Anti-NKp46 antibodies and uses thereof

Abstract

The invention relates to the technical field of antibodies, in particular to an anti-NKp46 antibody and application thereof. The technical problem to be solved by the invention is to provide a series of anti-NKp46 antibodies or fragments thereof. The anti-NKp46 antibody provided by the invention has good affinity and NKp46 signal activation activity, can promote NK cell activation and mediate PBMC to kill tumor cells, and has good application potential in preparation of antitumor drugs.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to an anti-NKp46 antibody and its application. 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 CDRs and the conformational polymorphisms of the loop. Because different antibodies have high structural similarity in their FR regions, 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 well, 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 used to replace the CDR of a human antibody through CDR grafting technology, thereby recombining it with the human FR region to form a humanized antibody. If the FR regions of the human and murine antibodies match well, then the antigen-binding ability can still be retained.

[0004] 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, making cancer treatment a hot research topic in the medical field. Cancer immunotherapy is a successful cancer treatment method, and the application of antibody drugs in cancer immunotherapy has been recognized by the medical community. Antibody-mediated cancer immunotherapy strategies currently focus on regulating T cells, such as anti-CD3 antibodies that directly activate T cells, and immune checkpoint antibodies (represented by targets such as PD-1 and CTLA-4) that relieve T cell inhibitory signals. However, these therapies also have some limitations. Anti-CD3 antibodies can easily cause cytokine release syndrome, which can lead to a cytokine storm in severe cases, threatening life. Immune checkpoint antibodies, except for a few tumors such as melanoma and Hodgkin's lymphoma, are not very effective against most tumors. Therefore, there is an urgent need to develop new cancer immunotherapy strategies to address unmet medical needs.

[0005] NK cells, as a type of tumor-killing immune cell other than T cells, possess several unique characteristics. For example, NK cell killing activity is not MHC-restricted; they kill target cells by sensing abnormal innate immune signals on the surface of tumor cells. Furthermore, the release of cytokines after NK cell activation is relatively mild, offering a potential safety advantage compared to T cells. The mechanisms by which NK cells kill tumor cells mainly include cytotoxic granules (such as perforin and granzyme B), death receptors (including TRAIL and FASL), and cytokines (such as TNF and IFN-γ). NK cell activity is regulated by various activating and inhibitory receptors on their surface. Activating receptors mainly include the NCR family (NKp46, NKp30, NKp44), CD16, NKG2D, and DNAM1, while inhibitory receptors mainly include cytotoxic cell immunoglobulin-like receptors (KIRs) and NKG2A. NKp46 is one of the most important activating receptors on the NK cell surface, playing a crucial role in NK cell recognition of tumor cells and initiating killing, making it an important research target for tumor immunotherapy. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an anti-NKp46 antibody or a fragment thereof, which has good affinity and NKp46 signaling activation activity, can promote NK cell activation, and mediate PBMC killing of tumor cells, and has application potential in the preparation of anti-tumor drugs.

[0007] The amino acid sequences of the heavy chain complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3 of the anti-NKp46 antibody (46-3) 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 complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3 are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region VH of the anti-NKp46 antibody (46-3) is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.5.

[0009] The amino acid sequences of the heavy chain complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3 of another anti-NKp46 antibody (46-5) provided by this invention are shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively; and the amino acid sequences of the light chain complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3 are shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region VH of the anti-NKp46 antibody (46-5) is shown in SEQ ID NO.9, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.13.

[0011] Furthermore, the CDR region of the aforementioned antibody or its fragment can be spliced ​​with the FR region of the human antibody frame region to form a humanized antibody.

[0012] The amino acid sequence of the heavy chain variable region VH of the humanized anti-NKp46 antibody (46-3) is from SEQ ID NO.19, SEQ ID NO.20 or SEQ ID NO.21, and the amino acid sequence of the light chain variable region VL is from SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 or SEQ ID NO.25.

[0013] Furthermore, the amino acid sequence of the heavy chain variable region VH of the humanized anti-NKp46 antibody (46-5) is from SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29 or SEQ ID NO.30, and the amino acid sequence of the light chain variable region VL is from SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33 or SEQ ID NO.34.

[0014] Furthermore, the heavy chain constant region of the aforementioned antibody or its fragment may be derived from the constant region of the heavy chain of human immunoglobulins IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, and IgD, and the light chain constant region may be derived from the constant region of the light chain of human immunoglobulins κ and λ.

[0015] Furthermore, the antibody fragments mentioned above can be Fab (antigen binding fragment) or scFv (single-chain fragment variable).

[0016] The amino acid sequence of the anti-NKp46 antibody (46-3) scFv is SEQ ID NO.17. The amino acid sequence of the anti-NKp46 antibody (46-5) scFv is SEQ ID NO.18.

[0017] The present invention also provides nucleic acid molecules encoding the above-mentioned anti-NKp46 antibody or fragments thereof.

[0018] The present invention also provides a recombinant vector comprising a nucleic acid molecule encoding an antibody or fragment thereof against NKp46, wherein the recombinant vector may be a plasmid or a viral vector.

[0019] This invention also provides cells comprising the above-described recombinant vector, wherein the cells may be eukaryotic cells or prokaryotic cells. This invention also provides the use of the above-described anti-NKp46 antibody or fragments thereof in the preparation of antitumor drugs.

[0020] Furthermore, the tumor mentioned in the above-mentioned uses is acute monocytic leukemia.

[0021] The antibodies described above in this invention can be prepared into various forms of pharmaceutical formulations using conventional pharmaceutical techniques. Liquid injections and freeze-dried injections are preferred.

[0022] The antibodies described above in this invention can form pharmaceutical compositions with other drugs, and these compositions can be used in conjunction with other treatment methods to treat diseases, including chemotherapy, radiotherapy, and biological therapy.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention provides a series of anti-NKp46 antibodies or fragments thereof, which have good affinity and NKp46 signaling activation activity, can promote NK cell activation, and mediate PBMC killing of tumor cells, and have application potential in the preparation of anti-tumor drugs. Attached Figure Description

[0025] Figure 1 Flow cytometry analysis was used to determine the binding activity of anti-NKp46 antibody to CHOK1-NKp46 cells.

[0026] Figure 2 Activation activity of anti-NKp46 antibody on NKp46 reporter cell lines.

[0027] Figure 3 The activation activity of anti-NKp46 antibody on primary NK cells.

[0028] Figure 4 Activation activity of anti-NKp46 antibody on PBMC cells.

[0029] Figure 5 Flow cytometry was used to detect the expression of FcR in THP-1 cells of acute monocytic leukemia.

[0030] Figure 6 Antibodies 46-3 and 46-5 mediate the killing of acute monocytic leukemia cells (THP-1) by PBMCs.

[0031] Figure 7 Flow cytometry analysis was performed on the binding activity of 46-3 scFv and 46-5 scFv to CHOK1-NKp46 cells. Detailed Implementation

[0032] This invention provides a series of anti-NKp46 antibodies or fragments thereof, which have good affinity and NKp46 signaling activation activity, can promote NK cell activation, and mediate PBMC killing of tumor cells, and have application potential in the preparation of anti-tumor drugs.

[0033] The anti-NKp46 antibody of this invention was obtained through hybridoma screening. Human NKp46 extracellular domain 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. Primitive clones of hybridoma cells with good NKp46 binding activity were screened using ELISA and flow cytometry, and activity was further confirmed during the subcloning stage.

[0034] To obtain the amino acid sequence of hybridoma antibodies, this invention sequenced the antibody mRNA of hybridoma cells to obtain the variable region sequence of the hybridoma antibody, constructed an expression vector, transfected HEK293 cells for transient expression, and prepared eight recombinant anti-NKp46 monoclonal antibodies (46-1, 46-2, 46-3, 46-4, 46-5, 46-6, 46-7, 46-8).

[0035] Flow cytometry analysis confirmed that these eight anti-NKp46 antibodies (46-1, 46-2, 46-3, 46-4, 46-5, 46-6, 46-7, and 46-8) had good binding activity to the CHOK1 cell line (CHOK1-NKp46) that stably expresses NKp46. SPR affinity analysis confirmed that seven of these anti-NKp46 antibodies (46-1, 46-2, 46-3, 46-4, 46-5, 46-6, and 46-8) had good affinity. Functional activity analysis revealed that eight anti-NKp46 antibodies (46-1, 46-2, 46-3, 46-4, 46-5, 46-6, 46-7, and 46-8) exhibited limited activation of the NKp46 reporter cell line (NKp46 / CD3zeta-NFAT Reporter Jurkat cells) in cell culture supernatant. However, two anti-NKp46 antibodies (46-3 and 46-5) showed significant activation of the NKp46 reporter cell line in the coated state. Further analysis using primary NK cells and PBMCs confirmed that anti-NKp46 antibodies 46-3 and 46-5 showed limited activation in cell culture supernatant but significant activation in the coated state, suggesting their potential as effector elements of bispecific anti-NKp46-TAA (Tumor-Associated Antigen). Tumor cell killing activity assays showed that anti-NKp46 antibodies 46-3 and 46-5 could mediate the killing of acute monocytic leukemia cells THP-1 by PBMCs, demonstrating the application potential of 46-3 and 46-5 in the preparation of anti-tumor drugs.

[0036] The single-chain antibody (scFv) modified with the variable regions of anti-NKp46 antibodies 46-3 and 46-5 exhibits good binding activity to CHOK1-NKp46, demonstrating the application potential of 46-3 scFv and 46-5 scFv as multispecific antibody elements.

[0037] The murine anti-NKp46 antibodies 46-3 and 46-5 were humanized using CDR transplantation technology. SPR affinity analysis showed that the humanized heavy chains derived from human antibody germline templates IGHV1-69*08, IGHV1-2*06, and IGHV1-46*01, and the humanized light chains derived from human antibody germline templates IGKV1-39*01, IGKV1-12*01, IGKV1-6*01, and IGKV1-27*01, constructed from these human antibody germline templates, exhibited good affinity for the humanized antibody h46-3. The humanized antibody h46-5 constructed from the humanized heavy chains derived from human antibody germline templates IGHV4-31*02, IGHV4-34*09, IGHV4-30-4*01, IGHV4-61*01, and IGHV4-38-2*01 and the humanized light chains derived from human antibody germline templates IGKV1-33*01, IGKV1-39*02, IGKV1-6*01, and IGKV3-15*01 exhibits good affinity.

[0038] The anti-NKp46 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.

[0039] The anti-NKp46 antibody of this invention can further enhance its 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 amino acids of the antigen through hydrogen bonds, ionic bonds, van der Waals forces, etc. By mutating the amino acids in the antibody 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] The anti-NKp46 antibody of the present invention can be used for the detection of NKp46, including ELISA and flow cytometry. The anti-NKp46 antibody of the present invention has good affinity and NKp46 signaling activation activity, which can promote NK cell activation and mediate PBMC killing of tumor cells, showing potential application in the preparation of anti-tumor drugs.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] Example 1: Hybridoma cell screening

[0050] The murine anti-NKp46 antibody of this invention was obtained through hybridoma screening. Human NKp46 extracellular domain 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, and cultured to obtain cell culture supernatant containing the antibody. Hybridoma cell clones with good NKp46 binding activity were screened using ELISA and flow cytometry. Activity was further confirmed during the subcloning stage; subcloned cells were collected for antibody variable region sequencing and preparation of recombinant monoclonal antibodies for further activity confirmation.

[0051] Example 2: Obtaining the antibody variable region sequence

[0052] RNA was extracted from hybridoma cells using the Trizol method, and cDNA was obtained by reverse transcription using the RNA as a template. PCR amplification of the variable regions of the antibody's heavy and light chains 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 a recombinant plasmid. The recombinant plasmid was 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 to obtain the gene sequences of the antibody's heavy and light chain variable regions.

[0053] Example 3: Construction and preparation of recombinant monoclonal antibody against NKp46

[0054] 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 and light chains, respectively, and the results were identified by sequencing. 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, followed by ultrafiltration to replace the supernatant with PBS. Antibody purity and concentration were determined using reducing SDS-PAGE and NanoDrop 2000, and the antibodies were aliquoted and stored at -80℃ for later use.

[0055] Example 4: Screening of cell binding activity of anti-NKp46 antibody

[0056] CHOK1 cell lines stably expressing NKp46 (CHOK1-NKp46) were collected, with 1×E6 cells per group. Cells were washed once with PBS and centrifuged at 1000 g for 3 min. The cell pellet was resuspended in 100 μL of anti-NKp46 antibody (final concentration 10 μg / mL), PBS, and mouse isotype antibody mIgG (final concentration 10 μg / mL), and incubated on ice for 60 min. After incubation, cells were collected by centrifugation and washed twice with 300 μL of PBS. 100 μL of FITC-labeled goat anti-mouse antibody (1:200 dilution) was added to the cell pellet, resuspended, and incubated on ice in the dark for 60 min. After incubation, cells were collected by centrifugation and washed twice with 300 μL of PBS. Finally, cells were resuspended in 300 μL of PBS for flow cytometry analysis. The results showed that all eight anti-NKp46 monoclonal antibodies (46-1, 46-2, 46-3, 46-4, 46-5, 46-6, 46-7, and 46-8) could bind well to CHOK1-NKp46 cells (see [link to results]). Figure 1 ).

[0057] Example 5: Affinity screening of anti-NKp46 antibodies

[0058] The appropriate conjugation amount was calculated using the formula RL = (Rmax × MWligand) / (Sm × MWanalyte), and the anti-mouse antibody was conjugated to the CM5 chip using an aminecoupling kit. Hybridoma cell culture supernatant was captured onto the chip, and the response value of NKp46-HSA-His 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. The results showed that all seven anti-NKp46 antibodies (46-1, 46-2, 46-3, 46-4, 46-5, 46-6, and 46-8) exhibited good affinity, while one anti-NKp46 antibody (46-7) failed to yield any detectable data (see Table 1).

[0059]

[0060] Example 6: Screening of the activation activity of anti-NKp46 antibody on NKp46 reporter cell lines

[0061] NKp46 reporter cell line (NKp46 / CD3zeta-NFAT ReporterJurkat cells) (5×E4 cells / 50μL / well) was added to 96-well cell culture plates. Then, anti-NKp46 antibody (20 μg / mL, 50μL / well), along with equal volumes and concentrations of PBS and mouse isotype antibody mIgG, were added to the wells. The plates were incubated at 37℃ and 5% CO2 for 8 h. Luciferase assay reagent was added, and the plates were incubated at room temperature for 5 min before detection using a multi-functional microplate reader. The results showed that the signal values ​​of the eight anti-NKp46 antibody strains (46-1, 46-2, 46-3, 46-4, 46-5, 46-6, 46-7, and 46-8) were similar to those of the PBS and mIgG groups, indicating that the eight anti-NKp46 antibodies had limited activation of NKp46 signaling in the cell culture supernatant, suggesting that they cannot directly activate NK cells (see...). Figure 2 ).

[0062] To determine whether the eight anti-NKp46 antibodies mentioned above possess the potential to act as effector elements of anti-NKp46-TAA (Tumor-Associated Antigen) bispecific antibodies, anti-NKp46 antibodies were coated onto cell culture plates to mimic the binding of anti-NKp46-TAA bispecific antibodies to the solid surface of tumor cells. The activity of anti-NKp46 antibodies coated on the solid surface in activating NKp46 signaling was then tested. First, 96-well white transparent-bottom cell culture plates were coated with anti-mouse antibody (400 ng / well, 100 μL / well) and incubated overnight at 4°C. The plates were washed twice with PBS, and then anti-NKp46 antibody (10 μg / mL, 100 μL / well), along with equal volumes and concentrations of PBS and mouse isotype antibody mIgG, were added. The plates were then incubated at 37°C, 5% CO2 for 1 h for antibody coating. Discard the supernatant, add NKp46 reporter cell line (5×E4 cells / 50 μL / well), and incubate at 37℃, 5% CO2 for 8 h. Add luciferase detection reagent, incubate at room temperature for 5 min, and then perform detection using a multi-functional microplate reader. The results showed that antibodies 46-3 and 46-5 exhibited strong signal values, indicating that antibodies 46-3 and 46-5 can activate NKp46 signaling on the solid-phase surface and have the potential to serve as effector elements of anti-NKp46-TAA bispecific antibodies (see...). Figure 2 ).

[0063] Example 7: Screening of the activation activity of anti-NKp46 antibody on primary NK cells

[0064] Human primary NK cells (5×E4 cells / 50 μL / well) were added to 96-well cell culture plates. Then, anti-NKp46 antibody (20 μg / mL, 50 μL / well), along with equal volumes and concentrations of PBS and mouse isotype antibody mIgG, were added to the wells. The plates were incubated at 37°C and 5% CO2 for 72 h. IFN-γ levels in the cell culture supernatant were detected using an ELISA kit. The results showed that none of the eight anti-NKp46 antibodies (46-1, 46-2, 46-3, 46-4, 46-5, 46-6, 46-7, and 46-8) significantly stimulated NK cells to secrete IFN-γ. The detected values ​​were similar to those in the PBS and mIgG groups, indicating that the eight antibodies had limited activation of NK cells in the cell culture supernatant, avoiding direct activation and demonstrating good safety, thus reducing the risk of cytokine storm (see [link to ELISA]). Figure 3 ).

[0065] In another antibody-coated system, 96-well cell culture plates were first coated with anti-mouse antibody (400 ng / well, 100 μL / well) and incubated overnight at 4°C. The plates were washed twice with PBS, and then anti-NKp46 antibody (10 μg / mL, 100 μL / well), along with equal volumes and concentrations of PBS and mouse isotype antibody mIgG, were added. The plates were then incubated at 37°C, 5% CO2 for 1 h for antibody coating. The supernatant was discarded, and primary NK cells (5×E4 cells / 50 μL / well) were added. The plates were incubated at 37°C, 5% CO2 for 72 h, and IFN-γ in the cell culture supernatant was detected using an ELISA kit. The results showed that antibodies 46-3 and 46-5 significantly stimulated NK cells to secrete IFN-γ, indicating that antibodies 46-3 and 46-5 can activate NK cells on the solid-phase surface and have the potential to serve as effector elements of anti-NKp46-TAA bispecific antibodies (see [link to ELISA]). Figure 3 ).

[0066] Example 8: Screening of the activation activity of anti-NKp46 antibody on PBMC cells

[0067] Human PBMC cells (5×E4 cells / 50 μL / well) were added to 96-well cell culture plates. Then, anti-NKp46 antibody (20 μg / mL, 50 μL / well), along with equal volumes and concentrations of PBS and mouse isotype antibody mIgG, were added to the wells. The plates were incubated at 37°C and 5% CO2 for 48 h. IFN-γ levels in the cell culture supernatant were detected using an ELISA kit. The results showed that none of the eight anti-NKp46 antibodies (46-1, 46-2, 46-3, 46-4, 46-5, 46-6, 46-7, and 46-8) effectively stimulated PBMC cells to secrete IFN-γ in the cell culture supernatant. The detected values ​​were similar to those in the PBS and mIgG groups, indicating that the eight anti-NKp46 antibodies had limited activation effects on PBMC cells in the cell culture supernatant, avoiding direct activation of PBMC cells and demonstrating good safety, thus reducing the risk of cytokine storm (see [link to ELISA]). Figure 4 ).

[0068] In another antibody-coated system, 96-well cell culture plates were first coated with anti-mouse antibody (400 ng / well, 100 μL / well) and incubated overnight at 4°C. The plates were washed twice with PBS, and then anti-NKp46 antibody (10 μg / mL, 100 μL / well), along with equal volumes and concentrations of PBS and mouse isotype antibody mIgG, were added. The plates were then incubated at 37°C, 5% CO2 for 1 h for antibody coating. The supernatant was discarded, and human PBMC cells (5×E4 cells / 50 μL / well) were added. The plates were incubated at 37°C, 5% CO2 for 48 h, and IFN-γ in the cell culture supernatant was detected using an ELISA kit. The results showed that antibodies 46-3 and 46-5 significantly stimulated PBMC cells to secrete IFN-γ, indicating that antibodies 46-3 and 46-5 can activate PBMC cells on the solid-phase surface and have the potential to serve as effector elements of anti-NKp46-TAA bispecific antibodies (see [link to ELISA]). Figure 4 ).

[0069] Example 9: Tumor cell killing activity mediated by anti-NKp46 antibodies 46-3 and 46-5

[0070] Stable mCherry-expressing acute monocytic leukemia cells THP-1 (THP-1-mCherry) were collected as model tumor cells. THP-1 cells are a mononuclear cell line whose surface-expressed Fc receptors (FcRs) can bind to the Fc of antibodies. In this embodiment, the binding of the Fc of anti-NKp46 antibody to the Fc of the Fc on the surface of THP-1 cells was used to mimic the tumor-associated antigen (TAA), thereby examining whether the anti-NKp46 antibody can exert a similar effect to the anti-NKp46-TAA bispecific antibody by binding to both NKp46 on the surface of NK cells and the FcR on the surface of THP-1 tumor cells.

[0071] First, flow cytometry was used to confirm FcR expression in THP-1 cells. The primary antibodies used were PBS, mouse isotype antibody mIgG, and anti-NKp46 antibodies 46-3 and 46-5. The secondary antibody was a FITC-labeled goat anti-mouse antibody. Flow cytometry results showed that THP-1 cells expressed FcR, could bind to mIgG, and could bind to antibodies 46-3 and 46-5. Figure 5 THP-1 cells were added to 96-well plates (5×E4 cells / 50 μL / well). 50 μL of antibody 46-3 (final concentration 10 μg / mL), antibody 46-5 (final concentration 10 μg / mL), mouse isotype antibody mIgG (final concentration 10 μg / mL), and 1640 medium were added to each well, mixed well, and incubated at 37°C in a 5% CO2 incubator for 30 min. After incubation, the cells were centrifuged for 5 min to remove the supernatant, and resuspended in 100 μL of medium. The ratio of effector cells (PBMCs) to target cells (THP-1 cells) was set at 10:T = 10:1. 100 μL of PBMC suspension (5×E5 cells / well) was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator. The fluorescence signal of THP-1 cells was observed under a 100x fluorescence microscope at 24 h, 48 h, and 72 h. The results showed that THP-1 cells in the antibody 46-3 and 46-5 groups began to decrease at 24 h, and the decreasing trend was more obvious at 72 h, while the THP-1 cells in the mIgG group and the 1640 medium group did not change much, indicating that antibodies 46-3 and 46-5 can mediate the killing of tumor cells THP-1 by PBMCs. Figure 6 The above results further demonstrate that antibodies 46-3 and 46-5 can exert effects similar to anti-NKp46-TAA bispecific antibodies by binding to both NKp46 on the surface of NK cells and FcR on the surface of THP-1 tumor cells, and can be further developed as effector elements for anti-NKp46-TAA bispecific antibodies. The amino acid sequence information of the variable regions of antibodies 46-3 and 46-5 is shown in Table 2.

[0072]

[0073] Example 10: Construction and preparation of single-chain antibodies 46-3 and 46-5

[0074] Single-chain antibodies (scFv) were constructed by fusing the C-terminus of the heavy chain variable region VH and the N-terminus of the light chain variable region VL of antibodies 46-3 and 46-5 respectively with a linker (GGGGSGGGGSGGGGSGGGGS). The amino acid sequences of these scFvs are shown in Table 3. The scFvs were further fused with the Fc fragment of human IgG1 to construct the scFv-Fc fusion protein. The scFv-Fc gene was synthesized and inserted into the pTT5 plasmid. The recombinant plasmid was transfected into HEK293 cells using the PEI method, followed by serum-free suspension culture for transient antibody expression. Cell supernatant was collected after 7 days of culture, filtered through a 0.22 μm filter, and purified by Protein A affinity chromatography. The antibody was ultrafiltered and replaced with PBS solution. Antibody purity was assessed by reducing SDS-PAGE, and concentration was determined using NanoDrop 2000. The purified antibody was then aliquoted and stored at -80℃ for later use. Flow cytometry was used to analyze whether 46-3 scFv and 46-5 scFv had binding activity against CHOK1-NKp46 cells. The primary antibodies used in each group were PBS (control), human isotype antibody hIgG (control), 46-3 scFv, and 46-5 scFv, respectively. The secondary antibody was FITC-labeled goat anti-human antibody. Flow cytometry results showed that 46-3 scFv and 46-5 scFv had good binding activity against CHOK1-NKp46 (see...). Figure 7 ).

[0075]

[0076] Example 11, Antibody Humanization

[0077] Antibody humanization employed CDR transplantation technology. The variable region of the murine antibody was aligned with the germline sequence of the human antibody. Based on factors such as sequence consistency, similarity, and conservation, a suitable human antibody germline sequence was selected as the humanization template. The murine antibody CDR replaced the CDR of the human antibody germline sequence, forming humanized heavy chain variable region (VH) and light chain variable region (VL) sequences. Five humanized VH sequences and four humanized VL sequences were designed for each murine antibody, resulting in 20 different VH / VL combinations and thus 20 humanized antibody variable regions. These humanized antibody variable regions were further spliced ​​with the human antibody constant regions, ultimately forming 20 complete IgG1κ type humanized antibodies. The designs of the 20 46-3 humanized antibodies (h46-3) are shown in Table 4, and the designs of the 20 46-5 humanized antibodies (h46-5) are shown in Table 5.

[0078]

[0079]

[0080] Example 12: Expression of humanized antibodies

[0081] The heavy and light chain genes of the synthesized antibody were inserted into the pTT5 plasmid, and the recombinant plasmid was transfected into HEK293 cells using the PEI method. The cells were then cultured in serum-free suspension for transient expression of the humanized antibody. The supernatant from the HEK293 cells cultured for 7 days was collected, filtered through a 0.22 μm filter membrane, and stored at 4°C for later use.

[0082] Example 13: Affinity Screening of Humanized Antibodies

[0083] The appropriate conjugation amount was calculated using the formula RL = (Rmax × MWligand) / (Sm × MWanalyte), and the anti-human antibody was conjugated to the CM5 chip using an aminecoupling kit. The expression supernatant of the humanized antibody was captured onto the chip, and the response value of NKp46-HSA-His flowing through the channel was detected using a Biacore 8K detector. Analysis of humanized antibodies in section 46-3 revealed that eight humanized antibodies with heavy chains derived from human germline genes IGHV1-69-2*01 and IGHV1-24*01 failed to produce a signal, while the remaining 12 humanized antibodies showed good affinity (see Table 6). Analysis of humanized antibodies in section 46-5 showed that all 20 humanized antibodies showed good affinity (see Table 7).

[0084]

[0085] .

[0086] sequence list

[0087] SEQ ID NO.1

[0088] 46-3 Heavy Chain Variable Region VH Amino Acid Sequence

[0089] EVQLQQSGPELVKPGASVKISCKTSGYSFTDYYIHWVKQSHVKGLEWVGRIKPINGATTNNQNFIDKASLTVDKSSNIAYMELHNLASEDSAVYYCARTFYDGHYAWFTYWGQGTPVTVSA

[0090] SEQ ID NO.2

[0091] 46-3 Heavy Chain Variable Region VH-CDR1 Amino Acid Sequence

[0092] GYSFTDYY

[0093] SEQ ID NO.3

[0094] 46-3 Heavy Chain Variable Region VH-CDR2 Amino Acid Sequence

[0095] IKPINGAT

[0096] SEQ ID NO.4

[0097] 46-3 Heavy Chain Variable Region VH-CDR3 Amino Acid Sequence

[0098] ARTFYDGHYAWFTY

[0099] SEQ ID NO.5

[0100] 46-3 Light chain variable region VL amino acid sequence

[0101] DIVMTQSPASLSVSVGETVTITCRASENIYSNLAWYQLKEGKSPQLLVYTATNLAAGVASRFSGSGSGTHFFLMINSLQSEDFGNYYCQHFWDTPWTFGGGTKLEIK

[0102] SEQ ID NO.6

[0103] 46-3 Light chain variable region VL-CDR1 amino acid sequence

[0104] ENIYSN

[0105] SEQ ID NO.7

[0106] 46-3 Light chain variable region VL-CDR2 amino acid sequence

[0107] TAT

[0108] SEQ ID NO.8

[0109] 46-3 Light chain variable region VL-CDR3 amino acid sequence

[0110] QHFWDTPWT

[0111] SEQ ID NO.9

[0112] 46-5 Heavy chain variable region VH amino acid sequence

[0113] EVKLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWSWIRQFPGDKLEWMGSISYDGNSNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKIYYDYDGDFDVWGAGTTVTVSS

[0114] SEQ ID NO.10

[0115] 46-5 Heavy chain variable region VH-CDR1 amino acid sequence

[0116] GYSITSGYY

[0117] SEQ ID NO.11

[0118] 46-5 Heavy chain variable region VH-CDR2 amino acid sequence

[0119] ISYDGNS

[0120] SEQ ID NO.12

[0121] 46-5 Heavy chain variable region VH-CDR3 amino acid sequence

[0122] AKIYYDYDGDFDV

[0123] SEQ ID NO.13

[0124] 46-5 Light chain variable region VL amino acid sequence

[0125] SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTVFTFTITTVQAEDLAVYFCLQDYSSPWTFGGGTKLEIK

[0126] SEQ ID NO.14

[0127] 46-5 Light chain variable region VL-CDR1 amino acid sequence

[0128] QSVSND

[0129] SEQ ID NO.15

[0130] 46-5 Light chain variable region VL-CDR2 amino acid sequence

[0131] YAS

[0132] SEQ ID NO.16

[0133] 46-5 Light chain variable region VL-CDR3 amino acid sequence

[0134] LQDYSSPWT

[0135] SEQ ID NO.17

[0136] 46-3 scFv amino acid sequence

[0137] EVQLQQSGPELVKPGASVKISCKTSGYSFTDYYIHWVKQSHVKGLEWVGRIKPINGATTNNQNFIDKASLTVDKSSNIAYMELHNLASEDSAVYYCARTFYDGHYAWFTYWGQGTPVTVSAGGG GSGGGGSGGGGSGGGGSDIVMTQSPASLSVSVGETVTITCRASENIYSNLAWYQLKEGKSPQLLVYTATNLAAGVASRFSGSGSGTHFFLMINSLQSEDFGNYYCQHFWDTPWTFGGGTKLEIK

[0138] SEQ ID NO.18

[0139] 46-5 scFv amino acid sequence

[0140] EVKLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWSWIRQFPGDKLEWMGSISYDGNSNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKIYYDYDGDFDVWGAGTTVTVSSGGG GSGGGGSGGGGSGGGGSSIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTVFTFTITTVQAEDLAVYFCLQDYSSPWTFGGGTKLEIK

[0141] SEQ ID NO.19

[0142] 46-3 Humanized antibody heavy chain variable region VH amino acid sequence (IGHV1-69*08)

[0143] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYYIHWVRQAPGQGLEWMGRIKPINGATTNNQNFIDRVTITADKSTSTAYMELSSLRSEDTAVYYCARTFYDGHYAWFTYWGQGTLVTVSS

[0144] SEQ ID NO.20

[0145] 46-3 Humanized antibody heavy chain variable region VH amino acid sequence (IGHV1-2*06)

[0146] QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYIHWVRQAPGQGLEWMGRIKPINGATTNNQNFIDRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTFYDGHYAWFTYWGQGTLVTVSS

[0147] SEQ ID NO.21

[0148] 46-3 Humanized antibody heavy chain variable region VH amino acid sequence (IGHV1-46*01)

[0149] QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYIHWVRQAPGQGLEWMGRIKPINGATTNNQNFIDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARTFYDGHYAWFTYWGQGTLVTVSS

[0150] SEQ ID NO.22

[0151] 46-3 Humanized antibody light chain variable region VL amino acid sequence (IGKV1-39*01)

[0152] DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLIYTATNLAAGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWDTPWTFGGGTKVEIK

[0153] SEQ ID NO.23

[0154] 46-3 Humanized antibody light chain variable region VL amino acid sequence (IGKV1-12*01)

[0155] DIQMTQSPSSVSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLIYTATNLAAGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWDTPWTFGGGTKVEIK

[0156] SEQ ID NO.24

[0157] 46-3 Humanized antibody light chain variable region VL amino acid sequence (IGKV1-6*01)

[0158] AIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLIYTATNLAAGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWDTPWTFGGGTKVEIK

[0159] SEQ ID NO.25

[0160] 46-3 Humanized antibody light chain variable region VL amino acid sequence (IGKV1-27*01)

[0161] DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKVPKLLIYTATNLAAGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHFWDTPWTFGGGTKVEIK

[0162] SEQ ID NO.26

[0163] 46-5 Humanized antibody heavy chain variable region VH amino acid sequence (IGHV4-31*02)

[0164] QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYYWSWIRQHPGKGLEWIGSISYDGNSNYNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIYYDYDGDFDVWGQGTLVTVSS

[0165] SEQ ID NO.27

[0166] 46-5 Humanized antibody heavy chain variable region VH amino acid sequence (IGHV4-34*09)

[0167] QVQLQESGPGLVKPSQTLSLTCAVYGYSITSGYYWSWIRQPPGKGLEWIGSISYDGNSNYNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIYYDYDGDFDVWGQGTLVTVSS

[0168] SEQ ID NO.28

[0169] 46-5 Humanized antibody heavy chain variable region VH amino acid sequence (IGHV4-30-4*01)

[0170] QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYYWSWIRQPPGKGLEWIGSISYDGNSNYNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIYYDYDGDFDVWGQGTLVTVSS

[0171] SEQ ID NO.29

[0172] 46-5 Humanized antibody heavy chain variable region VH amino acid sequence (IGHV4-61*01)

[0173] QVQLQESGPGLVKPSETLSLTCTVSGYSITSGYYWSWIRQPPGKGLEWIGSISYDGNSNYNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIYYDYDGDFDVWGQGTLVTVSS

[0174] SEQ ID NO.30

[0175] 46-5 Humanized antibody heavy chain variable region VH amino acid sequence (IGHV4-38-2*01)

[0176] QVQLQESGPGLVKPSETLSLTCAVSGYSITSGYYWSWIRQPPGKGLEWIGSISYDGNSNYNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIYYDYDGDFDVWGQGTLVTVSS

[0177] SEQ ID NO.31

[0178] 46-5 Humanized antibody light chain variable region VL amino acid sequence (IGKV1-33*01)

[0179] DIQMTQSPSSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYASNRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQDYSSPWTFGGGTKVEIK

[0180] SEQ ID NO.32

[0181] 46-5 Humanized antibody light chain variable region VL amino acid sequence (IGKV1-39*02)

[0182] DIQMTQSPSFLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYSSPWTFGGGTKVEIK

[0183] SEQ ID NO.33

[0184] 46-5 Humanized antibody light chain variable region VL amino acid sequence (IGKV1-6*01)

[0185] AIQMTQSPSSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYSSPWTFGGGTKVEIK

[0186] SEQ ID NO.34

[0187] 46-5 Humanized antibody light chain variable region VL amino acid sequence (IGKV3-15*01)

[0188] EIVMTQSPATLSVSPGERATLSCKASQSVSNDVAWYQQKPGQAPRLLIYYASNRYTGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCLQDYSSPWTFGGGTKVEIK

Claims

1. An anti-NKp46 antibody or a fragment thereof, characterized in that: The amino acid sequences of the heavy chain complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3 are shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively, and the amino acid sequences of the light chain complementarity-determining regions 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 anti-NKp46 antibody or a fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

5.

3. The anti-NKp46 antibody or a fragment thereof according to claim 1, characterized in that: The antibody is a humanized antibody, formed by splicing the complementarity-determining region (CDR) of the anti-NKp46 antibody according to claim 1 with the humanized antibody frame region (FR); further, the amino acid sequence of the heavy chain variable region (VH) of the humanized antibody is from SEQ ID NO.19, SEQ ID NO.20 or SEQ ID NO.21; the amino acid sequence of the light chain variable region (VL) is from SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 or SEQ ID NO.

25.

4. An anti-NKp46 antibody or a fragment thereof, characterized in that: The amino acid sequences of the heavy chain complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3 are shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively, and the amino acid sequences of the light chain complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3 are shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.

5. The anti-NKp46 antibody or a fragment thereof according to claim 4, characterized in that: The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.9, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

13.

6. The anti-NKp46 antibody or a fragment thereof according to claim 4, characterized in that: The antibody is a humanized antibody, formed by splicing the complementarity-determining region (CDR) of the anti-NKp46 antibody as described in claim 4 with the humanized antibody frame region (FR); furthermore, the amino acid sequence of the heavy chain variable region (VH) of the humanized antibody is from SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29 or SEQ ID NO.30; and the amino acid sequence of the light chain variable region (VL) is from SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33 or SEQ ID NO.

34.

7. The anti-NKp46 antibody or a fragment thereof according to any one of claims 1 to 6, characterized in that: The antibody fragment is scFv (single-chain fragment variable) or Fab (antigen binding fragment); further, when the antibody fragment is scFv, the amino acid sequence of the variable region is as shown in SEQ ID NO.17 or SEQ ID NO.

18.

8. The anti-NKp46 antibody or a fragment thereof according to any one of claims 1 to 7, characterized in that: The heavy chain constant region of the antibody is derived from the constant region of the heavy chain of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA or IgD, and the light chain constant region is derived from the constant region of the light chain of human immunoglobulin κ or λ.

9. A nucleic acid molecule encoding the anti-NKp46 antibody or a fragment thereof as described in any one of claims 1 to 8.

10. A recombinant vector comprising the nucleic acid molecule of claim 9.

11. Cells comprising the recombinant vector of claim 10.

12. The use of the anti-NKp46 antibody or fragment thereof according to any one of claims 1 to 8 in the preparation of an antitumor drug; further, the tumor is acute monocytic leukemia.

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