 3, 6, 9, and 12. The murine antibody B4-7C2 used was purified from mouse ascites fluid, with a murine isotype antibody (mIgG) as a control. Tumor volume was measured every 3 days (calculated as V = 1 / 2 × major axis × minor axis × minor axis), and mouse weight and condition were monitored. The results showed that the murine antibody B4-7C2 effectively inhibited tumor growth (see...). Figure 3 ).

[0101] Example 12: Antitumor effect of chimeric antibody B4-7C2-h in a human PBMC immune reconstitution mouse model

[0102] PBMCs (8×10) 6 (each / animal) and human acute myeloid leukemia cells THP-1 (4×10) 6 (Number of mice per mouse) were subcutaneously injected into the back of NOD mice until the tumor volume reached 50-100 mm. 3Mice were randomly assigned to groups. They were administered intraperitoneally (10 mg / kg) on ​​days 0, 3, 6, 9, and 12. The chimeric antibody B4-7C2-h used had a constant region of human (mouse-human chimeric antibody, constant region of human hIgG1κ type), with a human isotype antibody (hIgG) as a control. Tumor volume was measured every 3 days (calculated as V = 1 / 2 × major axis × minor axis × minor axis), and mouse weight and condition were monitored. Results showed that the chimeric antibody B4-7C2-h effectively inhibited tumor growth (see...). Figure 4 ).

[0103] Example 13: Humanization of mouse antibody B4-7C2

[0104] Homology modeling of the variable region of antibody B4-7C2 was performed based on antibody structures from the PDB database, and the CDR region was determined based on amino acid primary sequence characteristics and the spatial conformation of the variable region. The antibody variable region was aligned with the amino acid sequences encoded by human antibody germline genes V and J. Based on factors such as consistency, similarity, and conservation of the frame region (FR), five V genes and one J gene from the IGHV library were selected and spliced ​​with the heavy chain CDR to form five different humanized heavy chain variable regions. Similarly, four V genes and one J gene from the IGKV library were selected and spliced ​​with the light chain CDR to form four different humanized light chain variable regions. The humanized heavy and light chain variable regions were fused with the human IgG1κ heavy and light chain constant regions, respectively, to form complete heavy and light chains. The five heavy chains were combined with the four light chains to construct 20 different humanized antibodies. Biacore binding kinetic analysis showed that the humanized antibody hB4-7C2-14 exhibited good binding kinetics with both LILRB4 and LILRB3 (see Biacore). Figure 5 The binding kinetic parameters are shown in Table 2, and the amino acid sequence of its variable region is shown in Table 3.

[0105] Table 2. Combining dynamic parameters

[0106]

[0107] Table 3. Amino acid sequence of the variable region of the antibody

[0108]

[0109] Example 14: Analysis of the THP-1 cell binding activity of humanized antibody hB4-7C2-14 and chimeric antibody B4-A11-2-h.

[0110] Collect THP-1 cells, with 1 × 10⁻⁶ cells per group. 6Cell suspensions were transferred to U-shaped 96-well plates. Cells were washed once with PBS and centrifuged at 1200 rpm for 5 min. Humanized antibody hB4-7C2-14, chimeric antibody B4-A11-2-h, and isotype antibody hIgG solutions were prepared at concentrations of 80000 ng / mL, 40000 ng / mL, 20000 ng / mL, 10000 ng / mL, 5000 ng / mL, 2500 ng / mL, 1250 ng / mL, 625 ng / mL, 312.5 ng / mL, 156.25 ng / mL, 78.125 ng / mL, 39.0625 ng / mL, and 19.53125 ng / mL, respectively. 100 μL of each antibody solution was added to a 96-well plate containing THP-1 cells and incubated on ice for 60 min. After incubation, cells were collected by centrifugation and washed twice with 200 μL PBS. Add 100 μL of Alexa Fluor-488-labeled goat anti-mouse IgG (H+L) (1:200 dilution) to the cell wells, resuspend and mix, and incubate on ice in the dark for 40 min. After incubation, collect the cells by centrifugation and wash twice with 200 μL PBS. Finally, resuspend the cells in 130 μL PBS for flow cytometry analysis. Flow cytometry analysis showed that both the humanized antibody hB4-7C2-14 and antibody B4-A11-2-h (mouse-human chimeric antibody, constant region of human hIgG1κ type) had good cell-binding activity against THP-1 cells, exhibiting a concentration-dependent characteristic. Since the humanized antibody hB4-7C2-14 can simultaneously bind to 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...). Figure 6 ).

[0111] Example 15: ADCC Activity Analysis of Humanized Antibody hB4-7C2-14

[0112] THP-1 cells were collected as target cells and their density was adjusted to 1.1 × 10⁻⁶. 6 Cells / mL. 90 μL of cell suspension was added to each well of a 96-well plate, with three replicates per group. Humanized antibody hB4-7C2-14 and homologous antibody hIgG solutions were prepared at concentrations of 20000 ng / mL, 4000 ng / mL, 800 ng / mL, 160 ng / mL, 32 ng / mL, 6.4 ng / mL, 1.28 ng / mL, 0.256 ng / mL, 0.0512 ng / mL, and 0.01024 ng / mL. 20 μL of each antibody solution was added to a 96-well plate. A negative control group was also included. The Jurkat-Lucia-NFAT-CD16a luciferase reporter cell line was used as effector cells, and the cell density was adjusted to 2.2 × 10⁻⁶ cells / well.6 Cells / mL were counted, and 90 μL of cell suspension was added to a 96-well plate. The 96-well plate was mixed thoroughly and incubated in a cell culture incubator for 6 h. After incubation, the 96-well plate was removed and allowed to equilibrate to room temperature for 30 min. 20 μL of cell culture supernatant was aspirated and Quanti-LUC detection reagent (50 μL / well) was added. The reaction was allowed to proceed for 20 s, and the luminescent signal was detected using a multi-functional microplate reader. The results showed that the humanized antibody hB4-7C2-14 exhibited a significant ADCC signal (see...). Figure 7 ).

[0113] Example 16: In vitro tumor cell killing activity of humanized antibody hB4-7C2-14 and chimeric antibody B4-A11-2-h.

[0114] THP-1 cells were collected as target cells, and the cell suspension was added to 96-well plates at a density of 50 μL per well, 4.5 × 10⁻⁶. 4 Antibody solutions were prepared at concentrations of 0.2 μg / mL, 2 μg / mL, and 20 μg / mL. Homotyped antibody hIgG (20 μg / mL) was gently mixed with the target cell suspension and incubated at 37°C in a 5% CO2 incubator for 30 min. The ratio of effector cells (PBMCs) to target cells (THP-1) was E:T = 10:1. 100 μL of PBMC cell suspension (4.5 × 10⁻¹¹ cells) was added to each well. 5 Cells were collected (cells / well) and incubated at 37°C with 5% CO2 for 20 h. After incubation, cells were collected by centrifugation, washed with 1 mL PBS, and centrifuged at 400 g, 4°C for 5 min. 100 μL PBS was added to the cell pellet to resuspend the cells, along with 0.3 μL of Fixable Viability Stain 450. Cells were incubated at room temperature in the dark for 10 min. After incubation, cells were collected by centrifugation, washed with 1 mL PBS, and repeated twice. Finally, cells were resuspended in 200 μL PBS for flow cytometry analysis. The results showed that the humanized antibody hB4-7C2-14 (hIgG1κ type) (see...) Figure 8 ), B4-A11-2-h (mouse-human chimeric antibody, constant region is human hIgG1κ type) (see) Figure 9 All of them have good in vitro killing effects.

[0115] Example 17: Antitumor effects of humanized antibody hB4-7C2-14 and chimeric antibody B4-A11-2-h in a human PBMC immune reconstitution mouse model.

[0116] Human PBMC (2.7×10) 6(8 x 10^6 cells / mouse) were injected via the tail vein into 5-6 week old NSG mice. Seven days later, human acute myeloid leukemia cells THP-1 were subcutaneously inoculated into the back of the mice. 6 (One per tumor). When the tumor volume reaches 50-100 mm. 3 Mice were randomly assigned to groups and administered the drug intraperitoneally (10 mg / kg) on ​​days 0, 3, 7, 10, and 13. The humanized antibody hB4-7C2-14 (which recognizes both LILRB3 and 4) is human IgG1κ (hIgG1κ), and the constant region of antibody B4-A11-2-h (which recognizes only LILRB4) is human (mouse-human chimeric antibody, constant region is human hIgG1κ). Human isotype antibody (hIgG) was used as a control. Tumor volume was measured every 3 days (calculated as V = 1 / 2 × major axis × minor axis × minor axis), and mouse weight and condition were monitored. Results showed that the humanized antibody hB4-7C2-14 and the chimeric antibody B4-A11-2-h effectively inhibited the growth of THP-1 tumors, and hB4-7C2-14 showed a stronger inhibitory effect on THP-1 tumor growth than B4-A11-2-h (see [link to study]). Figure 10 ).

[0117] sequence list

[0118] SEQ ID NO.1

[0119] Antibody B4-A11-2 heavy chain variable region VH amino acid sequence

[0120] QVQLQQPGAELVKPGASVKLSCKASGYTFISYWMHWVKQRPGQGLEWIGEINPSNGRTNYNEKFKSKAT

[0121] LTVDKSSSTAYMQLSSLTSEDSAVYYCARPTYGNYWYLDVWGVGTTVTVSS

[0122] SEQ ID NO.2

[0123] The amino acid sequence of the VH-CDR1 variable region of the antibody B4-A11-2 heavy chain

[0124] GYTFISYWMH

[0125] SEQ ID NO.3

[0126] The amino acid sequence of the VH-CDR2 variable region of the antibody B4-A11-2 heavy chain

[0127] EINPSNGRTNYNEKFKS

[0128] SEQ ID NO.4

[0129] The amino acid sequence of the VH-CDR3 variable region of the antibody B4-A11-2 heavy chain

[0130] PTYGNYWYLDV

[0131] SEQ ID NO.5

[0132] Antibody B4-A11-2 light chain variable region VL amino acid sequence

[0133] DVQMTQTTSSLSASLGDRVTISCRASQDIGNYLNWYQQKPDGTFKLLIFYTSRLHSGVPSRFSGSGSGTDY

[0134] SLTISNLEHEDVATYFCQQGDTLPWTTFGGGTKLEIK

[0135] SEQ ID NO.6

[0136] The amino acid sequence of the VL-CDR1 variable region of the antibody B4-A11-2 light chain

[0137] RASQDIGNYLN

[0138] SEQ ID NO.7

[0139] The amino acid sequence of the VL-CDR2 variable region of the antibody B4-A11-2 light chain

[0140] YTSRLHS

[0141] SEQ ID NO.8

[0142] The amino acid sequence of the VL-CDR3 variable region of the antibody B4-A11-2 light chain

[0143] QQGDTLPWT

[0144] SEQ ID NO.9

[0145] Antibody B4-7C2 heavy chain variable region VH amino acid sequence

[0146] EVQLQQSGPELVKPGVSMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLIDPYNGVTNYNQKFKGKA

[0147] TLTVDKSSSTAYMELLSLLTSEDSAVYYCARNYGNYGGYGMDYWGQGTSVTVSS

[0148] SEQ ID NO.10

[0149] The amino acid sequence of the VH-CDR1 variable region of the antibody B4-7C2 heavy chain

[0150] GYSFTGYTMN

[0151] SEQ ID NO.11

[0152] The amino acid sequence of the VH-CDR2 variable region of the antibody B4-7C2 heavy chain

[0153] LIDPYNGVTNYNQKFKG

[0154] SEQ ID NO.12

[0155] The amino acid sequence of the VH-CDR3 variable region of the antibody B4-7C2 heavy chain

[0156] NYGNYGGYGMDY

[0157] SEQ ID NO.13

[0158] Antibody B4-7C2 light chain variable region VL amino acid sequence

[0159] DIVMTQSPSSLAVTAGEKVTMSCKSSQSLLWSVTQKNYLSWYQQKQRQPPKLLIYGASIRESWVPDRFTG

[0160] SGSGTDFTLTISSVHAEDLAVYYCQHNHGSFLPLTFGAGTKLELK

[0161] SEQ ID NO.14

[0162] The amino acid sequence of the VL-CDR1 variable region of the antibody B4-7C2 light chain

[0163] KSSQSLLWSVTQKNYLS

[0164] SEQ ID NO.15

[0165] The amino acid sequence of the VL-CDR2 variable region of the antibody B4-7C2 light chain

[0166] GASIRES

[0167] SEQ ID NO.16

[0168] The amino acid sequence of the VL-CDR3 variable region of the antibody B4-7C2 light chain

[0169] QHNHGSFLPLT

[0170] SEQ ID NO.17

[0171] Antibody B4-12A5 heavy chain variable region VH amino acid sequence

[0172] QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVNQRPGQGLEWIGEINPSNGRSNYNEKFKNKA

[0173] TLTVDKSSSTAYMQLSSLTSEDSAVYYCARSVYYGFDWYFDVWGAGTTVTVSS

[0174] SEQ ID NO.18

[0175] The amino acid sequence of the VH-CDR1 variable region of the antibody B4-12A5 heavy chain

[0176] GYTFTSYWMH

[0177] SEQ ID NO.19

[0178] The amino acid sequence of the VH-CDR2 variable region of the antibody B4-12A5 heavy chain

[0179] EINPSNGRSNYNEKFKN

[0180] SEQ ID NO.20

[0181] The amino acid sequence of the VH-CDR3 variable region of the antibody B4-12A5 heavy chain

[0182] SVYYGFDWYFDV

[0183] SEQ ID NO.21

[0184] Antibody B4-12A5 light chain variable region VL amino acid sequence

[0185] DIVMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDY

[0186] SLTINSLEYEDMGIYYCLQSDEFLTFGSGTKLEIK

[0187] SEQ ID NO.22

[0188] The amino acid sequence of the VL-CDR1 variable region of the antibody B4-12A5 light chain

[0189] KASQDINSYLS

[0190] SEQ ID NO.23

[0191] The amino acid sequence of the VL-CDR2 variable region of the antibody B4-12A5 light chain

[0192] RANRLVD

[0193] SEQ ID NO.24

[0194] The amino acid sequence of the VL-CDR3 variable region of the antibody B4-12A5 light chain

[0195] LQSDEFLT

[0196] SEQ ID NO.25

[0197] Antibody hB4-7C2-14 heavy chain variable region VH amino acid sequence

[0198] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLIDPYNGVTNYNQKFKGR

[0199] VTMTRDTSTSTVYMELSSLRSEDTAVYYCARNYGNYGGYGMDYWGQGTLVTVSS

[0200] SEQ ID NO.26

[0201] Antibody hB4-7C2-14 light chain variable region VL amino acid sequence

[0202] DIVMTQSPLSLPVTPGEPASISCKSSQSLLWSVTQKNYLSWYLQKPGQSPQLLIYGASIRESGVPDRFSGSG

[0203] SGTDFTLKISRVEAEDVGVYYCQHNHGSFLPLTFGGGTKVEIK

Claims

1. An antibody against LILRB4 or its antigen-binding fragment, characterized in that: The anti-LILRB4 antibody or its antigen-binding fragment includes a heavy chain complementarity-determining region (CDR) and a light chain complementarity-determining region (LCD). The amino acid sequences of the heavy chain CDRs VH-CDR1, VH-CDR2, and VH-CDR3 are shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. The amino acid sequences of the light chain CDRs VL-CDR1, VL-CDR2, and VL-CDR3 are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively.

2. The antibody against LILRB4 or its antigen-binding fragment according to claim 1, characterized in that... Meet at least one of the following: 1) The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.1; 2) The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

5.

3. The antibody against LILRB4 or its antigen-binding fragment according to any one of claims 1 or 2, characterized in that: The variable region of the antibody is formed by splicing the complementarity-determining region (CDR) of the corresponding antibody with the framework region (FR) of the human antibody.

4. The antibody against LILRB4 according to any one of claims 1 or 2, or its antigen-binding fragment, is characterized in that... Satisfy at least one of the following: 1) The constant region of the heavy chain of the antibody is selected from at least one of the constant regions of the heavy chains of human immunoglobulins IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA or IgD; 2) The constant region of the light chain of the antibody is selected from at least one of the constant regions of the light chain of human immunoglobulin κ or λ.

5. The antibody against LILRB4 according to any one of claims 1 or 2, or its antigen-binding fragment, is characterized in that: The antigen-binding fragment of the antibody is at least one of Fab (antigen binding fragment) or scFv (single-chain fragment variable).

6. A nucleic acid molecule encoding an antibody against LILRB4 as described in any one of claims 1 to 5, or an antigen-binding fragment thereof.

7. A recombinant vector comprising the nucleic acid molecule of claim 6.

8. Cells comprising the recombinant vector of claim 7.

9. Use of the anti-LILRB4 antibody or its antigen-binding fragment as described in any one of claims 1 to 5 in the preparation of a medicament for killing acute myeloid leukemia cells.

10. Use of the anti-LILRB4 antibody or its antigen-binding fragment as described in any one of claims 1 to 5 in the preparation of a medicament for treating acute myeloid leukemia.

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