Novel anti-NKP46 antibodies and uses thereof

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to NKp46, and nucleic acid molecules encoding said antibodies or antigen-binding fragments thereof. The antibody disclosed by the invention is combined with a natural killer cell specific activation receptor and NKp46 with high affinity, so that the immunocompetence of NK cells to various pathogenic substances is remarkably enhanced. The antibodies of the invention may also be used to provide reliable information about the presence and number of natural killer cells in a biological sample, or to sort them in a sample with high selectivity.

Description

Technical Field

[0001] The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to NKp46 (an activating receptor expressed on natural killer cells) and their therapeutic uses. This invention was supported by the National New Drug Creation Project of the National Science and ICT Research Foundation (RS-2023-00258675) and funded by the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, and the Ministry of Health and Welfare. Background Art

[0002] Cancer immunotherapy is performed by targeting cancer cells with antibodies specific for cancer cell surface antigens, killing cancer cells through fusion of antigen-presenting cells with cancer cells, or tumor-specific activation of immune cells such as natural killer cells and T cells.

[0003] Natural killer cells (NK cells) are lymphocytes that make up approximately 10% of blood cells and play a crucial role in immune responses. NK cells perform a variety of biological functions, but their ability to eliminate cancer cells or cells infected with foreign pathogens is particularly important for eliminating abnormal cells that have the potential to become pathological.

[0004] Under normal circumstances, most NK cells present in the body are in an inactivated state. However, because activated NK cells are required for therapeutic applications, active research is underway to effectively activate NK cells from healthy or patient blood.

[0005] Three activating receptors found on NK cells, NKp30, NKp44, and NKp46, are natural cytotoxicity receptors (NCRs) that play an important role in NK cell anti-tumor and antiviral defense. NKp46 is expressed exclusively by NK cells, making it not only an important activating receptor but also a valuable marker for identifying, isolating, or detecting NK cells. Its ligands range from viral ligands such as hemagglutinin (HA) and hemagglutinin-neuraminidase (HN) from influenza, Sendai, Newcastle disease, and poxviruses to bacterial ligands such as Fusobacterium nucleatum, as well as ligands expressed by tumors, adipocytes, and human pancreatic β cells.

[0006] Therefore, there is an increasing demand for the development of antibodies that specifically recognize NKp46, as they can be used to enhance the immune activity of NK cells against various pathogenic substances and to specifically detect and isolate NK cells from mixtures containing heterogeneous cell populations.

[0007] In this specification, many publications and patent documents are cited and cited. The cited publications and patent documents are incorporated herein by reference in their entirety to more clearly describe the state of the art and content of the present invention. Summary of the Invention

[0008] Technical issues

[0009] The present inventors have conducted intensive research to develop a highly effective anti-NKp46 antibody that binds with high affinity to NKp46, an activating receptor expressed on natural killer (NK) cells, promoting the activation of endogenous or exogenously administered NK cells and enhancing the expansion of NK cells ex vivo, thereby inducing a synergistic anti-tumor immune response. The results demonstrate that the present inventors have successfully identified a number of antibodies that recognize NKp46 with significant affinity and specificity and elucidated the structures of their antigen-binding regions, thereby completing the present invention.

[0010] Therefore, an object of the present invention is to provide antibodies or antigen-binding fragments thereof that specifically bind to NKp46 and nucleic acid molecules encoding the same.

[0011] Another object of the present invention is to provide a composition for preventing or treating cancer or infectious diseases, which composition comprises the antibody or its antigen-binding fragment, or a nucleic acid molecule encoding the antibody or its antigen-binding fragment as an active ingredient, a composition for activating immune cells, a composition for detecting NKp46-expressing cells, and a composition for diagnosing cancer.

[0012] Other objects and advantages of the present invention will become more apparent from the following detailed description, the appended claims and the accompanying drawings.

[0013] Technical Solution

[0014] In one aspect of the present invention, an antibody or antigen-binding fragment thereof that specifically binds to NKp46 is provided, comprising a heavy chain variable region comprising a HCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-38; a HCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-47; and a HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48-56.

[0015] The present inventors have conducted intensive research to develop a highly effective anti-NKp46 antibody that binds with high affinity to NKp46 (an activating receptor expressed on natural killer (NK) cells), promotes the activation of endogenous or exogenously administered NK cells, and enhances the ex vivo expansion of NK cells, thereby inducing a synergistic anti-tumor immune response. The results have shown that a number of antibodies that recognize NKp46 have been identified with significant affinity and specificity, as well as the structures of their antigen recognition sites.

[0016] As used herein, the term "antibody" refers to a peptide molecule that specifically recognizes and binds to a specific epitope of NKp46, including antigen-binding fragments (immunologically active fragments) of the antibody as well as the entire antibody molecule. The structure of a complete antibody has two full-length light chains and two full-length heavy chains, which are interconnected by disulfide bonds. The heavy chain constant region has γ (γ), mu (μ), α (α), delta (δ), and ε (ε) types, and is further divided into γ1 (γ1), γ2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0017] As used herein, the term "antigen-binding fragment of an antibody" refers to a polypeptide portion that has significant antigen-antibody binding function within the overall immunoglobulin structure. Examples of antigen-binding fragments include, but are not limited to, Fab, F(ab'), F(ab')2, Fv, and nanobodies.

[0018] Among antibody fragments, Fab is a structure with one antigen-binding site, which includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain.

[0019] Fab' differs from Fab in that it has a hinge domain that contains one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab').2 antibodies are produced by disulfide bonds formed by the cysteine ​​residues in the Fab' hinge. WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 0.7086 and WO88 / 09344 disclose recombinant technologies for producing Fv fragments with minimal antibody fragments having only heavy and light chain variable regions. A two-chain Fv has a heavy and light chain variable region linked by a non-covalent bond, while a single-chain Fv has a heavy and light chain variable region linked by a covalent bond (usually through a peptide linker or directly at the C-terminus), which can form a dimer-like structure.

[0020] As used herein, the term "light chain" refers to a full-length light chain and fragments thereof, including a variable region VL and a constant region CL, whose amino acid sequence has sufficient variable region sequence to confer antigen specificity.

[0021] As used herein, the term "heavy chain" refers to a full-length heavy chain and fragments thereof, comprising one variable region VH and three constant regions CH1, CH2 and CH3, wherein the amino acid sequence has sufficient variable region sequence to confer antigen specificity.

[0022] As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulins. The heavy chain (HCDR1, HCDR2, and HCDR3) and light chain (LCDR1, LCDR2, and LCDR3) each contain three CDRs that provide the primary contact residues for antibody binding to antigen or epitope.

[0023] The antibodies or antigen-binding fragments of the present invention include variants with conservative amino acid substitutions in the CDR regions. Furthermore, the antibodies or antigen-binding fragments of the present invention include amino acid variants of the sequences listed in the accompanying sequence listing, such that they are capable of specifically recognizing NKp46. For example, additional changes may be made to the antibody's amino acid sequence to further enhance its binding affinity and / or other biological properties. Such modifications include deletions, insertions, and / or substitutions of residues in the antibody's amino acid sequence. These amino acid modifications are based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine are similar in size; and phenylalanine, tryptophan, and tyrosine are similar in shape. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine are biologically functional equivalents.

[0024] In addition, amino acid substitutions in proteins that do not alter the overall activity of the molecule are known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common amino acid residue substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0025] Taking into account biologically equivalent variations, the amino acid sequences comprising the antibodies of the present invention may include sequences that have substantial identity thereto. Sequences that have substantial identity exhibit at least 61%, specifically at least 70%, more specifically at least 80%, and even more specifically at least 90% similarity to the amino acids of the present invention, as measured using one of the sequence comparison algorithms. Methods of sequence alignment for comparison are well-known in the art. Various alignment methods and algorithms are disclosed in Huang et al., Comp. Appl. BioSci 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994).

[0026] According to a specific embodiment, the antibody or antigen-binding fragment thereof further comprises a light chain variable region, wherein the light chain variable region comprises LCDR1 having an amino acid sequence selected from SEQ ID NOs: 1-6; LCDR2 having an amino acid sequence selected from SEQ ID NOs: 7-12; and LCDR3 having an amino acid sequence selected from SEQ ID NOs: 13-19.

[0027] According to one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of (a)-(m):

[0028] (a) an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 1, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 7, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 13; and a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 33, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 39, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 48;

[0029] (b) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 2, a LCDR2 having the amino acid sequence of SEQ ID NO: 8, and a LCDR3 having the amino acid sequence of SEQ ID NO: 14; and a HCDR1 having the amino acid sequence of SEQ ID NO: 34, a HCDR2 having the amino acid sequence of SEQ ID NO: 40, and a HCDR3 having the amino acid sequence of SEQ ID NO: 49;

[0030] (c) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 3, a LCDR2 having the amino acid sequence of SEQ ID NO: 9, and a LCDR3 having the amino acid sequence of SEQ ID NO: 15; and a HCDR1 having the amino acid sequence of SEQ ID NO: 35, a HCDR2 having the amino acid sequence of SEQ ID NO: 41, and a HCDR3 having the amino acid sequence of SEQ ID NO: 50;

[0031] (d) an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 4, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 10, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 16; and a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 35, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 42, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 51;

[0032] (e) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 5, a LCDR2 having the amino acid sequence of SEQ ID NO: 11, and a LCDR3 having the amino acid sequence of SEQ ID NO: 17; and a HCDR1 having the amino acid sequence of SEQ ID NO: 35, a HCDR2 having the amino acid sequence of SEQ ID NO: 42, and a HCDR3 having the amino acid sequence of SEQ ID NO: 52;

[0033] (f) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 5, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; and a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 35, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 43, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 52;

[0034] (g) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 5, a LCDR2 having the amino acid sequence of SEQ ID NO: 11, and a LCDR3 having the amino acid sequence of SEQ ID NO: 17; and a HCDR1 having the amino acid sequence of SEQ ID NO: 36, a HCDR2 having the amino acid sequence of SEQ ID NO: 44, and a HCDR3 having the amino acid sequence of SEQ ID NO: 53;

[0035] (h) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of SEQ ID NO: 10, and a LCDR3 having the amino acid sequence of SEQ ID NO: 19; and a HCDR1 having the amino acid sequence of SEQ ID NO: 37, a HCDR2 having the amino acid sequence of SEQ ID NO: 45, and a HCDR3 having the amino acid sequence of SEQ ID NO: 54;

[0036] (i) an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 5, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 17; and a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 38, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 42, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 55;

[0037] (j) an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 6, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; and a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 35, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 43, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 52;

[0038] (k) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 1, a LCDR2 having the amino acid sequence of SEQ ID NO: 7, and a LCDR3 having the amino acid sequence of SEQ ID NO: 13; and a HCDR1 having the amino acid sequence of SEQ ID NO: 33, a HCDR2 having the amino acid sequence of SEQ ID NO: 46, and a HCDR3 having the amino acid sequence of SEQ ID NO: 48;

[0039] (1) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 5, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 12, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 17; and a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 36, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 44, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 53; and

[0040] (m) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of SEQ ID NO: 10, and a LCDR3 having the amino acid sequence of SEQ ID NO: 19; and a HCDR1 having the amino acid sequence of SEQ ID NO: 37, a HCDR2 having the amino acid sequence of SEQ ID NO: 47, and a HCDR3 having the amino acid sequence of SEQ ID NO: 56.

[0041] More specifically, (a) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 40 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 92. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 105.

[0042] More specifically, (b) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 41 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 93. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 106.

[0043] More specifically, (c) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 42 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 94. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 107.

[0044] More specifically, (d) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 43 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 95. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 108.

[0045] More specifically, (e) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 44 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 96. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 109.

[0046] More specifically, (f) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 45 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 97. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 110.

[0047] More specifically, (g) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 46 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 98. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 111.

[0048] More specifically, (h) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 47 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 99. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 112.

[0049] More specifically, (i) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 48 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 100. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 113.

[0050] More specifically, (j) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 49 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 101. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 114.

[0051] More specifically, (k) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 50 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 102. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 115.

[0052] More specifically, (1) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 51 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 103. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 116.

[0053] More specifically, (m) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 52 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 104. Most specifically, (a) comprises the amino acid sequence of SEQ ID NO: 117.

[0054] In another aspect of the present invention, a nucleic acid molecule encoding the antibody or antigen-binding fragment of the present invention that specifically binds to NKp46 is provided.

[0055] As used herein, the term "nucleic acid molecule" refers to DNA (gDNA and cDNA) and RNA molecules. Nucleotides are the basic structural units of nucleic acid molecules and include not only natural nucleotides but also analogs with modified sugar or base moieties (Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)). For those skilled in the art, the nucleotide sequence encoding the amino acid sequence of a full-length antibody or its heavy chain or light chain variable region may involve modifications, including additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0056] The nucleic acid molecules of the present invention are interpreted as including nucleotide sequences that are substantially identical to any of the above-mentioned nucleotide sequences. Substantial identity refers to sequences that exhibit at least 80% homology, particularly at least 90% homology, and more particularly at least 95% homology, as determined by aligning the sequence of the present invention with any other sequence so as to correspond to each other as closely as possible and analyzing the aligned sequences using algorithms commonly used in the art.

[0057] According to a specific embodiment, the nucleic acid molecule used in the present invention can be an mRNA molecule, more specifically an in vitro transcribed (IVT) mRNA.

[0058] When mRNA is used as a nucleic acid molecule of the present invention, various modifications can be made to improve the expression (translation) efficiency of the antibody or antigen-binding fragment, including, for example, changing the length of the poly (A) tail or replacing certain adenine bases; modifying the 5' cap; using one or more modified nucleotides. Modified nucleotides include, for example, N1-methylpseudouridine, pseudouridine, 2-thiouridine, 5-methyluridine or 5-methylcytidine, 5-methylcytidine and 5-methoxyuracil, but are not limited thereto. Any modified nucleotide known in the art can also be used to reduce the immunogenicity of the mRNA molecule.

[0059] In yet another aspect of the present invention, a gene delivery vector comprising the nucleic acid molecule is provided.

[0060] According to the present invention, the antibody or antigen-binding fragment thereof of the present invention can be obtained recombinantly by expressing a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof in a host cell.

[0061] As used herein, the term "expression" refers to the artificial replication of a foreign gene or overexpression of an endogenous gene in a target cell as an extrachromosomal factor or through chromosomal integration in a target cell via a gene delivery system. Therefore, "expression" can be used interchangeably with "transformation," "transfection," or "transduction." More specifically, "expression" in the present invention refers to artificial expression of a foreign gene in a target cell.

[0062] As used herein, the term "gene delivery system" or "gene delivery vector" refers to any method for delivering a gene into a cell, and the term "gene delivery" has the same meaning as the intracellular transduction of a gene. At the cellular or tissue level, gene delivery has the same meaning as gene propagation. Therefore, the gene delivery system of the present invention can be referred to as a gene transduction system or a gene propagation system.

[0063] The gene delivery vector of the present invention can include an expression cassette, and its polynucleotide construct comprises all elements required for the autonomous expression of the gene to be introduced. The expression cassette generally includes a promoter, a transcription termination signal, a ribosome bind site and a translation termination signal effectively connected to the gene. The expression cassette can be in the form of a self-replicating expression vector. As used herein, the term "operably connected" refers to the functional connection between a nucleic acid expression regulatory sequence (for example, a promoter, a signal sequence or a transcription regulatory factor binding site array) and a target nucleic acid sequence. By connection, the regulatory sequence regulates transcription and / or translation of the target nucleic acid sequence.

[0064] The recombinant vector system of the present invention can be constructed by various methods known in the art, and the specific methods are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001).

[0065] The vector of the present invention can generally be constructed as a cloning vector or an expression vector. In addition, the vector of the present invention can be constructed as a host in a prokaryotic or eukaryotic cell. For example, if the vector of the present invention is an expression vector with a prokaryotic cell as a host, a strong promoter capable of driving transcription (for example, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiating detoxification and a transcription / detoxification termination sequence can be used. When Escherichia coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthesis pathway (Yanofsky, CJ. Bacteriol. 158: 1018-1024 (1984)) and the left-handed promoter of bacteriophage lambda (pLλ promoter, Herskowitz, I. and Hagen, D. Ann. Rev. Genet. 14: 399-445 (1980)) can be used as a regulatory site. If Bacillus is used as a host cell, Bacillus churrigensis (Appl. Environ. Microbiol. 64: 3932-3938 (1998); Mol. Gen. Genet. 250: 734-741 (1996)) or any promoter that can be expressed in Bacillus can be used as a regulatory site.

[0066] The recombinant vector of the present invention can be constructed by using a plasmid (for example, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19, etc.), a phage (for example: λgt4-λB, λ-Charon, λΔz1 and M13, etc.) or a virus (for example, SV40, etc.).

[0067] On the other hand, if the vector of the present invention is an expression vector and is located in a eukaryotic cell, it may contain a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Roos Sakoma virus (RSV) promoter). These vectors usually have a polyadenylation sequence as a transcription termination sequence.

[0068] The recombinant vectors of the present invention can also be fused with other sequences to facilitate purification of the antibodies expressed therefrom. Fusion sequences can include, for example, glutathione S-transferase (Pharmacia, USA); maltose binding protein (NEB, USA); FLAG (IBI, USA); marker sequences such as 6x His (hexahistidine; Quiagen, USA), Pre-S1, c-Myc; and leader sequences such as OmpA and PeIB. Furthermore, since the protein expressed by the vectors of the present invention is an antibody, the expressed antibody can be easily purified using a protein A column or the like without the need for additional purification sequences.

[0069] The recombinant vector of the present invention may also include antibiotic resistance genes conventionally used as selection markers in the art, such as resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0070] The vector expressing the antibody of the present invention can be a vector system in which the light chain and the heavy chain are expressed simultaneously in a single vector, or it can be a system in which the light chain and the heavy chain are expressed in a separate vector. In the latter case, the two vectors are introduced into the host cell by co-transformation and targeted transformation. Co-transformation comprises introducing the corresponding vector DNA encoding the light chain and the heavy chain into the host cell simultaneously, and then selecting the cell expressing the light chain and the heavy chain simultaneously. Targeted transfection is a method for selecting cells transformed with a vector containing a light chain (or heavy chain), and using a vector containing a heavy chain (or light chain) to transform the selected cells expressing the light chain again, ultimately selecting the cell expressing the light chain and the heavy chain simultaneously.

[0071] The gene delivery vector of the present invention can be any gene delivery system conventionally used for gene insertion, including but not limited to plasmids, adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpes simplex virus, bacillus virus, liposomes, nanosomes and lipid nanoparticles.

[0072] In another aspect of the present invention, a host cell transformed with the gene delivery vector of the present invention is provided.

[0073] In one embodiment, the host cell is a cell transformed with a recombinant vector containing a gene encoding an antibody of the present invention. Any host cell known in the art can be used to stably and continuously clone and express the vector of the present invention. For example, in the case of prokaryotes, host cells include but are not limited to strains of Escherichia coli, Bacillus, such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (such as Pseudomonas putida), Proteus mirabilis and / or Staphylococcus aureus (such as Staphylococcus carnosus).

[0074] Suitable eukaryotic host cells that can be used in the present invention include, for example, fungi (such as Aspergillus), yeast (such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe) and Neurospora crassa, other lower eukaryotic cells, cells from higher eukaryotic organisms (such as insect-derived cells), and cells from plants or mammals.

[0075] As used herein, the term "transfection" refers to the introduction of a target gene into a host cell by a gene delivery vector of the present invention, and includes any method by which a nucleic acid is introduced into an organism, cell, tissue, or organ. Transfection can be carried out by selecting standard techniques for suitable host cells known in the art. These methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, silicon carbide fiber stirring, agrobacterium-mediated transfection, PEG, dextran sulfate, liposomes, and drying / inhibition-mediated transfection.

[0076] The culture of host cells for producing antibodies or their antigen-binding fragments can be accomplished using any suitable culture medium and culture conditions known in the art. These culture procedures can be easily adapted to selected strains by those skilled in the art, and examples of specific culture methods are disclosed in the literature (e.g., James M. Lee, Biochemical Engineering, Prentice-Hall International Editions, 138-176). The antibodies obtained by host cell culture can be used in an unpurified state or can be further purified using various conventional methods, such as dialysis, salt precipitation, and chromatography. When chromatography is used, the type and order of the columns can be selected from ion exchange chromatography, size exclusion chromatography, affinity chromatography, etc., according to the properties of the antibody and the method for host cell culture.

[0077] In another aspect of the present invention, provided is a composition for preventing or treating cancer or infectious diseases, which comprises as an active ingredient the antibody or antigen-binding fragment thereof, nucleic acid or gene delivery vector of the present invention.

[0078] In yet another aspect of the present invention, provided is a method for preventing or treating cancer or infectious diseases, comprising administering the antibody or antigen-binding fragment thereof, nucleic acid or gene delivery vector of the present invention to a subject in need thereof.

[0079] As used herein, the term "preventing" refers to inhibiting the occurrence of a condition or disease in a subject who has never been diagnosed with the condition or disease, but is at risk of developing the condition or disease.

[0080] As used herein, the term "treatment" refers to (a) inhibiting the progression of a disorder, disease or symptom; (b) alleviating a disorder, disease or symptom; or (c) eliminating a disorder, disease or symptom. When the composition of the present invention is administered to a subject, it inhibits the progression of the disease, eliminates or alleviates the symptoms caused by the disease by inducing the activation of endogenous NK cells or exogenous autologous / allogeneic NK cells as therapeutic cells, thereby significantly enhancing the immune response to tumors or pathogenic strains. Therefore, the composition of the present invention can itself be a cell therapy composition, or it can be used as a therapeutic adjuvant for a disease by administering it together with other active ingredients. Therefore, the term "treatment" or "therapeutic agent" in this specification includes the meaning of "therapeutic adjuvant" or "therapeutic adjuvant".

[0081] As used herein, the term "administering" or "administering" refers to administering a therapeutically effective amount of a composition of the present invention directly to a subject so that an equivalent amount is formed in the subject's body.

[0082] As used herein, the term "therapeutically effective amount" refers to an amount of the composition of the present invention sufficient to provide a therapeutic or preventive effect to a subject to which the composition is administered, and thus includes the meaning of "prophylactically effective amount."

[0083] The term "subject" as used herein includes, but is not limited to, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Specifically, the subject of the present invention is a human.

[0084] The compositions of the present invention effectively activate immune cells, particularly NK cells, and therefore can be applied to the treatment of tumors and infectious diseases. The antibodies of the present invention can be applied to all types of tumors, including solid cancers and blood cancers. Solid cancer refers to cancers that form lumps in organs, which, unlike blood cancers, include most cancers that occur in organs. Cancers that can be treated using the antibodies of the present invention may include, but are not limited to, gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumors, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. Infectious diseases that can be treated by immune cells activated by the antibodies of the present invention are diseases caused by viral or pathogen infection, including all diseases that can be transmitted and infected through the respiratory tract, blood, skin contact, and other routes. Such infectious diseases include, for example, hepatitis B and C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratory diseases, and influenza, but are not limited thereto.

[0085] According to a specific embodiment, the composition further comprises immune cells.

[0086] As used herein, the term "immune cell" refers to any cell involved in the stimulation or promotion of an immune response, more specifically, an immune effector cell. Immune cells include, but are not limited to, for example, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, and mast cells. More specifically, the immune cell is a natural killer cell.

[0087] In yet another aspect of the present invention, provided is a composition for activating immune cells, comprising as an active ingredient the antibody or antigen-binding fragment thereof, nucleic acid or gene delivery vector of the present invention.

[0088] In yet another aspect of the present invention, a method for activating immune cells is provided, comprising administering the antibody or antigen-binding fragment thereof, nucleic acid or gene delivery vector of the present invention to a subject in need thereof.

[0089] In yet another aspect of the present invention, a composition for detecting cells expressing NKp46 is provided, the composition comprising the antibody or antigen-binding fragment thereof of the present invention.

[0090] In another aspect of the present invention, a method for detecting cells expressing NKp46 is provided, comprising contacting the antibody or antigen-binding fragment thereof of the present invention with a sample to be analyzed.

[0091] According to the present invention, the antibodies of the present invention specifically recognize NKp46, which is expressed only on natural killer cells, and can therefore be used to selectively isolate, detect, and quantify natural killer cells in biological samples containing various cell populations. Therefore, the terms "detection" and "isolation," "classification," and "quantification" can be used interchangeably.

[0092] Cells expressing NKp46 in a biological sample can be detected by detecting the formation of an antigen-antibody complex using colorimetry, electrochemistry, fluorescence, luminescence, particle counting, visual assessment, or scintillation counting. As used herein, "detection" refers to the detection of an antigen-antibody complex, and detection can be performed using a variety of labeling agents. Specific examples of labeling agents include enzymes, fluorophores, ligands, luminescent agents, microparticles, or radioisotopes.

[0093] Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase and β-latamase; fluorescent groups include fluorescein, Eu(3+), Eu(3+) chelates or cryptands; ligands include biotin derivatives; luminescent agents include acridinium esters and isorhamnetin derivatives; microparticles include colloidal gold and colored latex; radioisotopes include 57 Co、 3 H. 125 I and 125 I-Bonton Hunter reagent; radioisotopes include 57 Co、 3 H. 125 I-BontonHunter reagent.

[0094] According to one embodiment, an enzyme-linked immunosorbent assay (ELISA) can be used to detect antigen-antibody complexes. Enzyme-linked immunosorbent assays include direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support; indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in an antibody complex of an antigen attached to a solid support; direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody bound to a solid support; and indirect sandwich ELISA using a labeled secondary antibody that recognizes an antigen in a complex of an antibody bound to a solid support. The antibodies of the present invention may have a detection label. If not, the antigen of the present invention can be identified by treating it with another antibody that can capture the antibody and has a detection label.

[0095] As used herein, the term "biological sample" refers to any sample obtained from a mammal (including a human) containing cells expressing NKp46, including but not limited to tissue, cells, whole blood, serum, plasma, saliva, urine, lymph, spinal fluid, tissue autopsy specimens (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, disrupted eukaryotic cells, and bacterial expression systems. More specifically, the sample is tissue, cells, whole blood, serum, plasma, saliva, urine, lymph, or spinal fluid, and more specifically, whole blood, serum, or plasma.

[0096] In yet another aspect of the present invention, provided is a composition for diagnosing cancer, the composition comprising the antibody or antigen-binding fragment thereof of the present invention as an active ingredient.

[0097] In yet another aspect of the present invention, provided is a method for diagnosing cancer, comprising administering the antibody or antigen-binding fragment thereof of the present invention to a subject in need thereof.

[0098] As used herein, the term "diagnosis" includes determining a subject's susceptibility to a particular disease, determining whether a subject currently has a particular disease, and determining a subject's prognosis for having a particular disease.

[0099] As used herein, the term "composition for diagnosis" refers to an integrated mixture or device that contains means for measuring NKp46 protein expression to determine the presence or predict the likelihood of a pathological condition associated with NKp46 expression status (such as cancer), and may also be expressed as a "diagnostic kit."

[0100] Beneficial effects

[0101] The features and advantages of the present invention are summarized as follows:

[0102] (a) The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to NKp46, and nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof.

[0103] (b) The antibodies of the present invention bind to NKp46 (a specific activating receptor for natural killer cells) with high affinity, thereby significantly enhancing the immune activity of NK cells against a variety of pathogens. The antibodies of the present invention can also be used to provide reliable information about the presence and number of natural killer cells in biological samples, or to sort them with high selectivity in samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 The results of ELISA validation of the binding of antibodies obtained from the sera of mice immunized with NKp46 antigen to human NKp46 protein and monkey NKp46 protein are shown.

[0105] Figure 2 shows the interaction of five mouse monoclonal antibodies screened by the Beacon method with human NKp46 protein ( Figure 2a ) and monkey NKp46 protein ( Figure 2b ) were verified by ELISA.

[0106] Figure 3 Shown are the results of SDS-PAGE analysis of the size and purity of five mouse monoclonal antibodies selected by the Beacon method and produced as mouse / human chimeric IgG4 antibodies.

[0107] Figures 4a to 4b Shown is flow cytometric analysis of binding between five mouse monoclonal antibodies selected by the beacon method and human CHO-K1 cells expressing NKp46.

[0108] Figure 5 shows the results of ELISA analysis of 8 mouse monoclonal antibodies selected by hybridoma method and human NKp46 protein ( Figure 5a ) and monkey NKp46 protein ( Figure 5b ) combination.

[0109] Figures 6a to 6b Shown are the results of SDS-PAGE analysis of the size and purity of eight mouse monoclonal antibodies selected and produced as mouse / human chimeric IgG4 antibodies by the hybridoma method.

[0110] Figures 7a to 7c Shown are the results of flow cytometric analysis of the binding between eight mouse monoclonal antibodies selected by the beacon method and human CHO-K1 cells expressing NKp46.

[0111] Figure 8 Shown is a schematic diagram of the surface plasmon resonance (SPR) procedure for measuring the binding of anti-NKp46 antibodies to human NKp46 protein.

[0112] Figures 9a to 9b Shown are surface plasmon resonance (SPR) results of 10 mouse monoclonal antibodies binding to human NKp46 protein.

[0113] Figure 10 Surface plasmon resonance (SPR) results of three humanized antibodies binding to human NKp46 protein are shown. DETAILED DESCRIPTION

[0114] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only used to illustrate the present invention in more detail, and it is clear to those skilled in the art that the scope of the present invention according to the subject matter of the present invention is not limited by these examples.

[0115] Example

[0116] Example 1: Screening of anti-NKp46 antibodies

[0117] ELISA confirmed that mice immunized with human NKp46 antigen produced antibodies against human and monkey NKp46 ( Figure 1 ), plasma cells were isolated from mouse spleens using a CD138 kit, and antibodies specifically binding to human and monkey NKp46 were screened using two screening methods: beacon screening and hybridoma screening. First, plasma cells specifically binding to human and monkey NKp46 were isolated using a beacon device. Then, antibody sequences that bound to NKp46 were obtained by sequence analysis of the mouse variable regions.

[0118] The candidate antibody was recombinantly expressed and purified as a chimeric anti-human NKp46 antibody that combines the variable region of a mouse-derived monoclonal antibody with the constant region of a human antibody. 3 μg of the purified anti-human NKp46 antibody was separated on SDS-PAGE and the protein was stained with Coomassie Brilliant Blue solution to confirm the size and purity of the protein ( Figure 3 6 ). It was confirmed that the size of the candidate antibody was an antibody dimer of approximately 150 kDa under non-reducing conditions, and the light chain and heavy chain antibodies were approximately 25 kDa and 50 kDa, respectively, under reducing conditions.

[0119] Specifically, human CHO-K1 cells expressing NKp46 in co-culture were centrifuged to remove the culture medium, and the remaining cells were washed twice with an appropriate amount of PBS. 1x10 5 The cells were treated with 10 μg / mL anti-human NKp46 antibody and then incubated with stirring for 40 minutes. After the reaction, each well was washed twice with 150 μL PBS and then incubated with 1 μg / mL fluorescent conjugated secondary antibody (Alexa Fluor The cells were treated with 647AffiniPure Fcγ-specific Gort anti-human IgG (min X Bov, Hrs, Ms Sr Prot) (Jackson, 109-605-098) and stirred for 40 minutes. After the reaction, each well was washed twice with 150 μL PBS, and then 150 μL PBS was added. Binding to human NKp46 was confirmed by flow cytometry (Figure 4).

[0120] Human NKp46 and monkey NKp46 proteins were added to a 96-well plate at a concentration of 0.5 μg / mL and incubated at 37°C for 1 hour. The plates were then washed three times with an appropriate amount of washing buffer (0.05% Tween 20 in PBS) and incubated with 150 μL of blocking buffer (1% BSA in PBS) at 37°C for 1 hour. After washing three times with an appropriate amount of washing buffer (0.05% Tween 20 in PBS), the plates were treated with three serial dilutions of an anti-human NKp46 antibody at a maximum concentration of 1 μg / mL and incubated at 37°C for 1 hour. Following the reaction, each well was washed three times with 150 μL of washing buffer and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (human IgG (H&L), Rackland, 309-103-123) at a concentration of 0.1 μg / mL. The plates were incubated at 37°C for 30 minutes and then washed three times with washing buffer. The degree of antigen-antibody binding was determined using a 3',5,5'-tetramethylbenzidine solution. Finally, the reaction was terminated by adding 1 M HCl, and the absorbance was measured at 450 nm using an ELISA reader (ThermoFisher Scientific). Four antibodies (1-2, 1-4-2, 1-8, and 1-10) that specifically bind to human NKp46 and monkey NKp46 were selected using these two binding assays.

[0121] To utilize hybridoma screening as an alternative screening method, hybridoma cells were generated by fusing mouse plasma cells with mouse myeloma cells. Among the mouse antibodies produced by the hybridoma cells, antibody clones that specifically bound to human NKp46 and monkey NKp46 were selected, and the variable region sequences of the mouse antibodies were analyzed. Following the production of mouse / human chimeric antibodies, six antibodies (2-1, 2-2, 2-3, 2-5, 2-7, and 2-10) that specifically bound to human NKp46 and monkey NKp46 were further screened by ELISA analysis (Figure 5) and flow cytometry (Figure 7).

[0122] By identifying the binding ability and epitope of the 10 antibodies obtained by the above two methods, the inventors screened out the three antibodies with the best binding ability (1-2, 2-3 and 2-5) among the antibodies binding to the same epitope, and then changed the variable region of the mouse antibody except the CDR site to a humanized sequence to produce the final three humanized antibodies (Hu1-2, Hu 2-3 and Hu 2-5).

[0123] Example 2: Measurement of affinity

[0124] Experimental methods

[0125] The affinity of each antibody for its target antigen was assessed using Biacore 8K. The samples, equipment, and reagents used are summarized in Tables 1 and 2.

[0126] [Table 1]

[0127]

[0128] [Table 2]

[0129]

[0130] Prepare the running buffer by diluting the 10x buffer with 9 volumes of degassed, filtered MilliQ water. Prepare the regeneration buffer (10 mM glycine) by dissolving an aliquot of glycine in MilliQ water and adjusting the pH to 1.5-1.7. Perform the assay at 25°C using HBS-EP+ running buffer. Inject the antibody to capture Protein A on the S-series sensor chip. In the binding step, the antigen is diluted to various concentrations and injected onto the surface of flow cells 1 and 2, followed by the addition of running buffer in the separation step. Figure 8 The bonding schematic is shown, and Table 3 summarizes the operating parameters.

[0131] [Table 3]

[0132]

[0133] result

[0134] All data were processed using Biacore 8K Evaluation Software version 3.0. In each cycle, a blank injection of flow cell 1 and buffer was used as a double reference to subtract response units (RU). Figure 9a and 9b As shown in Figure 2, it was found that the binding levels of the 10 antibodies of the present invention to human NKp46 were in the range of 0.24-30 nM (based on KD). The study also found that the binding levels of humanized antibodies Hu1-2, Hu2-3 and Hu2-5 to human NKp46 were in the range of 0.42-8.7 nM (based on KD). Figure 10 The KD values ​​of the three humanized antibodies did not vary by more than 2-fold compared to the non-humanized antibodies. The calculated affinities for each antibody are summarized in Table 4.

[0135] [Table 4]

[0136]

[0137] Example 3: Identification of epitopes

[0138] The target antigen human NKp46 recombinant protein (His) was coated at a concentration of 0.50 μg / ml in 100 μl / well, and PBS (pH 7.4) was used as the coating buffer. The antibody of the present invention was used as the detection antibody, and streptavidin-HRP was used as the secondary antibody.

[0139] [Table 5]

[0140]

[0141]

[0142] Antibodies targeting epitope 1 were identified as 2-1, 2-2, 2-3, 2-7, 2-10, and 1-10; antibodies targeting epitope 2 were identified as 1-2 and 1-4-2; and antibodies targeting epitope 3 were identified as 2-5 and 1-8.

[0143] [Table 6]

[0144] SEQ ID number of the amino acid sequence of each antibody.

[0145]

[0146] [Table 7]

[0147] The amino acid sequence of the variable region of each antibody.

[0148]

[0149]

[0150]

[0151]

[0152]

[0153] Having described the specific embodiments of the present invention in detail above, it should be understood that variations and modifications within the scope of the invention may become apparent to those skilled in the art, and the scope of the invention will be determined by the appended claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to NKp46, comprising a heavy chain variable region comprising a HCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-38; a HCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-47; and a HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48-56.

2. The antibody or antigen-binding fragment thereof of claim 1, further comprising a light chain variable region comprising LCDR1 having an amino acid sequence of SEQ ID NOs: 1-6; LCDR2 having an amino acid sequence selected from SEQ ID NOs: 7-12; and LCDR3 having an amino acid sequence selected from SEQ ID NOs: 13-19.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein The antibody or antigen-binding fragment thereof is selected from the group consisting of (a)-(m): (a) an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 1, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 7, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 13; and a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 33, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 39, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 48; (b) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 2, a LCDR2 having the amino acid sequence of SEQ ID NO: 8, and a LCDR3 having the amino acid sequence of SEQ ID NO: 14; and a HCDR1 having the amino acid sequence of SEQ ID NO: 34, a HCDR2 having the amino acid sequence of SEQ ID NO: 40, and a HCDR3 having the amino acid sequence of SEQ ID NO: 49; (c) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 3, a LCDR2 having the amino acid sequence of SEQ ID NO: 9, and a LCDR3 having the amino acid sequence of SEQ ID NO: 15; and a HCDR1 having the amino acid sequence of SEQ ID NO: 35, a HCDR2 having the amino acid sequence of SEQ ID NO: 41, and a HCDR3 having the amino acid sequence of SEQ ID NO: 50; (d) an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 4, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 10, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 16; and a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 35, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 42, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 51; (e) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 5, a LCDR2 having the amino acid sequence of SEQ ID NO: 11, and a LCDR3 having the amino acid sequence of SEQ ID NO: 17; and a HCDR1 having the amino acid sequence of SEQ ID NO: 35, a HCDR2 having the amino acid sequence of SEQ ID NO: 42, and a HCDR3 having the amino acid sequence of SEQ ID NO: 52; (f) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 5, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; and a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 35, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 43, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 52; (g) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 5, a LCDR2 having the amino acid sequence of SEQ ID NO: 11, and a LCDR3 having the amino acid sequence of SEQ ID NO: 17; and a HCDR1 having the amino acid sequence of SEQ ID NO: 36, a HCDR2 having the amino acid sequence of SEQ ID NO: 44, and a HCDR3 having the amino acid sequence of SEQ ID NO: 53; (h) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of SEQ ID NO: 10, and a LCDR3 having the amino acid sequence of SEQ ID NO: 19; and a HCDR1 having the amino acid sequence of SEQ ID NO: 37, a HCDR2 having the amino acid sequence of SEQ ID NO: 45, and a HCDR3 having the amino acid sequence of SEQ ID NO: 54; (i) an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 5, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 17; and a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 38, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 42, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 55; (j) an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 6, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; and a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 35, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 43, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 52; (k) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 1, a LCDR2 having the amino acid sequence of SEQ ID NO: 7, and a LCDR3 having the amino acid sequence of SEQ ID NO: 13; and a HCDR1 having the amino acid sequence of SEQ ID NO: 33, a HCDR2 having the amino acid sequence of SEQ ID NO: 46, and a HCDR3 having the amino acid sequence of SEQ ID NO: 48; (1) an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 5, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 12, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 17; and a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 36, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 44, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 53; and (m) an antibody or antigen-binding fragment thereof, comprising a light chain variable region having a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of SEQ ID NO: 10, and a LCDR3 having the amino acid sequence of SEQ ID NO: 19; and a HCDR1 having the amino acid sequence of SEQ ID NO: 37, a HCDR2 having the amino acid sequence of SEQ ID NO: 47, and a HCDR3 having the amino acid sequence of SEQ ID NO:

56.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (a) comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 20 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:

57.

5. The antibody or antigen-binding fragment thereof according to claim 3, wherein (b) comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 21 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:

58.

6. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (c) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 22 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

59.

7. The antibody or antigen-binding fragment thereof according to claim 3, wherein (d) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

60.

8. The antibody or antigen-binding fragment thereof according to claim 3, wherein (e) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 24 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

61.

9. The antibody or antigen-binding fragment thereof according to claim 3, wherein (f) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

62.

10. The antibody or antigen-binding fragment thereof according to claim 3, wherein (g) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 26 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

63.

11. The antibody or antigen-binding fragment thereof according to claim 3, wherein (h) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 27 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

64.

12. The antibody or antigen-binding fragment thereof according to claim 3, wherein (i) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 28 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

65.

13. The antibody or antigen-binding fragment thereof according to claim 3, wherein (j) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 29 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

66.

14. The antibody or antigen-binding fragment thereof according to claim 3, wherein (k) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 30 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

67.

15. The antibody or antigen-binding fragment thereof according to claim 3, wherein (1) comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 31 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:

68.

16. The antibody or antigen-binding fragment thereof according to claim 3, wherein: (m) comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 32 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

69.

17. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to claim 1. A gene delivery vector comprising the nucleic acid molecule according to claim 17 .

19. A host cell transformed with the gene delivery vector according to claim 18.

20. A composition for preventing or treating cancer or infectious diseases, comprising as an active ingredient the antibody or antigen-binding fragment thereof according to claim 1, the nucleic acid molecule according to claim 17, or the gene delivery vector according to claim 18.

21. The composition of claim 20, further comprising immune cells.

22. A composition for activating immune cells, comprising as an active ingredient the antibody or antigen-binding fragment thereof according to claim 1, the nucleic acid molecule according to claim 17, or the gene delivery vector according to claim 18.

23. A composition for detecting cells expressing NKp46, comprising the antibody or antigen-binding fragment thereof according to claim 1 as an active ingredient.

24. A composition for diagnosing cancer, comprising the antibody or antigen-binding fragment thereof according to claim 1 as an active ingredient.

Images