Novel anti-NKp46 antibody and its use

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to NKp46, and nucleic acid molecules encoding them. The antibodies of the present invention not only significantly enhance the immune activity of NK cells against various pathogenic substances by binding with high affinity to NKp46, a natural killer cell-specific activation receptor, but also provide highly reliable information on the presence and quantity of natural killer cells in biological samples or are useful as a means for specifically sorting natural killer cells within samples.

Description

[Technical Field]

[0001] The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to NKp46, an activation receptor expressed in natural killer cells, and therapeutic uses thereof. This invention was made with support from the National New Drug Development Project (RS-2023-00258675) of the National New Drug Development Corporation of Japan, funded by the Ministry of Science and ICT, the Ministry of Industry, Trade and Energy, and the Ministry of Health and Welfare. [Background technology]

[0002] Tumor immunotherapy is carried out through targeting cancer cells with antibodies specific to cancer cell surface antigens, killing cancer cells by fusion of cancer cells with antigen-presenting cells, or tumor-specific activation of immune cells, including natural killer cells and T cells.

[0003] Natural killer cells (NK cells) are lymphocyte cells that account for approximately 10% of blood cells and play an important role in immune responses. NK cells perform a variety of biological functions, particularly the ability to kill cancer cells and cells infected with exogenous pathogens, and to eliminate abnormal cells that may become diseased.

[0004] Most NK cells in the body exist in an inactivated state at steady state, but to use them for therapeutic purposes, activated NK cells are required. Therefore, research into activating NK cells from normal blood or patient blood is actively underway.

[0005] Three activating receptors found on NK cells, NKp30, NKp44, and NKp46, function as natural cytotoxicity receptors (NCRs) and play important roles in NK cell antitumor and antiviral defense. Because NKp46 is expressed exclusively by NK cells, it is not only an important activating receptor but also an important marker for identifying, isolating, or detecting NK cells. Its ligands include a wide range of ligands, from viral ligands such as hemagglutinin (HA) and hemagglutinin-neuraminidase (HN) of influenza virus, Sendai virus, Newcastle disease virus, and poxviruses to ligands expressed by bacteria such as Fusobacterium nucleatum, tumors, adipocytes, and human pancreatic β cells.

[0006] Therefore, there is an increasing demand for the development of antibodies that specifically recognize NKp46, which can enhance the immune activity of NK cells against various pathogenic substances and can be useful for specifically detecting and separating NK cells from mixtures containing heterogeneous cell populations.

[0007] Numerous papers and patent documents are referenced and citations are provided throughout this specification, the disclosures of which are hereby incorporated by reference in their entirety into this specification to more clearly describe the state of the art and the content of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have conducted extensive research to discover effective anti-NKp46 antibodies that can bind with high affinity to NKp46, an activation receptor expressed in natural killer (NK) cells, thereby inducing activation of endogenous or exogenously injected NK cells, in vitro proliferation of NK cells, and thereby synergistic anti-cancer immune responses. As a result, they have discovered a number of antibodies that recognize NKp46 with remarkable affinity and specificity, and have completed the present invention by clearly elucidating the structure of their antigen recognition site.

[0009] Therefore, an object of the present invention is to provide an antibody or antigen-binding fragment thereof that specifically binds to NKp46, and nucleic acid molecules encoding the same.

[0010] Another object of the present invention is to provide a composition for preventing or treating cancer or infectious diseases, a composition for activating immune cells, a composition for detecting NKp46-expressing cells, and a composition for diagnosing cancer, each of which contains the antibody or its antigen-binding fragment and a nucleic acid molecule encoding the antibody or its antigen-binding fragment as an active ingredient.

[0011] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings. [Means for solving the problem]

[0012] According to one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to NKp46, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region including: an HCDR1 region selected from the group consisting of sequences 33 to 38 of the Sequence Listing; an HCDR2 region selected from the group consisting of sequences 39 to 47 of the Sequence Listing; and an HCDR3 region selected from the group consisting of sequences 48 to 56 of the Sequence Listing.

[0013] The present inventors have conducted extensive research to discover effective anti-NKp46 antibodies that can bind with high affinity to NKp46, an activation receptor expressed in natural killer (NK) cells, thereby inducing activation of endogenous or exogenously injected NK cells, in vitro proliferation of NK cells, and thereby synergistic anti-cancer immune responses. As a result, they have discovered a number of antibodies that recognize NKp46 with remarkable affinity and specificity, and have completed the present invention by clearly elucidating the structure of their antigen recognition site.

[0014] The term "antibody" as used herein refers to an antibody against NKp46 and a peptide that specifically recognizes and binds to a specific epitope thereof, and includes not only intact antibodies but also antigen-binding fragments (antibody fragments) of antibody molecules. An intact antibody has two full-length light chains and two full-length heavy chains, each of which is linked to a heavy chain by a disulfide bond. Heavy chain constant regions include gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with subclasses of gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). Light chain constant regions include kappa (κ) and lambda (λ) types.

[0015] As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment that has significant antigen-antibody binding function within a whole antibody molecule, and includes Fab, F(ab'), F(ab')2, Fv, nanobody (or sybody), etc.

[0016] Among antibody fragments, Fab has a structure comprising light chain and heavy chain variable regions, a light chain constant region and the first heavy chain constant region (CH1), and has one antigen-binding site.

[0017] Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are generated by disulfide bond formation between cysteine ​​residues in the hinge region of Fab'. Fv is the minimum antibody fragment containing only the heavy chain variable region and the light chain variable region, and recombinant techniques for generating Fv fragments are disclosed in WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086, and WO88 / 09344. In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, while in a single-chain Fv, the heavy chain variable region and the single chain variable region are generally linked by a covalent bond via a peptide linker or directly at the C-terminus, and can form a dimer-like structure like a two-chain Fv.

[0018] As used herein, the term "light chain" refers to both a full-length light chain and fragments thereof, including a variable region domain VL and a constant region domain CL, which contain amino acid sequences with sufficient variable region sequence to confer specificity for an antigen.

[0019] As used herein, the term "heavy chain" refers to both a full-length heavy chain and fragments thereof, including a variable region domain VH and three constant region domains CH1, CH2, and CH3, which contain amino acid sequences with sufficient variable region sequence to confer specificity for an antigen.

[0020] As used herein, the term "CDR (complementarity determining region)" refers to the amino acid sequences of the hypervariable regions of immunoglobulin heavy and light chains. Each heavy chain (HCDR1, HCDR2, and HCDR3) and light chain (LCDR1, LCDR2, and LCDR3) contains three CDRs, which provide the main contact residues for antibody binding to an antigen or epitope.

[0021] The scope of the antibodies or antigen-binding fragments of the present invention includes variants with conservative amino acid substitutions in the CDR regions. Furthermore, the antibodies or antigen-binding fragments of the present invention may include variants of the amino acid sequences set forth in the attached sequence listings, as long as they are capable of specifically recognizing NKp46. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the antibody's binding affinity and / or other biological properties. Such modifications include, for example, deletion, insertion, and / or substitution of residues in the antibody's amino acid sequence. Such amino acid mutations are made based on the relative similarity of amino acid side-chain substitutions, such as hydrophobicity, hydrophobicity, charge, and size. Analysis of the size, pattern, and type of amino acid side-chain substitutions reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar patterns. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine are biologically functional equivalents.

[0022] Additionally, amino acid substitutions in proteins that do not globally alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are between amino acid residues 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.

[0023] Considering the aforementioned biologically equivalent mutations, the amino acid sequences constituting the antibodies of the present invention are also understood to include sequences that show substantial identity to the sequences set forth in the sequence listing. The term "substantial identity" refers to a sequence that shows at least 61% identity, in one specific example, 70% identity, in another specific example, 80% identity, and in another specific example, 90% identity when the sequences of the present invention are aligned with any other sequence for maximum correspondence and analyzed using an algorithm commonly used in the art. Alignment methods and algorithms for comparing sequences are disclosed, for example, in Huang et al. Comp. Appl. BioSci. (1992) 8:155-65 and Pearson et al. Meth. Mol. Biol. (1994) 24:307-31.

[0024] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof of the present invention further comprises a light chain variable region comprising an LCDR1 region selected from the group consisting of Sequences 1 to 6 of the Sequence Listing; an LCDR2 region selected from the group consisting of Sequences 7 to 12 of the Sequence Listing; and an LCDR3 region selected from the group consisting of Sequences 13 to 19 of the Sequence Listing.

[0025] According to a more specific embodiment of the present invention, the antibody or antigen-binding fragment thereof is any one selected from the following (a) Sequence List (a) to (m):

[0026] (a) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 1 of the Sequence Listing, an LCDR2 of sequence 7 of the Sequence Listing, and an LCDR3 of sequence 13 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 33 of the Sequence Listing, an HCDR2 of sequence 39 of the Sequence Listing, and an HCDR3 of sequence 48 of the Sequence Listing;

[0027] (b) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 2 of the Sequence Listing, an LCDR2 of sequence 8 of the Sequence Listing, and an LCDR3 of sequence 14 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 34 of the Sequence Listing, an HCDR2 of sequence 40 of the Sequence Listing, and an HCDR3 of sequence 49 of the Sequence Listing;

[0028] (c) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 3 of the Sequence Listing, an LCDR2 of sequence 9 of the Sequence Listing, and an LCDR3 of sequence 15 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 35 of the Sequence Listing, an HCDR2 of sequence 41 of the Sequence Listing, and an HCDR3 of sequence 50 of the Sequence Listing;

[0029] (d) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 4 of the Sequence Listing, an LCDR2 of sequence 10 of the Sequence Listing, and an LCDR3 of sequence 16 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 35 of the Sequence Listing, an HCDR2 of sequence 42 of the Sequence Listing, and an HCDR3 of sequence 51 of the Sequence Listing;

[0030] (e) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 5 of the Sequence Listing, an LCDR2 of sequence 11 of the Sequence Listing, and an LCDR3 of sequence 17 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 35 of the Sequence Listing, an HCDR2 of sequence 42 of the Sequence Listing, and an HCDR3 of sequence 52 of the Sequence Listing;

[0031] (f) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 5 of the Sequence Listing, an LCDR2 of sequence 11 of the Sequence Listing, and an LCDR3 of sequence 18 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 35 of the Sequence Listing, an HCDR2 of sequence 43 of the Sequence Listing, and an HCDR3 of sequence 52 of the Sequence Listing;

[0032] (g) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 5 of the Sequence Listing, an LCDR2 of sequence 11 of the Sequence Listing, and an LCDR3 of sequence 17 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 33 of the Sequence Listing, an HCDR2 of sequence 44 of the Sequence Listing, and an HCDR3 of sequence 53 of the Sequence Listing;

[0033] (h) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 4 of the Sequence Listing, an LCDR2 of sequence 10 of the Sequence Listing, and an LCDR3 of sequence 19 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 37 of the Sequence Listing, an HCDR2 of sequence 45 of the Sequence Listing, and an HCDR3 of sequence 54 of the Sequence Listing;

[0034] (i) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence No. 5 in the Sequence Listing, an LCDR2 of sequence No. 11 in the Sequence Listing, and an LCDR3 of sequence No. 17 in the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence No. 38 in the Sequence Listing, an HCDR2 of sequence No. 42 in the Sequence Listing, and an HCDR3 of sequence No. 55 in the Sequence Listing;

[0035] (j) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 6 of the Sequence Listing, an LCDR2 of sequence 11 of the Sequence Listing, and an LCDR3 of sequence 18 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 35 of the Sequence Listing, an HCDR2 of sequence 43 of the Sequence Listing, and an HCDR3 of sequence 52 of the Sequence Listing;

[0036] (k) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 1 of the Sequence Listing, an LCDR2 of sequence 7 of the Sequence Listing, and an LCDR3 of sequence 13 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 33 of the Sequence Listing, an HCDR2 of sequence 46 of the Sequence Listing, and an HCDR3 of sequence 48 of the Sequence Listing;

[0037] (l) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 5 of the Sequence Listing, an LCDR2 of sequence 12 of the Sequence Listing, and an LCDR3 of sequence 17 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 36 of the Sequence Listing, an HCDR2 of sequence 44 of the Sequence Listing, and an HCDR3 of sequence 53 of the Sequence Listing; and

[0038] (m) An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LCDR1 of sequence No. 4 of the Sequence Listing, LCDR2 of sequence No. 10 of the Sequence Listing, and LCDR3 of sequence No. 19 of the Sequence Listing; and a heavy chain variable region comprising HCDR1 of sequence No. 37 of the Sequence Listing, HCDR2 of sequence No. 47 of the Sequence Listing, and HCDR3 of sequence No. 56 of the Sequence Listing.

[0039] More specifically, (a) comprises a light chain variable region of sequence 40 and a heavy chain variable region of sequence 92. Most specifically, (a) comprises sequence 105.

[0040] More specifically, (b) comprises a light chain variable region of sequence 41 and a heavy chain variable region of sequence 93. Most specifically, (b) comprises sequence 106.

[0041] More specifically, (c) comprises a light chain variable region of sequence 42 and a heavy chain variable region of sequence 94. Most specifically, (c) comprises sequence 107.

[0042] More specifically, (d) comprises a light chain variable region of sequence 43 and a heavy chain variable region of sequence 95. Most specifically, (d) comprises sequence 108.

[0043] More specifically, (e) comprises a light chain variable region of sequence 44 and a heavy chain variable region of sequence 96. Most specifically, (e) comprises sequence 109.

[0044] More specifically, (f) comprises a light chain variable region of sequence 45 and a heavy chain variable region of sequence 97. Most specifically, (f) comprises sequence 110.

[0045] More specifically, (g) comprises a light chain variable region of sequence 46 and a heavy chain variable region of sequence 98. Most specifically, (g) comprises sequence 111.

[0046] More specifically, (h) comprises a light chain variable region of sequence 47 and a heavy chain variable region of sequence 99. Most specifically, (h) comprises sequence 112.

[0047] More specifically, (i) comprises a light chain variable region of sequence 48 of the Sequence Listing and a heavy chain variable region of sequence 100 of the Sequence Listing. Most specifically, (i) comprises sequence 113 of the Sequence Listing.

[0048] More specifically, (j) comprises a light chain variable region of sequence 49 and a heavy chain variable region of sequence 101. Most specifically, (j) comprises sequence 114.

[0049] More specifically, (k) comprises the light chain variable region of sequence No. 50 in the Sequence Listing and the heavy chain variable region of sequence No. 102 in the Sequence Listing. Most specifically, (k) comprises sequence No. 115 in the Sequence Listing.

[0050] More specifically, (1) comprises a light chain variable region of sequence 51 of the Sequence Listing and a heavy chain variable region of sequence 103 of the Sequence Listing. Most specifically, (1) comprises sequence 116 of the Sequence Listing.

[0051] More specifically, (m) comprises a light chain variable region of sequence 52 of the Sequence Listing and a heavy chain variable region of sequence 104 of the Sequence Listing. Most specifically, (m) comprises sequence 117 of the Sequence Listing.

[0052] According to another aspect of the present invention, there is provided a nucleic acid molecule encoding the above-described antibody or antigen-binding fragment thereof that specifically binds to NKp46 of the present invention.

[0053] The term "nucleic acid molecule" as used herein is intended to encompass DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base moiety is modified (Uhlman et al., Chemical Reviews (1990) 90:543-584). The sequences of nucleic acid molecules encoding the full-length antibodies or heavy and light chain variable regions of the present invention may be modified, and such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0054] The nucleic acid molecules of the present invention are also understood to include nucleotide sequences that exhibit substantial identity to the nucleotide sequences described above, where substantial identity means a nucleotide sequence that exhibits at least 80% homology, in one particular example, at least 90% homology, and in another particular example, at least 95% homology, when the nucleotide sequences of the present invention are aligned with any other sequence for maximum correspondence and the aligned sequences are analyzed using algorithms commonly used in the art.

[0055] According to a specific embodiment of the present invention, the nucleic acid molecule used in the present invention may be an mRNA molecule, more specifically, an IVT (in vitro transcribed) mRNA.

[0056] When mRNA is used as the nucleic acid molecule of the present invention, various modifications may be made to improve the expression (translation) efficiency of the antibody or antigen-binding fragment of the present invention, such as changing the length of the poly(A) tail or substituting some adenine bases, modifying the 5' cap, or using one or more modified nucleosides. Modified nucleosides that can be used include, but are not limited to, N1-methylpseudouridine, pseudouridine, 2-thiouridine, 5-methyluridine, 5-methylcytidine, and 5-methoxyuridine. Any modified nucleoside known in the art to reduce the immunogenicity of mRNA molecules can be used.

[0057] In yet another aspect, the present invention provides a gene delivery vehicle comprising the nucleic acid molecule.

[0058] According to the present invention, the antibodies or antigen-binding fragments thereof of the invention can be obtained recombinantly by expressing nucleic acid molecules encoding same in host cells.

[0059] As used herein, the term "express" refers to expressing an exogenous gene in a target cell or artificially introducing an endogenous gene using a gene carrier to increase the natural expression level of the gene, thereby making the gene replicable in the target cell as an extrachromosomal element or by chromosomal integration. Therefore, the term "expression" has the same meaning as "transformation," "transfection," or "transduction." More specifically, in the present invention, "expression" refers to artificially expressing an exogenous gene in a target cell.

[0060] The term "gene carrier" as used herein refers to any means for transporting a gene into a cell, and gene delivery has the same meaning as intracellular gene transduction, and at the tissue level, gene delivery has the same meaning as gene spread. Therefore, the gene delivery system of the present invention may also be referred to as a gene delivery system or a gene spread system.

[0061] The gene delivery vehicle of the present invention may contain a gene to be introduced in the form of an expression cassette, which is a polynucleoside structure containing all elements necessary for the autonomous expression of the gene. The expression cassette typically contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the gene. The expression cassette may also be in the form of an expression vector capable of autonomous replication. As used herein, the term "operably linked" refers to the functional connection between a nucleic acid expression regulatory sequence, such as a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites, and another nucleic acid sequence, such that the expression regulatory sequence regulates the transcription and / or translation of the other nucleic acid sequence.

[0062] The recombinant vector system of the present invention can be constructed by various methods known in the art, and specific methods therefor are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001).

[0063] The vectors of the present invention can typically be constructed as vectors for cloning or expression. Furthermore, the vectors of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vectors of the present invention are expression vectors and prokaryotic cells are used as hosts, they generally contain a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, CJBacteriol. 158:1018-1024 (1984)) and the phage lambda leftward promoter (pLλ promoter, Herskowitz, I. and Hagen, D. Ann. Rev. Genet. 14:399-445 (1980)) can be used as a regulatory site. When Bacillus is used as a host cell, the promoter of the Bacillus thuringiensis toxin protein gene (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.

[0064] Meanwhile, the recombinant vector of the present invention may be constructed by manipulating a plasmid (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), a phage (e.g., λgt4 λB, λ-Charon, λΔz1, M13, etc.), or a virus (e.g., SV40, etc.) that is commonly used in the art.

[0065] On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as the host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) may be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0066] The recombinant vectors of the present invention may be fused with other sequences to facilitate purification of antibodies expressed therefrom. Examples of fused sequences include glutathione S-transferase (Pharmacia, USA); maltose-binding protein (NEB, USA); FLAG (IBI, USA); tag sequences such as 6xHis (hexahistidine; Quiagen, USA), Pre-S1, and c-Myc; and leader sequences such as OmpA and PelB. Furthermore, because the proteins expressed by the vectors of the present invention are antibodies, the expressed antibodies can be easily purified through a protein A column or the like, even without additional sequences for purification.

[0067] On the other hand, the recombinant vector of the present invention may contain an antibiotic resistance gene commonly used in the art as a selection marker, such as resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0068] The vectors expressing the antibodies of the present invention can be either a vector system in which the light chain and heavy chain are co-expressed in a single vector, or a system in which the light chain and heavy chain are each expressed in separate vectors. In the latter case, the two vectors are introduced into host cells through co-transformation and targeted transformation. Co-transformation is a method in which vector DNA encoding the light chain and heavy chain is simultaneously introduced into host cells, followed by selection of cells expressing both the light chain and the heavy chain. Targeted transformation is a method in which cells transformed with a vector containing the light chain (or heavy chain) are selected, and the selected cells expressing the light chain are then transformed again with a vector containing the heavy chain (or light chain), followed by final selection of cells expressing both the light chain and the heavy chain.

[0069] The gene delivery vehicle used in the present invention may be any gene delivery system commonly used for inserting genes, including, but not limited to, plasmids, adenoviruses, adeno-associated viruses (AAV), retroviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, liposomes, niosomes, and lipid nanoparticles.

[0070] In yet another aspect, the present invention provides a host cell transformed with the gene carrier.

[0071] In one embodiment, the host cell is a cell transformed with a recombinant vector into which a gene encoding the antibody of the present invention has been inserted. Host cells capable of stably and continuously cloning and expressing the vectors of the present invention may be any host cell known in the art, including, but not limited to, prokaryotic cells such as Escherichia coli, Bacillus strains (e.g., Bacillus subtilis and Bacillus thuringiensis), Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, or Staphylococcus (e.g., Staphylococcus carnosus).

[0072] Suitable eukaryotic host cells for use in the present invention include, for example, fungi such as Aspergillus species, yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, and Neurospora crassa, other lower eukaryotic cells, higher eukaryotic cells such as insect-derived cells, and cells of plant or mammalian origin.

[0073] As used herein, the term "transformation" refers to the introduction of a gene of interest into a host cell using a gene delivery construct of the present invention and includes any method for introducing nucleic acids into an organism, cell, tissue, or organ, and can be performed by selecting standard techniques appropriate for the host cell as known in the art. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO) precipitation, calcium chloride (CaCl) precipitation, silicon carbide fiber agitation, Agrobacterium-mediated transformation, PEG, dextran sulfate, lipofectamine, and desiccation / repression-mediated transformation.

[0074] Host cell culture for producing antibodies or antigen-binding fragments thereof can be carried out using appropriate media and culture conditions known in the art. Those skilled in the art can easily adjust and use such culture processes depending on the selected bacterial strain, and specific examples of culture methods are disclosed in various publications (e.g., James M. Lee, Biochemical Engineering, Prentice-Hall International Editions, pp. 138-176). Antibodies obtained by culturing host cells can be used without purification, or can be purified to a high purity using various conventional methods, such as dialysis, salt precipitation, and chromatography. When chromatography is used, the type and order of columns can be selected from ion exchange chromatography, size exclusion chromatography, affinity chromatography, etc., depending on the characteristics of the antibody and the host cell culture method.

[0075] In yet another aspect, the present invention provides a composition for preventing or treating cancer or infectious diseases, comprising the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid molecule, or gene carrier of the present invention as an active ingredient.

[0076] According to yet another aspect, the present invention provides a method for preventing or treating cancer or an infectious disease, comprising the step of administering to a subject the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid molecule, or gene carrier of the present invention.

[0077] As used herein, the term "prevention" means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to such disease or condition.

[0078] As used herein, the term "treatment" refers to (a) inhibiting the development of a disease, disorder, or symptom; (b) alleviating a disease, disorder, or symptom; or (c) eliminating a disease, disorder, or symptom. Administration of the compositions of the present invention to a subject significantly enhances the immune response against tumors or pathogenic strains by inducing the activation of endogenous NK cells or exogenous autologous / allogeneic NK cells injected as therapeutic cells, thereby inhibiting the progression, eliminating, or alleviating the symptoms associated with these diseases. Therefore, the compositions of the present invention can be used by themselves to treat these diseases, or can be administered together with other pharmacological ingredients and used as an adjunct to the treatment of the diseases. Therefore, the terms "treatment" or "therapeutic agent" as used herein also include the meaning of "adjunct therapy" or "adjunct therapy."

[0079] As used herein, the term "administration" or "administering" refers to administering a therapeutically effective amount of a composition of the present invention directly to a subject so that the same amount is formed in the subject's body.

[0080] The term "therapeutically effective amount" as used herein means the content of the composition in an amount sufficient to provide a therapeutic or prophylactic effect to an individual to whom the pharmaceutical composition of the present invention is to be administered, and includes a "prophylactically effective amount."

[0081] As used herein, the term "subject" includes, but is not limited to, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus monkey. Specifically, the subject of the present invention is a human.

[0082] The compositions of the present invention efficiently activate immune cells, particularly NK cells, and can therefore be used to treat tumors and infectious diseases. The antibodies of the present invention are applicable to all types of tumors, including solid cancers and blood cancers. Unlike blood cancers, solid cancer refers to cancers that form as masses in organs and include cancers that occur in most organs. Tumors that can be treated using the antibodies of the present invention include, but are not limited to, gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. Infectious diseases that can be treated using immune cells activated by the antibodies of the present invention are diseases caused by infection with viruses or pathogenic bacteria and include all diseases that can be transmitted through the respiratory tract, blood, or skin contact. Such infectious diseases include, but are not limited to, hepatitis B and C, human papilloma virus (HPV) infection, cytomegalovirus infection, viral respiratory diseases, and influenza.

[0083] According to a specific embodiment of the present invention, the composition further comprises immune cells.

[0084] The term "immune cells" as used herein refers to any cell involved in the initiation or promotion of an immune response, and more specifically, refers to immune effector cells. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, and mast cells. More specifically, the immune cells are natural killer cells.

[0085] In another aspect, the present invention provides a composition for activating immune cells, comprising the above-described antibody or antigen-binding fragment thereof, nucleic acid molecule or gene carrier of the present invention as an active ingredient.

[0086] According to yet another aspect of the present invention, there is provided a method for activating immune cells, comprising the step of administering to a subject the above-described antibody or antigen-binding fragment thereof, nucleic acid molecule or gene carrier of the present invention.

[0087] In yet another aspect, the present invention provides a composition for detecting NKp46-expressing cells, which comprises the above-described antibody of the present invention or its antigen-binding fragment as an active ingredient.

[0088] According to yet another aspect, the present invention provides a method for detecting NKp46-expressing cells, comprising the step of contacting the above-described antibody of the present invention or its antigen-binding fragment with a sample to be analyzed.

[0089] According to the present invention, the antibodies of the present invention specifically recognize NKp46, which is exclusively expressed in natural killer cells, and can therefore be useful for selectively isolating, detecting, and quantifying natural killer cells in biological samples containing diverse cell populations. Therefore, the term "detection" is used interchangeably with "isolation," "sorting," and "quantification."

[0090] In biological samples, NKp46-expressing cells can be detected by detecting antigen-antibody complex formation using colorimetric, electrochemical, fluorimetric, luminometric, particle counting, visual assessment, or scintillation counting methods. As used herein, "detection" refers to the detection of antigen-antibody complexes and can be performed using various labels. Specific examples of labels include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, and radioisotopes.

[0091] Enzymes that can be used as detection labels include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, and β-lactamase, and fluorescent substances include fluorescein, Eu 3+ ,EU 3+ Examples of the ligand include biotin derivatives, etc.; examples of the luminescent material include acridinium esters and isoluminol derivatives, etc.; examples of the microparticles include colloidal gold and colored latex, etc.; examples of the radioisotopes include 57 Co, 3 H, 125 I and 125 I-Bolton Hunter reagent etc.

[0092] According to one embodiment of the present invention, antigen-antibody complexes can be detected using enzyme-linked immunosorbent assays (ELISAs). Enzyme-linked immunosorbent assays include various ELISA methods, such as direct ELISA, which uses a labeled antibody that recognizes the antigen attached to a solid support; indirect ELISA, which uses a labeled secondary antibody that recognizes the capture antibody in an antibody complex that recognizes the antigen attached to a solid support; direct sandwich ELISA, which uses a labeled or other antibody that recognizes the antigen in an antibody-antigen complex attached to a solid support; and indirect sandwich ELISA, which reacts an antibody-antigen complex attached to a solid support with another antibody that recognizes the antigen and then uses a labeled secondary antibody that recognizes this antibody. The antibodies of the present invention may have a detection label; or, if they do not have a detection label, they can be captured and confirmed by treating them with another antibody that has a detection label.

[0093] The term "biological sample" as used herein refers to any sample containing cells expressing CD16 obtained from a mammal, including a human, including, but not limited to, tissue, cells, whole blood, serum, plasma, saliva, urine, lymph, spinal fluid, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells, and bacterial expression systems. Specifically, the sample is tissue, cells, whole blood, serum, plasma, saliva, urine, lymph, or spinal fluid, more specifically, whole blood, serum, or plasma.

[0094] In yet another aspect, the present invention provides a composition for diagnosing cancer, comprising the above-described antibody of the present invention or its antigen-binding fragment as an active ingredient.

[0095] According to yet another aspect, the present invention provides a method for diagnosing cancer, comprising the step of administering to a subject the above-described antibody of the present invention or its antigen-binding fragment.

[0096] As used herein, the term "diagnosis" includes determining an individual's susceptibility to a particular disease, determining whether an individual currently has a particular disease, and determining the prognosis of a subject with a particular disease.

[0097] The term "diagnostic composition" as used herein means an integrated mixture or device containing a means for measuring the expression level of NKp46 protein in order to determine the presence or absence of a pathological condition associated with the expression state of NKp46, such as cancer, or to predict the possibility of onset, and may also be referred to as a "diagnostic kit." [Effects of the Invention]

[0098] The features and advantages of the present invention can be summarized as follows:

[0099] (a) The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to NKp46, and nucleic acid molecules encoding these antibodies or antigen-binding fragments thereof.

[0100] (b) The antibodies of the present invention not only significantly enhance the immune activity of NK cells against various pathogenic substances by binding with high affinity to NKp46, a natural killer cell-specific activation receptor, but also provide highly reliable information on the presence and number of natural killer cells in biological samples or can be useful as a means for specifically sorting natural killer cells within samples. [Brief explanation of the drawings]

[0101] [Figure 1] FIG. 1 shows the results of ELISA confirming the binding between antibodies obtained from the serum of mice immunized with the NKp46 antigen and human and monkey NKp46 proteins.

[0102] [Figure 2a]FIG. 2 shows the results of ELISA confirming the binding between five mouse monoclonal antibodies selected by the beacon method and human NKp46 protein (FIG. 2a) and monkey NKp46 protein (FIG. 2b).

[0103] [Figure 2b] Same as above.

[0104] [Figure 3] FIG. 3 shows the results of SDS-PAGE analysis of the protein size and purity after producing mouse / human chimeric IgG4 antibodies from five mouse monoclonal antibodies selected using the beacon method.

[0105] [Figure 4a] FIG. 4a shows the results of flow cytometry confirming the binding between five mouse monoclonal antibodies selected by the beacon method and human NKp46-expressing CHO-K1 cells.

[0106] [Figure 4b] FIG. 4b shows the results of flow cytometry confirming the binding between five mouse monoclonal antibodies selected by the beacon method and human NKp46-expressing CHO-K1 cells.

[0107] [Figure 5a] FIG. 5 shows the results of ELISA confirming the binding between eight mouse monoclonal antibodies selected by the hybridoma method and human NKp46 protein (FIG. 5a) and monkey NKp46 protein (FIG. 5b).

[0108] [Figure 5b] Same as above.

[0109] [Figure 6a] FIG. 6a shows the results of SDS-PAGE analysis of the protein size and purity after producing mouse / human chimeric IgG4 antibodies from eight mouse monoclonal antibodies selected by the hybridoma method.

[0110] [Figure 6b] FIG. 6b shows the results of SDS-PAGE analysis of the protein size and purity after producing mouse / human chimeric IgG4 antibodies from eight mouse monoclonal antibodies selected by the hybridoma method.

[0111] [Figure 7a] FIG. 7a shows the results of flow cytometry confirming the binding between eight mouse monoclonal antibodies selected by the beacon method and human NKp46-expressing CHO-K1 cells.

[0112] [Figure 7b] FIG. 7b shows the results of flow cytometry confirming the binding between eight mouse monoclonal antibodies selected by the beacon method and human NKp46-expressing CHO-K1 cells.

[0113] [Figure 7c] FIG. 7c shows the results of flow cytometry confirming the binding between eight mouse monoclonal antibodies selected by the beacon method and human NKp46-expressing CHO-K1 cells.

[0114] [Figure 8] FIG. 8 is a schematic diagram illustrating the SPR (surface plasmon resonance) procedure for measuring the binding strength between an NKp46 antibody and human NKp46 protein.

[0115] [Figure 9a] FIG. 9a shows the results of SPR (surface plasmon resonance) for measuring the binding affinity of 10 mouse monoclonal antibodies to human NKp46 protein.

[0116] [Figure 9b]FIG. 9b shows the results of SPR (surface plasmon resonance) for measuring the binding affinity of 10 mouse monoclonal antibodies to human NKp46 protein.

[0117] [Figure 10] FIG. 10 shows the results of SPR (surface plasmon resonance) for measuring the binding strength of three types of humanized antibodies to human NKp46 protein. DETAILED DESCRIPTION OF THE INVENTION

[0118] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are provided to more specifically illustrate the present invention, and that the scope of the present invention is not limited to these examples according to the gist of the present invention. [Example]

[0119] Example

[0120] Example 1: Screening of anti-NKp46 antibodies

[0121] After immunizing mice with human NKp46 antigen, the production of antibodies against human NKp46 and monkey NKp46 was confirmed by ELISA (Figure 1). Plasma cells from the mouse spleen were isolated using a CD138 kit and antibodies that specifically bound to human NKp46 and monkey NKp46 were selected using two screening methods: beacon screening and hybridoma screening. Among the isolated plasma cells, those that specifically bound to human NKp46 and monkey NKp46 were screened using a beacon kit, and the sequences of antibodies that bound to NKp46 were identified through sequence analysis of the mouse variable regions.

[0122] The candidate antibody was 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, and was recombinantly expressed and purified. Three micrograms of the purified anti-human NKp46 antibody was separated on SDS-PAGE, and the protein was stained with Coomassie Brilliant Blue to confirm the size and purity of the protein (Figures 3 and 6). The protein size of the candidate antibody was confirmed to be approximately 150 kDa for the antibody dimer form under non-reducing conditions, and approximately 25 and 50 kDa for the light and heavy chain antibodies under reducing conditions.

[0123] Specifically, human NKp46-expressing CHO-K1 cells in subculture were centrifuged, the culture medium was removed, and the remaining cells were washed twice with an appropriate amount of PBS. 5 The cells were plated in each well of a 96-well plate, treated with an anti-human NKp46 antibody at a concentration of 10 μg / mL, and incubated for 40 minutes with stirring. After incubation, each well was washed twice with 150 μL of PBS and then treated with a fluorescently conjugated secondary antibody (Alexa Fluor® 647 AffiniPure Fcγ fragment-specific goat anti-human IgG (min x Bov, Hrs, Ms, Sr, Prot) (Jackson, 109-605-098)) at a concentration of 1 μg / mL and incubated for 40 minutes with stirring. After incubation, each well was washed twice with 150 μL of PBS, and 150 μL of PBS was added. Binding to human NKp46 was confirmed by flow cytometry (Figure 4).

[0124] Human NKp46 protein and monkey NKp46 protein were added to a 96-well plate at a concentration of 0.5 μg / mL and incubated for 1 hour at 37°C. After washing three times with an appropriate amount of washing solution (0.05% Tween 20, PBS), the plate was treated with 150 μL of blocking solution (1% BSA, PBS) and incubated for 1 hour at 37°C. After washing three times with an appropriate amount of washing solution (0.05% Tween 20, PBS), the plate was treated with a solution of anti-human NKp46 antibody (1 μg / mL) serially diluted 3-fold from the highest concentration, and incubated for 1 hour at 37°C. After the reaction, each well was washed three times with 150 μL of wash solution and then treated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Human IgG (H&L) Secondary Antibody Peroxidase Conjugated, Rackland, 309-103-123) at a concentration of 0.1 μg / mL. The reaction was incubated at 37°C for 30 minutes and then washed three times with wash solution. The degree of antigen-antibody binding was detected using 3',5,5'-tetramethylbenzidine solution. Finally, the reaction was stopped by adding 1 M HCl, and the absorbance was measured at 450 nm using an ELISA reader (Thermo Fisher Scientific). Through this dual-binding assay, four antibodies (1-2, 1-4-2, 1-8, and 1-10) that specifically bound to human NKp46 and monkey NKp46 were selected.

[0125] As another screening method, hybridoma cells were produced by fusing mouse plasma cells with mouse myeloma cells to conduct hybridoma screening. Among the mouse antibodies produced by the hybridoma cells, clones that specifically bound to human NKp46 and monkey NKp46 were selected, and the variable region sequences of the mouse antibodies were analyzed. After producing 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 selected through ELISA analysis (Figure 5) and flow cytometry (Figure 7).

[0126] From the ten antibodies obtained by the above two methods, three antibodies (1-2, 2-3, and 2-5) with the best binding ability were selected from the antibodies that bind to the same epitope through epitope identification. The regions of the variable regions of the mouse antibodies, excluding the CDR regions, were then modified to humanized sequences to produce the three final humanized antibodies (Hu1-2, Hu2-3, and Hu2-5). [Example]

[0127] Example 2: Affinity Measurement

[0128] Experimental Method

[0129] The present inventors used Biacore 8K to evaluate the affinity of each antibody for the target antigen, and the samples, equipment, and reagents used are summarized in Tables 1 and 2 below.

[0130] [Table 1]

[0131] [Table 2]

[0132] A running buffer was prepared by diluting 10x buffer with 9 volumes of degassed, filtered Milli-Q water. A regeneration buffer (10 mM glycine) was prepared by dissolving a fixed amount of glycine in Milli-Q water and adjusting the pH to 1.5-1.7. Tests were performed at 25°C using HBS-EP + running buffer. Antibodies were injected for capture on the Series S Sensor Chip Protein A. For the binding step, antigens were diluted at various concentrations and then injected onto the surfaces of flow cells 1 and 2. Subsequently, running buffer was injected for the separation step. A binding diagram is shown in Figure 8, and running parameters are summarized in Table 3 below.

[0133] [Table 3]

[0134] result

[0135] All data were processed using Biacore 8K Evaluation software version 3.0. Injections of flow cell 1 and buffer blanks were used as double references for RU (Response Units) subtraction in every cycle. As shown in Figures 9a and 9b, the binding levels of the 10 antibodies of the present invention to human NKp46 were found to be in the range of 0.24-30 nM based on KD. Furthermore, the binding levels of the humanized antibodies Hu 1-2, Hu 2-3, and Hu 2-5 to human NKp46 were confirmed to be in the range of 0.42-8.7 nM based on KD (Figure 10). The KD values ​​of the three humanized antibodies were confirmed to be no more than two-fold higher than those of the unhumanized antibodies. The calculated affinity of each antibody is summarized in Table 4 below.

[0136] [Table 4]

[0137] Example 3: Epitope Identification

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

[0139] [Table 5]

[0140] 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.

[0141] [Table 6]

[0142] [Table 7-1] [Table 7-2] [Table 7-3]

[0143] Although certain aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is to be defined 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 including an HCDR1 region selected from the group consisting of sequences 33 to 38 of the Sequence Listing; an HCDR2 region selected from the group consisting of sequences 39 to 47 of the Sequence Listing; and an HCDR3 region selected from the group consisting of sequences 48 to 56 of the Sequence Listing.

2. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a light chain variable region comprising an LCDR1 region selected from the group consisting of sequences 1 to 6 of the sequence listing; an LCDR2 region selected from the group consisting of sequences 7 to 12 of the sequence listing; and an LCDR3 region selected from the group consisting of sequences 13 to 19 of the sequence listing.

3. The antibody or antigen-binding fragment thereof is any one selected from the following (a) sequence list (a) to (m): (a) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 1 of the Sequence Listing, an LCDR2 of sequence 7 of the Sequence Listing, and an LCDR3 of sequence 13 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 33 of the Sequence Listing, an HCDR2 of sequence 39 of the Sequence Listing, and an HCDR3 of sequence 48 of the Sequence Listing; (b) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 2 of the Sequence Listing, an LCDR2 of sequence 8 of the Sequence Listing, and an LCDR3 of sequence 14 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 34 of the Sequence Listing, an HCDR2 of sequence 40 of the Sequence Listing, and an HCDR3 of sequence 49 of the Sequence Listing; (c) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 3 of the Sequence Listing, an LCDR2 of sequence 9 of the Sequence Listing, and an LCDR3 of sequence 15 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 35 of the Sequence Listing, an HCDR2 of sequence 41 of the Sequence Listing, and an HCDR3 of sequence 50 of the Sequence Listing; (d) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 4 of the Sequence Listing, an LCDR2 of sequence 10 of the Sequence Listing, and an LCDR3 of sequence 16 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 35 of the Sequence Listing, an HCDR2 of sequence 42 of the Sequence Listing, and an HCDR3 of sequence 51 of the Sequence Listing; (e) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 5 of the Sequence Listing, an LCDR2 of sequence 11 of the Sequence Listing, and an LCDR3 of sequence 17 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 35 of the Sequence Listing, an HCDR2 of sequence 42 of the Sequence Listing, and an HCDR3 of sequence 52 of the Sequence Listing; (f) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence No. 5 in the Sequence Listing, an LCDR2 of sequence No. 11 in the Sequence Listing, and an LCDR3 of sequence No. 18 in the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence No. 35 in the Sequence Listing, an HCDR2 of sequence No. 43 in the Sequence Listing, and an HCDR3 of sequence No. 52 in the Sequence Listing; (g) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence No. 5 in the Sequence Listing, an LCDR2 of sequence No. 11 in the Sequence Listing, and an LCDR3 of sequence No. 17 in the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence No. 36 in the Sequence Listing, an HCDR2 of sequence No. 44 in the Sequence Listing, and an HCDR3 of sequence No. 53 in the Sequence Listing; (h) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 4 of the Sequence Listing, an LCDR2 of sequence 10 of the Sequence Listing, and an LCDR3 of sequence 19 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 37 of the Sequence Listing, an HCDR2 of sequence 45 of the Sequence Listing, and an HCDR3 of sequence 54 of the Sequence Listing; (i) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence No. 5 in the Sequence Listing, an LCDR2 of sequence No. 11 in the Sequence Listing, and an LCDR3 of sequence No. 17 in the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence No. 38 in the Sequence Listing, an HCDR2 of sequence No. 42 in the Sequence Listing, and an HCDR3 of sequence No. 55 in the Sequence Listing; (j) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence No. 6 in the Sequence Listing, an LCDR2 of sequence No. 11 in the Sequence Listing, and an LCDR3 of sequence No. 18 in the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence No. 35 in the Sequence Listing, an HCDR2 of sequence No. 43 in the Sequence Listing, and an HCDR3 of sequence No. 52 in the Sequence Listing; (k) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 1 of the Sequence Listing, an LCDR2 of sequence 7 of the Sequence Listing, and an LCDR3 of sequence 13 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 33 of the Sequence Listing, an HCDR2 of sequence 46 of the Sequence Listing, and an HCDR3 of sequence 48 of the Sequence Listing; (l) an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an LCDR1 of sequence 5 of the Sequence Listing, an LCDR2 of sequence 12 of the Sequence Listing, and an LCDR3 of sequence 17 of the Sequence Listing; and a heavy chain variable region comprising an HCDR1 of sequence 36 of the Sequence Listing, an HCDR2 of sequence 44 of the Sequence Listing, and an HCDR3 of sequence 53 of the Sequence Listing; and (m) a light chain variable region comprising an LCDR1 of sequence No. 4 of the sequence listing, an LCDR2 of sequence No. 10 of the sequence listing, and an LCDR3 of sequence No. 19 of the sequence listing; and a heavy chain variable region comprising an HCDR1 of sequence No. 37 of the sequence listing, an HCDR2 of sequence No. 47 of the sequence listing, and an HCDR3 of sequence No. 56 of the sequence listing.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (a) comprises a light chain variable region of sequence 20 of the sequence listing and a heavy chain variable region of sequence 57 of the sequence listing.

5. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (b) comprises a light chain variable region of sequence No. 21 of the sequence listing and a heavy chain variable region of sequence No. 58 of the sequence listing.

6. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (c) comprises a light chain variable region of sequence No. 22 of the sequence listing and a heavy chain variable region of sequence No. 59 of the sequence listing.

7. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (d) comprises a light chain variable region of sequence No. 23 in the sequence listing and a heavy chain variable region of sequence No. 60 in the sequence listing.

8. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (e) comprises a light chain variable region of sequence No. 24 in the sequence listing and a heavy chain variable region of sequence No. 61 in the sequence listing.

9. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (f) comprises a light chain variable region of sequence No. 25 in the sequence listing and a heavy chain variable region of sequence No. 62 in the sequence listing.

10. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (g) comprises a light chain variable region of sequence No. 26 of the sequence listing and a heavy chain variable region of sequence No. 63 of the sequence listing.

11. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (h) comprises a light chain variable region of sequence No. 27 in the sequence listing and a heavy chain variable region of sequence No. 64 in the sequence listing.

12. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (i) comprises a light chain variable region of sequence No. 28 of the sequence listing and a heavy chain variable region of sequence No. 65 of the sequence listing.

13. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (j) comprises a light chain variable region of sequence No. 29 in the sequence listing and a heavy chain variable region of sequence No. 66 in the sequence listing.

14. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (k) comprises a light chain variable region of sequence No. 30 in the sequence listing and a heavy chain variable region of sequence No. 67 in the sequence listing.

15. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (l) comprises a light chain variable region of sequence No. 31 of the sequence listing and a heavy chain variable region of sequence No. 68 of the sequence listing.

16. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that (m) comprises a light chain variable region of sequence 32 of the sequence listing and a heavy chain variable region of sequence 69 of the sequence listing.

17. A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to NKp46 according to claim 1.

18. A gene delivery vehicle comprising the nucleic acid molecule of claim 17.

19. A host cell transformed with the gene carrier of claim 18.

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

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

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

23. A composition for detecting NKp46-expressing cells, 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.

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