NKp46 antibodies and uses thereof

Abstract

The present invention proposes an antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region CDR having the amino acid sequence of SEQ ID NOs: 1-3, 7-9, and 13-15, or a conservatively modified form thereof, and at least one CDR selected from the light chain variable region CDRs having the amino acid sequence of SEQ ID NOs: 4-6, 10-12, and 16-18, or a conservatively modified form thereof.

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceutical technology. Specifically, the present invention relates to NKp46 antibodies and uses thereof. More specifically, the present invention relates to antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells, pharmaceutical compositions, conjugates, kits and uses thereof. [Background technology]

[0002] NK cells are innate lymphoid cells of great importance in the body, playing important roles in antiviral and antitumor functions, and are also deeply involved in the onset and progression of autoimmune diseases, transplant rejection, sterile inflammation, and excessive inflammation caused by pathogenic microbial infection.

[0003] The surface of NK cells express a variety of receptors, including activating receptors such as NKp46, NKp30, and NKG2D, as well as inhibitory receptors such as NKG2A, KIR2DL3, TIGIT, and PVRIG. Ligands for activating receptors, such as B7H6 and MICA / B, are upregulated in conditions such as malignant cell transformation (tumor), autoimmune inflammation, and infectious inflammation. NK cells recognize and bind to the corresponding ligands via activating receptors encoded by germline genes, and then recognize and kill tumor cells.

[0004] NKp46 is an important NK cell-activating receptor that, upon binding to ligands on the surface of tumor cells, transmits activation signals and promotes NK cell killing of tumors. Currently, the only patents related to NKp46 antibodies published worldwide are US20190367609A1 and US10716864. Investigations have shown that the NKp46 patent disclosed in US10716864 does not bind to monkey NKp46, which is unfavorable for drug safety evaluation in non-human primates.

[0005] Therefore, there is a strong need to develop antibodies that bind to human and monkey NKp46 and promote the activation of NK cells. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention solves at least one of the technical problems existing in the prior art, at least to some extent, by providing an antibody or antigen-binding fragment capable of binding to NKp46, which can bind to NKp46 and promote the activation of NK cells. [Means for solving the problem]

[0007] In one aspect, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises a heavy chain variable region CDR having the amino acid sequence of SEQ ID NOs: 1-3, 7-9, and 13-15, or a conservatively modified form thereof, and at least one CDR selected from the group consisting of a light chain variable region CDR having the amino acid sequence of SEQ ID NOs: 4-6, 10-12, and 16-18, or a conservatively modified form thereof. The antibody or antigen-binding fragment according to an embodiment of the present invention can bind to NKp46 and promote NK cell activation, and can be used for the prevention or treatment of tumors or cancer.

[0008] In another aspect, the present invention provides a recombinant protein. According to an embodiment of the present invention, the recombinant protein comprises the above-described antibody or antigen-binding fragment. The recombinant protein according to an embodiment of the present invention can bind to NKp46 and promote NK cell activation, and can be used for the prevention or treatment of tumors or cancer.

[0009] In a further aspect, the present invention provides a multispecific antibody. According to an embodiment of the present invention, the multispecific antibody comprises a first binding domain comprising the aforementioned antibody or antigen-binding fragment, and a second binding domain having a first molecule binding activity. As can be seen from the above, the aforementioned antibody or antigen-binding fragment can bind to NKp46 and promote NK cell activation, and thus a multispecific antibody containing the aforementioned antibody or antigen-binding fragment can bind to NKp46 and promote NK cell activation, and can be used for the prevention or treatment of tumors or cancer.

[0010] In a further aspect, the invention provides a nucleic acid molecule, which according to an embodiment of the invention encodes the antibody or antigen-binding fragment, the recombinant protein or the multispecific antibody as described above. The nucleic acid molecule according to an embodiment of the invention encodes the antibody or antigen-binding fragment, the recombinant protein or the multispecific antibody as described above.

[0011] In a further aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described above, thereby enabling efficient expression of the antibody or antigen-binding fragment, the recombinant protein, or the multispecific antibody, and further enabling the ex vivo production of large amounts of the antibody or antigen-binding fragment, the recombinant protein, or the multispecific antibody.

[0012] In a further aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the nucleic acid molecule or the expression vector described above, or expresses the antibody or antigen-binding fragment, the recombinant protein, or the multispecific antibody described above. The recombinant cell can be used to effectively express the antibody or antigen-binding fragment, the recombinant protein, or the multispecific antibody in cells under appropriate conditions.

[0013] In a further aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the above-mentioned antibody or antigen-binding fragment, the above-mentioned recombinant protein, the above-mentioned multispecific antibody, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, or the above-mentioned recombinant cell. The pharmaceutical composition of the present invention can bind to NKp46 and promote NK cell activation, and can be used for the prevention or treatment of tumors or cancer.

[0014] In a further aspect, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, or the aforementioned multispecific antibody, and a conjugation moiety attached to the antibody or antigen-binding fragment, recombinant protein, or multispecific antibody. The conjugate of the present invention can bind to NKp46 and promote NK cell activation, and can be used for the prevention or treatment of tumors or cancer.

[0015] In a further aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the above-mentioned antibody or antigen-binding fragment, the above-mentioned recombinant protein, the above-mentioned multispecific antibody, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, or the above-mentioned recombinant cell. The kit according to the present invention can effectively bind to NKp46 and can be used to effectively detect NKp46.

[0016] In a further aspect, the present invention proposes the use of said antibody or antigen-binding fragment, said recombinant protein, said multispecific antibody, said pharmaceutical composition or said conjugate in the preparation of a medicament for the prevention and / or treatment of an NKp46-mediated disease.

[0017] In a further aspect, the present invention proposes the use of said antibody or antigen-binding fragment, said recombinant protein, said multispecific antibody, said pharmaceutical composition or said conjugate in the prevention and / or treatment of NKp46-mediated diseases or in the detection of NKp46.

[0018] In a further aspect, the present invention proposes an antibody or antigen-binding fragment as described above, a recombinant protein as described above, a multispecific antibody as described above, a pharmaceutical composition as described above or a conjugate as described above for preventing and / or treating an NKp46-mediated disease or for detecting NKp46.

[0019] In a further aspect, the present invention provides a method for detecting NKp46. According to an embodiment of the present invention, the method includes the steps of contacting the antibody or antigen-binding fragment, the recombinant protein, the multispecific antibody, the pharmaceutical composition, the conjugate, or the kit with a sample to be detected to form an immune complex, and determining whether the sample to be detected contains NKp46 or the amount of NKp46 based on the signal of the immune complex.

[0020] In a further aspect, the present invention provides a method for treating or preventing cancer or tumors, which, according to an embodiment of the present invention, comprises administering to a subject a pharmaceutically acceptable amount of the antibody or antigen-binding fragment, the recombinant protein, the multispecific antibody, the pharmaceutical composition, or the conjugate.

[0021] Additional aspects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. [Brief explanation of the drawings]

[0022] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the following description of the embodiments taken in conjunction with the drawings. [Figure 1] 1 shows the results of binding between mouse-derived NKp46 antibody and NKp46 protein detected by ELISA in Example 2 of the present invention. [Figure 2]1 shows the results of binding between mouse-derived NKp46 antibody and PBMC cells detected using flow ceteometry in Example 3 of the present invention. [Figure 3] 1 shows the results of binding between a human-mouse chimeric NKp46 antibody and NKp46 protein detected by ELISA in Example 4 of the present invention. [Figure 4] 1 shows the results of binding between a human-mouse chimeric NKp46 antibody and PBMC cells detected using flow ceteometry in Example 5 of the present invention. [Figure 5] 1 shows the results of binding between a humanized NKp46 antibody and an NKp46 protein detected by ELISA in Example 7 of the present invention. [Figure 6] 1 shows a schematic diagram of the structure of a mouse-derived NKp46×MICA bispecific antibody detected by ELISA in Example 8 of the present invention, and the results of its binding to NKp46 protein. [Figure 7] 1 shows the binding results of the mouse-derived NKp46×MICA bispecific antibody bridge NKp46 and MICA detected by ELISA in Example 9 of the present invention. [Figure 8] 1 shows the results of the mouse-derived NKp46×MICA bispecific antibody promoting tumor cell killing by PBMCs, as detected by ELISA in Example 10 of the present invention. [Figure 9] 1 shows a schematic diagram of the structure of a humanized NKp46×MICA bispecific antibody detected by ELISA in Example 11 of the present invention, and the results of its binding to NKp46 protein. [Figure 10] 1 shows the results of the enhancement of tumor cell killing by PBMCs by a humanized NKp46×MICA bispecific antibody as detected by ELISA in Example 12 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0023] Examples of the present invention will be described in detail below. The examples described below are illustrative and are used only to explain the present invention, and should not be understood as limitations on the present invention.

[0024] It should be noted that the terms "first," "second," etc. are for descriptive purposes only and cannot be considered to indicate or imply relative importance or the express number of technical features. Thus, a feature qualified as "first" or "second" may expressly or imply the inclusion of one or more of said features. Furthermore, in the description of this invention, unless specifically stated otherwise, "plurality" means two or more.

[0025] In this specification, the terms "comprises" or "comprises" are open-ended expressions, i.e., include the subject matter set forth in the present invention, but do not exclude the subject matter of other embodiments.

[0026] As used herein, the terms "optionally," "optional," or "optional" generally mean that a described event or circumstance may occur, but does not necessarily occur, and the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur.

[0027] As used herein, "fragment" refers to a target protein or polypeptide, and to a target protein or polypeptide having an N-terminal (N-terminus) or C-terminal (C-terminus) truncation, and / or an internal deletion.

[0028] As used herein, the term "knob-into-hole structure" refers to a knob-hole mutation formed in the CH3 region of an antibody heavy chain constant region, which facilitates the mating of heavy chains to form heterodimers, and is achieved, for example, by amino acids in the CH3 domain of the human IgG1 heavy chain constant region (T366S, L368A, Y407V, Y349C mutations, i.e., "hole" in one chain, and T366W, S354C mutations, i.e., "knob" in the other chain).

[0029] As used herein, the terms "identity," "homology," or "similarity," when describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, refer to the conventional determination of the percentage of identical amino acids or nucleotides between two amino acid or nucleic acid sequences.

[0030] As used herein, "at least 90% identical" means having at least 90% identity to the respective reference sequence, which may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%.

[0031] As used herein, the amino acids in the IgG1 Fc portion are numbered according to the EU numbering system. For example, position 366 refers to position 366 according to the EU numbering system, "T366W" means that the threonine at position 366 as numbered according to the EU numbering system is substituted with tryptophan, and "L368A" means that the leucine at position 368 as numbered according to the EU numbering system is substituted with alanine.

[0032] As used herein, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into an appropriate host and self-replicate, transferring the inserted nucleic acid molecule into and / or between host cells. Expression vectors include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcribing and / or translating DNA or RNA. Expression vectors further include vectors with various of the above functions. The expression vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into an appropriate host cell. Generally, the expression vector can produce a desired expression product by culturing an appropriate host cell containing the expression vector.

[0033] As used herein, the term "recombinant cell" refers to a cell that has a unique trait with stable inheritance, typically by modifying or recombining the genetic material of a host cell using genetic engineering or cell fusion techniques. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformation" and "transfect," as used herein, refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the present invention and used to express and / or secrete target proteins. Examples of suitable host cells for use in the present invention include permanent hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, CaP cells (human amniotic fluid-derived cells), insect cells, PER.C6 cells, and CoS cells.

[0034] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or more accessory ingredients. Typically, the compositions are prepared by uniformly and intimately combining the active antibody or antigen-binding fragment with a liquid carrier, a finely divided solid carrier, or both.

[0035] As used herein, the term "pharmaceutically acceptable adjuvant" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. To the extent that any conventional adjuvant is incompatible with the antibodies or antigen-binding fragments of the invention, e.g., results in any adverse biological effect or interacts in a deleterious manner with any other component of the pharmaceutically acceptable composition, it is also contemplated by the present invention.

[0036] As used herein, the term "administration" means introducing a predetermined amount of a substance into a patient in a suitable manner. The antibody or antigen-binding fragment, recombinant protein, multispecific antibody, or drug composition of the present invention can be administered by any common method as long as it can reach the desired tissue. Various modes of administration are possible, such as peritoneal, intravenous, intramuscular, and subcutaneous injection, but the present invention is not limited to these exemplary modes of administration. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.

[0037] As used herein, the term "treatment" means the use of a drug to obtain a desired pharmacological and / or physiological effect. The effect may be prophylactic, meaning that a disease or its symptoms are completely or partially prevented, and / or therapeutic, meaning that a disease and / or its adverse effects are partially or completely cured. As used herein, "treatment" covers mammalian, particularly human, diseases and includes (a) preventing the disease or occurrence of a disease in an individual who is susceptible to the disease but has not yet been diagnosed with the disease, (b) suppressing the disease, e.g., preventing the progression of the disease, or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" includes any administration of a fusion protein, pharmaceutical composition, or drug to an individual to treat, cure, alleviate, ameliorate, mitigate, or prevent a disease in an individual, including, but not limited to, administering a drug comprising a fusion protein described herein to an individual in need thereof.

[0038] The present invention provides antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells, pharmaceutical compositions, conjugates, kits and uses thereof, each of which is described in detail below.

[0039] Antibodies or antigen-binding fragments

[0040] In one aspect, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises at least one CDR selected from the heavy chain variable region CDR: amino acid sequence of SEQ ID NOs: 1-3, 7-9, 13-15, or conservatively modified forms thereof, and the light chain variable region CDR: amino acid sequence of SEQ ID NOs: 4-6, 10-12, 16-18, or conservatively modified forms thereof. The antibody or antigen-binding fragment according to an embodiment of the present invention can bind to NKp46 and promote NK cell activation, and can be used for the prevention or treatment of tumors or cancer.

[0041] As used herein, the term "antibody" is used in the broadest sense and can encompass full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies. The specific structure is not limited as long as they exhibit the desired biological activity. Antibody molecules typically comprise a light chain with a low molecular weight and a heavy chain with a high molecular weight, with the heavy (H) and light (L) chains connected by disulfide bonds. The amino-terminal (N-terminal) amino acid sequence of the peptide chain is highly variable and is called the variable region (V region), while the carboxyl-terminal (C-terminal) amino acid sequence is relatively stable and less variable and is called the constant region (C region). The V regions of the L and H chains are called VL and VH, respectively. As used herein, the terms "complementarity-determining region," "CDR," or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulin proteins, and include one or more, and ultimately all, of the major amino acid residues that contribute to the binding affinity of an antibody or its functional fragment to the antigen or epitope it recognizes. In a specific embodiment of the present disclosure, CDRs refer to the hypervariable regions of the heavy and light chains of said antibody.

[0042] As used herein, the term "antigen-binding fragment" refers to a fragment containing part or all of an antibody, which lacks at least a portion of the amino acids present in at least a portion of the full-length chain, yet still retains the ability to specifically bind to an antigen; for example, the fragment may contain part or all of the antibody CDRs. Such fragments bind to antigens and compete with other antigen-binding molecules (including complete antibodies) to bind to a predetermined epitope, and thus retain biological activity. Such fragments are selected from Fab, Fv, scFv, and single-domain antibodies. Such fragments can be produced by recombinant nucleic acid technology or by enzymatic or chemical degradation of antigen-binding molecules (including complete antibodies).

[0043] As used herein, a "conservatively modified amino acid sequence" refers to an amino acid modification that does not substantially affect or alter the binding characteristics of an antibody containing that amino acid sequence, and includes amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of antibodies of the invention can be substituted with another amino acid residue from the same side chain family, and the resulting antibody is tested for retention of function using the functional assays described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0044] According to an embodiment of the present invention, the above antibody or antigen-binding fragment may further comprise at least one of the following additional technical features:

[0045] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises heavy chain variable region CDR1, CDR2, CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or heavy chain variable region CDR1, CDR2, CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or heavy chain variable region CDR1, CDR2, CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively.

[0046] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises the light chain variable region CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or the light chain variable region CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, or the light chain variable region CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively.

[0047] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable region CDR1, CDR2, CDR3 sequences set forth in the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and light chain variable region CDR1, CDR2, CDR3 sequences set forth in the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively, and light chain variable region CDR1, CDR2, CDR3 sequences set forth in the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively.

[0048] According to an embodiment of the invention, the antibody or antigen-binding fragment comprises a heavy chain framework region and / or a light chain framework region.

[0049] According to an embodiment of the present invention, at least a portion of the heavy chain framework region and / or the light chain framework region is at least one selected from mouse-derived antibodies, human-derived antibodies, primate-derived antibodies, bovine antibodies, equine antibodies, cow-derived antibodies, porcine antibodies, ovine antibodies, goat-derived antibodies, canine antibodies, feline antibodies, rabbit-derived antibodies, camel-derived antibodies, donkey-derived antibodies, deer-derived antibodies, marten-derived antibodies, chicken-derived antibodies, duck-derived antibodies, goose-derived antibodies, turkey-derived antibodies, gamecock-derived antibodies, or variants thereof, and is preferably at least one of mouse-derived antibodies, human-derived antibodies, and primate-derived antibodies.

[0050] According to an embodiment of the invention, the antibody or antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25, or an amino acid sequence having at least 90% identity thereto, and / or a light chain variable region having an amino acid sequence set forth in any one of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26, or an amino acid sequence having at least 90% identity thereto.

[0051] According to an embodiment of the invention, the antibody or antigen-binding fragment comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:19 or an amino acid sequence at least 90% identical thereto, and a light chain variable region having the amino acid sequence set forth in any of SEQ ID NOs:20 or an amino acid sequence at least 90% identical thereto, or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:21 or an amino acid sequence at least 90% identical thereto, and a light chain variable region having the amino acid sequence set forth in any of SEQ ID NOs:22 or an amino acid sequence at least 90% identical thereto, or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:23 or an amino acid sequence at least 90% identical thereto, and a light chain variable region having the amino acid sequence set forth in any of SEQ ID NOs:24 or an amino acid sequence at least 90% identical thereto, or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:25 or an amino acid sequence at least 90% identical thereto, and a light chain variable region having the amino acid sequence set forth in any of SEQ ID NOs: It comprises a light chain variable region of an amino acid sequence set forth in any one of SEQ ID NO:26 or an amino acid sequence having at least 90% homology thereto.

[0052] According to an embodiment of the invention, the antibody or antigen-binding fragment further comprises a constant region, the constant region comprising at least one of a heavy chain constant region and a light chain constant region.

[0053] According to an embodiment of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant region is selected from at least one of mouse-derived antibodies, human-derived antibodies, primate-derived antibodies, bovine antibodies, equine antibodies, cow-derived antibodies, porcine antibodies, ovine antibodies, caprine antibodies, canine antibodies, feline antibodies, rabbit-derived antibodies, camel-derived antibodies, donkey-derived antibodies, deer-derived antibodies, marten-derived antibodies, chicken-derived antibodies, duck-derived antibodies, goose-derived antibodies, turkey-derived antibodies, gamecock-derived antibodies, or variants thereof.

[0054] According to an embodiment of the present invention, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD, or the light chain constant region is selected from a κ-type or λ-type light chain constant region.

[0055] According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both selected from a mouse-derived antibody or a mutant thereof, or a human-derived antibody or a mutant thereof.

[0056] According to an embodiment of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and / or the N-terminus of the light chain constant region is connected to the N-terminus of the light chain variable region.

[0057] According to an embodiment of the invention, the heavy chain constant region comprises or is the heavy chain constant region set forth in SEQ ID NO:27 or an amino acid sequence having at least 80% identity thereto, or the heavy chain constant region set forth in SEQ ID NO:28 or an amino acid sequence having at least 80% identity thereto, and / or the light chain constant region comprises or is the light chain constant region set forth in SEQ ID NO:55 or an amino acid sequence having at least 80% identity thereto, or the light chain constant region comprises or is the light chain constant region set forth in SEQ ID NO:56 or an amino acid sequence having at least 80% identity thereto.

[0058] According to embodiments of the invention, the antibody or antigen-binding fragment comprises a heavy chain having the amino acid sequence set forth in any one of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, or SEQ ID NO:41, or an amino acid sequence having at least 90% identity thereto, and / or a light chain having the amino acid sequence set forth in any one of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42, or an amino acid sequence having at least 90% identity thereto.

[0059] According to an embodiment of the invention, the antibody or antigen-binding fragment comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:29 or an amino acid sequence having at least 80% identity thereto, and a light chain having the amino acid sequence set forth in any of SEQ ID NOs:30 or an amino acid sequence having at least 80% identity thereto, or a heavy chain having the amino acid sequence set forth in any of SEQ ID NOs:31 or an amino acid sequence having at least 80% identity thereto, and a light chain having the amino acid sequence set forth in any of SEQ ID NOs:32 or an amino acid sequence having at least 80% identity thereto, or a heavy chain having the amino acid sequence set forth in any of SEQ ID NOs:33 or an amino acid sequence having at least 80% identity thereto, and a light chain having the amino acid sequence set forth in any of SEQ ID NOs:34 or an amino acid sequence having at least 80% identity thereto, or a heavy chain having the amino acid sequence set forth in any of SEQ ID NOs:35 or an amino acid sequence having at least 80% identity thereto, and a light chain having the amino acid sequence set forth in any of SEQ ID NOs: and a light chain of the amino acid sequence set forth in any one of SEQ ID NOs: 31 and 32 or an amino acid sequence having at least 80% identity thereto, or a heavy chain of the amino acid sequence set forth in any one of SEQ ID NOs: 32 and 33 or an amino acid sequence having at least 80% identity thereto, or a light chain of the amino acid sequence set forth in any one of SEQ ID NOs: 33 and 34 or an amino acid sequence having at least 80% identity thereto, or a heavy chain of the amino acid sequence set forth in any one of SEQ ID NOs: 34 and 35 or an amino acid sequence having at least 80% identity thereto, or a heavy chain of the amino acid sequence set forth in any one of SEQ ID NOs: 35 and 36 or an amino acid sequence having at least 80% identity thereto, or a heavy chain of the amino acid sequence set forth in any one of SEQ ID NOs: 36 and 36 or an amino acid sequence having at least 80% identity thereto, or a heavy chain of the amino acid sequence set forth in any one of SEQ ID NOs: 37 and 37 or an amino acid sequence having at least 80% identity thereto, and a light chain of the amino acid sequence set forth in any one of SEQ ID NOs: 38 and 38 or an amino acid sequence having at least 80% identity thereto, or a heavy chain of the amino acid sequence set forth in any one of SEQ ID NOs: 39 and 39 or an amino acid sequence having at least 80% identity thereto, and a light chain of the amino acid sequence set forth in any one of SEQ ID NOs: 40 and 40 or an amino acid sequence having at least 80% identity thereto, or a heavy chain of the amino acid sequence set forth in any one of SEQ ID NOs: 41 and 41 or an amino acid sequence having at least 80% identity thereto, and a light chain of the amino acid sequence set forth in any one of SEQ ID NOs: 42 and 4

[0060] According to an embodiment of the present invention, the antibody comprises at least one selected from a polyclonal antibody, a full-length monoclonal antibody, a Fab antibody, a Fab' antibody, a F(ab')2 antibody, an Fv antibody, a single-chain antibody, a single-domain antibody, and a minimal recognition unit, or the antigen-binding fragment comprises at least one selected from a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, an Fv fragment, an scFv fragment, an scFv-Fc fusion protein, an scFv-Fv fusion protein, and a minimal recognition unit.

[0061] As used herein, the terms "full length antibody," "full length monoclonal antibody," or "full length monoclonal antibody" refer to any antibody formed by at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0062] As used herein, the terms "polyclonal antibody" and "multispecific antibody" are synonymous and refer to antibodies capable of recognizing multiple antigen epitopes, such as antibodies capable of recognizing two antigen epitopes (bispecific antibodies, abbreviated as "biantibodies"), antibodies capable of recognizing three antigen epitopes, or antibodies capable of recognizing four antigen epitopes. These terms are broadly interpreted and the specific structure is not limited, as long as they can recognize multiple antigen epitopes. In the present invention, at least one of the multiple antigen epitopes is derived from NKp46.

[0063] As used herein, the terms "single-domain antibody," "nanobody," and "VHH antibody" can be used interchangeably and are originally described as "heavy chain antibodies" (i.e., "antibodies lacking light chains") that comprise the antigen-binding immunoglobulin protein (variable) domains of a "heavy chain antibody" (i.e., an antibody lacking light chains) (Hamers-Casterman C, Atharhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), which comprise a heavy chain variable region (VH) and conventional CH2 and CH3 regions and specifically bind to an antigen protein (e.g., NKp46) via the heavy chain variable region.

[0064] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which consists of the VH and CH1 of a heavy chain and an intact light chain, with a single disulfide bond connecting the light and heavy chains.

[0065] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen-binding F(ab') portions connected by disulfide bonds.

[0066] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment in which the light chain variable region (VL) and heavy chain variable region (VH) are non-covalently connected, and is the smallest functional fragment of an antibody molecule that retains the complete antigen-binding site.

[0067] As used herein, the terms "single-chain antibody" and "scFv fragment" refer to an antibody or fragment in which the antibody heavy chain variable region and light chain variable region are connected via a short peptide.

[0068] As used herein, the terms "minimal recognition unit" and "MRU" refer to an antibody or fragment consisting of only one CDR, and whose molecular weight is small enough to account for about 1% of the molecular weight of a complete antibody.

[0069] Recombinant proteins

[0070] In another aspect, the present invention provides a recombinant protein. According to an embodiment of the present invention, the recombinant protein comprises the above-described antibody or antigen-binding fragment. The recombinant protein according to an embodiment of the present invention can bind to NKp46 and promote NK cell activation, and can be used for the prevention or treatment of tumors or cancer.

[0071] According to an embodiment of the present invention, the composition comprises at least one selected from a biologically active protein or a fragment thereof, a biologically active polypeptide or a fragment thereof.

[0072] According to an embodiment of the present invention, the biologically active protein or fragment thereof comprises at least one selected from a protein tag, a protein toxin or fragment thereof, a tumor necrosis factor or fragment thereof, an interferon or fragment thereof, a biological response modifier or fragment thereof, and an Fc fragment.

[0073] As used herein, the term "protein tag" generally refers to a polypeptide or protein fused with a target protein (antibody or antigen-binding fragment) and can be used for the expression, detection, tracking, or purification of the target protein. Examples of such tags include, but are not limited to, His tag (also known as His-Tag, sequence: HHHHHH), Flag tag (also known as Flag-Tag, sequence: DYKDDDDK), GST tag (also known as GST-Tag or transferase tag), MBP tag (also known as MBP-Tag, maltose-binding protein tag), SUMO tag, and c-Myc tag.

[0074] As used herein, the term "toxin" generally refers to a substance that is toxic to a host, and includes protein toxins and non-protein toxins. Protein toxins include, but are not limited to, ricinus toxin protein, castor toxin protein A, Pseudomonas aeruginosa exotoxin, and diphtheria toxin. In the present invention, the protein toxin is preferably a protein toxin with enzymatic activity.

[0075] As used herein, "tumor necrosis factor" generally refers to substances that cause hemorrhagic necrosis in a variety of tumors, including, but not limited to, TNF-α and TNF-β.

[0076] As used herein, "interferon" generally refers to glycoproteins that directly kill or inhibit viruses, including, but not limited to, IFN-α, IFN-β, and IFN-γ.

[0077] As used herein, the term "biological response modifier" generally refers to a type of protein substance that directly or indirectly enhances the anti-tumor effect of the body via the immune system, including, but not limited to, lymphokines, IL-2, IL-6, IL-10, and GM-CSF.

[0078] As used herein, the term "Fc fragment" generally refers to the Fc region selected from IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM, and includes the CH2 and CH3 regions and an optional hinge region. Preferably, the IgG, IgA1, IgA2, IgD, IgE, or IgM is derived from a mouse, human, primate, or alpaca.

[0079] multispecific antibody

[0080] In a further aspect, the present invention provides a multispecific antibody. According to an embodiment of the present invention, the multispecific antibody comprises a first binding domain comprising the aforementioned antibody or antigen-binding fragment, and a second binding domain having a first molecule binding activity. As can be seen from the above, the aforementioned antibody or antigen-binding fragment can bind to NKp46 and promote NK cell activation, and thus a multispecific antibody containing the aforementioned antibody or antigen-binding fragment can bind to NKp46 and promote NK cell activation, and can be used to prevent or treat tumors or cancer.

[0081] According to an embodiment of the present invention, the multispecific antibody may further include at least one of the following additional technical features:

[0082] According to an embodiment of the present invention, the multispecific antibody comprises at least one selected from a bispecific antibody, a trispecific antibody, and a tetraspecific antibody, and is preferably a bispecific antibody.

[0083] The number of second binding regions may be one or more. If the multispecific antibody is a bispecific antibody, it will have one second binding region; if the multispecific antibody is a trispecific antibody, it will have two second binding regions, each of which can bind to two different first molecules; and if the multispecific antibody is a tetraspecific antibody, it will have three second binding regions, each of which can bind to three different first molecules.

[0084] According to an embodiment of the present invention, the first molecule comprises at least one selected from a tumor-associated antigen, a virus, a bacterium, an endotoxin, a cytokine, and a cytokine receptor, and is preferably a tumor antigen.

[0085] As used herein, the term "tumor-associated antigen" should be understood in a broad sense and refers to proteins that are differentially highly expressed in tumor cells compared to normal tissue cells, including, but not limited to, Her2, EpCAM, PD-L1, CEA, GD2, and GD3.

[0086] As used herein, the term "cytokine" should be understood broadly and can refer to proteins or small polypeptides that can transmit information between cells and have immunoregulatory and effector functions, including, but not limited to, IL-10, VEGF, EpCAM, GM2, and RANKL.

[0087] As used herein, the term "cytokine receptor" should be understood in a broad sense and can refer to a receptor on a cell surface that can bind to a cytokine, including, but not limited to, Her2, EGFR, and IL-10R.

[0088] According to an embodiment of the present invention, the first molecule includes, but is not limited to, B7H6, MICA, MICB, PDL1, etc., and is preferably MICA.

[0089] According to an embodiment of the present invention, the second binding domain is a binding protein or a fragment thereof of the first molecule.

[0090] The term "binding protein or fragment thereof of a first molecule" generally refers to a protein or protein fragment capable of binding to the first molecule. When the first molecule is a protein receptor, the binding protein or fragment thereof of the first molecule may be a cytokine or a fragment thereof, or an antibody or fragment thereof that binds to the protein receptor. When the first molecule is a protein other than a protein receptor, the binding protein or fragment thereof of the first molecule may be a protein receptor or a fragment thereof, or an antibody or fragment thereof that binds to the protein.

[0091] According to an embodiment of the present invention, the first binding region comprises a first heavy chain variable region and a first light chain variable region, and the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first light chain variable region, or the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region.

[0092] According to an embodiment of the present invention, the first binding region further comprises a first connecting peptide, wherein the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first connecting peptide and the C-terminus of the first connecting peptide is connected to the N-terminus of the first light chain variable region, or the C-terminus of the first light chain variable region is connected to the N-terminus of the first connecting peptide and the C-terminus of the first connecting peptide is connected to the N-terminus of the first heavy chain variable region.

[0093] According to an embodiment of the invention, the first heavy chain variable region is a heavy chain variable region defined by the aforementioned antibody or antigen-binding fragment thereof, and / or the first light chain variable region is a light chain variable region defined by the aforementioned antibody or antigen-binding fragment thereof.

[0094] According to an embodiment of the present invention, the first binding region further comprises a first Fc fragment, wherein the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the first light chain variable region, and the C-terminus of the first light chain variable region is connected to the N-terminus of the first Fc fragment; or the C-terminus of the first light chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the first heavy chain variable region, and the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first Fc fragment.

[0095] According to an embodiment of the present invention, the first Fc fragment has at least one of the following mutation sites compared to the amino acid sequence of the Fc fragment of human-derived wild-type IgG1: C220S, L234A, L235A, T366W, and S354C.

[0096] According to an embodiment of the present invention, the first Fc fragment has the amino acid sequence shown in SEQ ID NO:43.

[0097] According to an embodiment of the present invention, the first binding domain further comprises a second connecting peptide.

[0098] According to an embodiment of the present invention, the N-terminus of the second connecting peptide is connected to the C-terminus of the first light chain variable region and the C-terminus of the second connecting peptide is connected to the N-terminus of the first Fc fragment, or the N-terminus of the second connecting peptide is connected to the C-terminus of the first heavy chain variable region and the C-terminus of the second connecting peptide is connected to the N-terminus of the first Fc fragment.

[0099] According to an embodiment of the present invention, the amino acid sequence of the first connecting peptide and / or the second connecting peptide is (GGGGS) n wherein n is an integer of 1 or more, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; According to an embodiment of the present invention, the first connecting peptide has the amino acid sequence shown in SEQ ID NO:44.

[0100] According to an embodiment of the present invention, the second connecting peptide is GGGGS (SEQ ID NO:68).

[0101] According to an embodiment of the present invention, the first binding domain comprises an amino acid sequence shown in any of SEQ ID NOs: 45, 47, 57 and 59.

[0102] According to an embodiment of the present invention, the first binding domain comprises an amino acid sequence shown in any of SEQ ID NOs: 46, 48, 58 and 60.

[0103] According to an embodiment of the present invention, the second binding region comprises a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide being connected by an interchain disulfide bond, the first polypeptide comprising a second heavy chain variable region, a CH1 region and a second Fc fragment, the C-terminus of the second heavy chain variable region being connected to the N-terminus of the CH1 region and the C-terminus of the CH1 region being connected to the N-terminus of the second Fc fragment, and the second polypeptide comprising a second light chain variable region and a CL region, the C-terminus of the second light chain variable region being connected to the N-terminus of the CL region.

[0104] According to an embodiment of the present invention, the CH1 region is selected from the CH1 regions of wild-type IgG1 of human, primate or mouse origin, and is preferably the CH1 region of wild-type IgG1 of human origin.

[0105] Exemplarily, the CH1 region of a human-derived wild-type IgG1 has the amino acid sequence shown in SEQ ID NO:66.

[0106] According to an embodiment of the present invention, the second Fc fragment has at least one of the following mutation sites: L234A, L235A, Y349C, T366S, L368A, and Y407V, relative to the amino acid sequence of the Fc fragment of human-derived wild-type IgG1.

[0107] According to an embodiment of the present invention, the second Fc fragment has the amino acid sequence shown in SEQ ID NO:49.

[0108] According to an embodiment of the present invention, the CL region comprises a wild-type CL region of human, primate or mouse origin, preferably a wild-type CL region of human origin.

[0109] Exemplary, a wild-type CL region from humans has the amino acid sequence shown in SEQ ID NO:67.

[0110] According to an embodiment of the invention, the second heavy chain variable region has the amino acid sequence shown in SEQ ID NO:51.

[0111] According to an embodiment of the invention, the second light chain variable region has the amino acid sequence shown in SEQ ID NO:52.

[0112] According to an embodiment of the present invention, the first polypeptide has the amino acid sequence shown in SEQ ID NO:53.

[0113] According to an embodiment of the invention, the second polypeptide has the amino acid sequence shown in SEQ ID NO:54.

[0114] According to an embodiment of the present invention, the first coupling region and the second coupling region are connected by a knob-into-hole structure.

[0115] Nucleic acid molecules, expression vectors and recombinant cells

[0116] In a further aspect, the invention provides a nucleic acid molecule, which according to an embodiment of the invention encodes the antibody or antigen-binding fragment, the recombinant protein or the multispecific antibody as described above. The nucleic acid molecule according to an embodiment of the invention encodes the antibody or antigen-binding fragment, the recombinant protein or the multispecific antibody as described above.

[0117] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0118] It should be understood by those skilled in the art that the nucleic acid molecules referred to herein actually include either or both of the complementary strands. For convenience, in many cases, only one strand is shown in this description and claims, but the complementary strand is also disclosed. Also, references to nucleic acid sequences in this application include DNA and RNA forms, and disclosure of one means disclosure of the other.

[0119] In a further aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule. When the nucleic acid molecule is connected to a vector, the nucleic acid molecule can be directly or indirectly connected to control elements on the vector, as long as these control elements control the translation and expression of the nucleic acid molecule. Of course, these control elements may be derived directly from the vector itself or may be exogenous, i.e., not necessarily derived from the vector itself. Of course, the nucleic acid molecule only needs to be operably connected to the control elements. As used herein, "operably connected" refers to connecting an exogenous gene to a vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can function to regulate the transcription and translation of the exogenous gene. Commonly used vectors include plasmids and bacteriophages. After introducing an expression vector according to some specific embodiments of the present invention into an appropriate recipient cell, the expression of the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, or the aforementioned multispecific antibody can be effectively achieved through the control system, and large amounts of the antibody or antigen-binding fragment, the recombinant protein, or the multispecific antibody can be obtained in vitro.

[0120] According to an embodiment of the present invention, the expression vector is selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0121] According to an embodiment of the present invention, the expression vector is a plasmid expression vector.

[0122] In a further aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the nucleic acid molecule or the expression vector described above, or expresses the antibody or antigen-binding fragment, the recombinant protein, or the multispecific antibody described above. The recombinant cell can be used under appropriate conditions to effectively express the antibody or antigen-binding fragment, the recombinant protein, or the multispecific antibody within the cell.

[0123] The term "appropriate conditions" used in the present specification refers to conditions suitable for the expression of an antibody or antigen-binding fragment, recombinant protein, or multispecific antibody described in the present invention. Those skilled in the art should understand that suitable conditions for the expression of the antibody or antigen-binding fragment, recombinant protein, or multispecific antibody include, but are not limited to, a suitable transformation or transfection method, suitable transformation or transfection conditions, a healthy host cell state, a suitable host cell density, a suitable cell culture environment, and a suitable cell culture time. The term "appropriate conditions" is not particularly limited, and those skilled in the art will optimize the optimal conditions for the expression of the antibody or antigen-binding fragment, recombinant protein, or multispecific antibody according to the specific laboratory environment.

[0124] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the above-mentioned expression vector into a host cell.

[0125] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell, preferably the recombinant cell is a mammalian cell.

[0126] Drug Compositions, Conjugates, and Kits

[0127] In a further aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. As can be seen from the above, the aforementioned antibody or antigen-binding fragment can bind to NKp46, and multispecific antibodies containing the aforementioned antibody or antigen-binding fragment can bind to NKp46. Therefore, pharmaceutical compositions containing the aforementioned antibody or antigen-binding fragment and multispecific antibodies can also bind to NKp46 and promote NK cell activation, and can be used for the prevention or treatment of tumors or cancer.

[0128] According to an embodiment of the present invention, the composition further comprises a pharmaceutically acceptable adjuvant.

[0129] The pharmaceutical compositions of the present invention can be administered by any acceptable administration method. The pharmaceutical compositions of the present invention can be formulated as solid, semi-solid, liquid, or gaseous preparations, such as injections and lyophilized powders, and current methods for preparing these dosage forms are known or apparent to those skilled in the art. Typical routes for administering such pharmaceutical compositions include oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, and intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are prepared so that the biologically active ingredients contained therein are bioavailable after administration of the combination to a patient.

[0130] In a further aspect, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, or the aforementioned multispecific antibody, and a conjugation moiety attached to the antibody or antigen-binding fragment, recombinant protein, or multispecific antibody. As can be seen from the above, the aforementioned antibody or antigen-binding fragment can bind to NKp46, and a multispecific antibody containing the aforementioned antibody or antigen-binding fragment can bind to NKp46. Therefore, a conjugate containing the aforementioned antibody or antigen-binding fragment and multispecific antibody can also bind to NKp46 and promote NK cell activation, and can be used for the prevention or treatment of tumors or cancer.

[0131] According to an embodiment of the present invention, the conjugation moiety comprises at least one selected from a vector, a drug, a toxin, a cytokine, a protein tag, a modifier, and a chemotherapeutic agent.

[0132] As used herein, the vector may be a substance that can be suspended or dispersed in a liquid phase (e.g., a solid-phase vector such as a particle or a magnetic bead), or a solid phase that can accommodate or carry a liquid phase (e.g., a support such as a plate, a membrane, a test tube, and a container such as a well plate, a microfluidic chip, a glass capillary tube, a nanocolumn, or a monolith column), and may be a labeling vector for labeling an antibody or antigen-binding fragment, a recombinant protein, or a multispecific antibody, such as an enzyme (e.g., peroxidase, alkaline phosphatase, leucine, etc.). luciferin, β-galactosidase), affinity substances (e.g., streptavidin and biotin, or complementary sense and antisense strand nucleic acids), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol, etc.), radioisotopes (e.g., 3 H, 14 C. 32 P, 35 S, 125 I) and gold colloid.

[0133] According to an embodiment of the present invention, the drug is a small molecule drug capable of binding to an antibody or antigen-binding fragment, a recombinant protein, or a multispecific antibody.

[0134] According to an embodiment of the present invention, the protein tag includes, but is not limited to, a His tag, a Flag tag, a GST tag, a MBP tag, a SUMO tag, and a C-Myc tag.

[0135] According to an embodiment of the present invention, the modification should be broadly understood and may refer to a substance for modifying a protein, for example, polyethylene glycol or a derivative thereof.

[0136] According to an embodiment of the present invention, the chemotherapeutic agent includes, but is not limited to, albumin-bound paclitaxel, cyclophosphamide, ifosfamide, melphalan, methotrexate, fluorouracil, actinomycin D, and vincristine.

[0137] The conjugation moiety can be bound to an antibody or antigen-binding fragment, recombinant protein, or multispecific antibody by any method known in the art, including physical adsorption, covalent binding, affinity substance (e.g., biotin, streptavidin), and ionic binding.

[0138] In a further aspect, the present invention proposes a kit. According to an embodiment of the present invention, the kit includes the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. As can be seen from the above, the aforementioned antibody or antigen-binding fragment can bind to NKp46, and the aforementioned recombinant protein and multispecific antibody containing the aforementioned antibody or antigen-binding fragment can bind to NKp46. Furthermore, under appropriate conditions, the aforementioned nucleic acid molecule, expression vector, or recombinant cell can all express the aforementioned antibody or antigen-binding fragment. Furthermore, a kit containing the above materials can effectively bind to NKp46 and can be used to effectively detect NKp46. The kit can be used in scientific research, such as qualitatively or quantitatively detecting NKp46 in a biological sample. For example, after obtaining the individual's NKp46 level, it can be used to assess the individual's condition, such as determining whether the NKp46 level is excessively higher or lower than normal. The biological sample can be cells, tissue, blood, or the like.

[0139] use

[0140] In a further aspect, the present invention proposes the use of said antibody or antigen-binding fragment, said recombinant protein, said multispecific antibody, said pharmaceutical composition or said conjugate in the preparation of a medicament for the prevention and / or treatment of an NKp46-mediated disease.

[0141] In a further aspect, the present invention proposes the use of said antibody or antigen-binding fragment, said recombinant protein, said multispecific antibody, said pharmaceutical composition or said conjugate in the prevention and / or treatment of NKp46-mediated diseases or in the detection of NKp46.

[0142] In a further aspect of the present invention, the present invention proposes an antibody or antigen-binding fragment as described above, a recombinant protein as described above, a multispecific antibody as described above, a pharmaceutical composition as described above or a conjugate as described above for preventing and / or treating an NKp46-mediated disease or detecting NKp46.

[0143] According to an embodiment of the present invention, the above use may further include at least one of the following additional technical features:

[0144] According to an embodiment of the present invention, the NKp46-mediated disease includes at least one selected from tumors or cancers, diseases associated with transplant rejection, autoimmune diseases, and infectious diseases.

[0145] According to an embodiment of the present invention, the cancer includes at least one selected from lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0146] In a further aspect, the present invention proposes the use of said antibody or antigen-binding fragment, said recombinant protein, said multispecific antibody, said conjugate or said kit in the preparation of a product for detecting NKp46.

[0147] method

[0148] In a further aspect, the present invention provides a method for treating or preventing cancer or tumors. According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the above-mentioned antibody or antigen-binding fragment, the above-mentioned recombinant protein, the above-mentioned multispecific antibody, the above-mentioned pharmaceutical composition, or the above-mentioned conjugate. The method of the present invention can effectively treat or prevent cancer or tumors.

[0149] The effective amount of the antibody or antigen-binding fragment, recombinant protein, multispecific antibody, pharmaceutical composition, or conjugate described herein can vary depending on the mode of administration and the severity of the disease being treated. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., clinical trials). These factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life, the severity of the disease being treated in the patient, the patient's body weight, the patient's immune status, and the route of administration. For example, the drug may be administered in divided doses daily or the dose may be proportionally reduced depending on the exigencies of the therapeutic situation.

[0150] The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, drug compositions, or conjugates of the present invention can be formulated into medicaments suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular). These medicaments can be prepared in a variety of dosage forms, including liquid, semi-solid, and solid dosage forms, including, but not limited to, liquid solutions (e.g., injection and infusion solutions) or lyophilized powders. Typical medicaments are in the form of injection or infusion solutions. The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, drug compositions, or conjugates can be administered by intravenous infusion or injection, or intramuscular or subcutaneous injection.

[0151] According to an embodiment of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0152] In a further aspect, the present invention provides a method for detecting NKp46, which, according to an embodiment of the present invention, comprises contacting a sample to be detected with the antibody or antigen-binding fragment thereof, the recombinant protein thereof, the multispecific antibody thereof, the conjugate thereof or the kit thereof to form an immune complex.

[0153] According to an embodiment of the present invention, whether the sample to be detected contains NKp46 or the amount of NKp46 is determined based on the signal of the immune complex.

[0154] According to an embodiment of the present invention, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

[0155] According to an embodiment of the present invention, the immune complex further comprises a second antibody, wherein the second antibody binds to NKp46.

[0156] Details of the sequences relevant to the present invention are given in the table below: [Table 1(1)] [Table 1(2)] [Table 1(3)] [Table 1(4)] [Table 1(5)] [Table 1(6)] [Table 1(7)] [Table 1(8)] [Table 1(9)] [Table 1 (10)]

[0157] The following examples are used in combination to illustrate the present invention. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If specific techniques or conditions are not specified in the examples, they should be carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. If the manufacturer of the reagents or equipment used is not specified, they are conventional products that can be purchased commercially.

[0158] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art.

[0159] In the embodiments of the present invention, the nucleotide sequence used to prepare the expression vector can be obtained based on the amino acid sequence using conventional methods or conventional software (e.g., the online program Vectorbuilder (website: https: / / www.vectorbuilder.cn / tool / codon-optimization.html), the GeneOptimizer online program, etc.).

[0160] Example 1: Preparation of antibodies

[0161] A mouse-derived monoclonal antibody against human NKp46 was generated, and purified recombinant NKp46 extracellular domain His-tagged fusion protein (NKp46-His) (purchased from Acro) was used as an antigen to immunize Balb / c mice (9 weeks old, purchased from Shanghai Resc, weighing approximately 20 g).

[0162] Mice were immunized three times with purified antigen and complete Freund's adjuvant, and immune responses were detected after blood collection from the tail vein. Serum was screened by ELISA and flow cytometry to obtain mice with anti-human NKp46 immunoglobulin protein. Splenocytes were collected from the mice with the highest anti-NKp46 immunoglobulin protein levels and fused with mouse myeloma cells SP2 / 0 (ATCC No. CRL-1581). Antibody screening was performed on the fused hybridoma cells to obtain mouse monoclonal antibodies.

[0163] A total of 10 candidate hybridoma cells 6 The cells were cultured until they reached 100 cells / ml, then centrifuged at 800 rpm for 10 minutes to harvest. Total RNA was extracted using a Trizol kit (Invitrogen). A cDNA library (Invitrogen) was synthesized by reverse transcription using the total RNA as a template. The cDNA was then used as a template for PCR amplification of the variable region nucleic acid sequences corresponding to the hybridoma cells. Primer sequences used in the PCR amplification reaction were complementary to the first framework region or signal peptide region and constant region of the antibody variable region. To a 50 μL reaction mixture, 2 μL of cDNA, 5 μL of 10x PCR buffer, 2 μL (5 μmol) of upstream and downstream primers, 2 μL of dNTPs, 1 μL of Taq enzyme (Takara, Ex Taq), and 38 μL of HO were added. Initial denaturation was performed at 95°C for 5 minutes, followed by temperature cycling and PCR amplification. Reaction conditions: 30 seconds at 94°C for denaturation, 45 seconds at 58°C for annealing, and 50 seconds at 72°C for extension, repeating a total of 32 cycles, followed by 7 minutes of extension at 72°C. After sequencing the amplified product, the sequences of the heavy and light chain variable regions of the mouse monoclonal antibody were obtained.

[0164] In this example, three mouse monoclonal antibodies were obtained: mouse-derived 10D10 antibody (abbreviated as 10D10 antibody), mouse-derived 10E10 antibody (abbreviated as 10E10 antibody), and mouse-derived 3B9 antibody (abbreviated as 3B9 antibody). The 10D10 antibody has HCDRs shown in the amino acid sequences of SEQ ID NOs: 1 to 3 and LCDRs shown in the amino acid sequences of SEQ ID NOs: 4 to 6, a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 21, a light chain variable region shown in the amino acid sequence of SEQ ID NO: 22, a heavy chain shown in the amino acid sequence of SEQ ID NO: 33, and a light chain shown in the amino acid sequence of SEQ ID NO: 34. The 10E10 antibody has HCDRs shown in the amino acid sequences of SEQ ID NOs: 7 to 9 and LCDRs shown in the amino acid sequences of SEQ ID NOs: 10 to 12, a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 23, and a light chain shown in the amino acid sequence of SEQ ID NO: 34. The 3B9 antibody has HCDRs represented by the amino acid sequences of SEQ ID NOs:13-15 and LCDRs represented by the amino acid sequences of SEQ ID NOs:16-18, a heavy chain variable region represented by the amino acid sequence of SEQ ID NO:25 and a light chain variable region represented by the amino acid sequence of SEQ ID NO:26, a heavy chain represented by the amino acid sequence of SEQ ID NO:41, and a light chain represented by the amino acid sequence of SEQ ID NO:42.

[0165] Example 2: Mouse-derived NKp46 antibody ELISA binding experiment

[0166] ELISA experiments were performed to detect the binding properties of the three NKp46 antibodies (10D10, 10E10, and 3B9) obtained in Example 1. NKp46 extracellular domain His-tagged fusion protein (NKp46-His) was coated onto a 96-well plate, and the signal intensity after antibody addition was used to determine the binding properties of the antibody to NKp46. The specific steps are as follows:

[0167] Human NKp46-His fusion protein (purchased from Acro) and cynomolgus monkey-His fusion protein (purchased from Acro) were diluted to 1 μg / mL in PBS buffer, added to a 96-well plate at a volume of 100 μL per well, and incubated overnight at 4°C. The PBS buffer was removed from the 96-well plate, and the plate was washed six times with PBST (pH 7.2, 0.1% Tween 20). 200 μL of PBS / 10% BSA was added per well and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed six times with PBST. 100 μL of the NKp46 antibody to be measured, diluted to the appropriate concentration in PBST / 0.05% BSA, was added per well and incubated at 37°C for 1 hour. The reaction mixture was removed, and the plate was washed six times with PBST. An HRP (horseradish peroxidase)-conjugated anti-mouse secondary antibody was diluted in PBST / 0.05% BSA at 100 μL / well and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added, and the plate was incubated at room temperature for 3 minutes. To stop the reaction, 80 μL / well of 4M sulfuric acid was added. The absorbance at 450 nm was read using a microplate reader, and the results are shown in Figure 1.

[0168] As can be seen from the results, all of the mouse-derived NKp46 antibodies 10D10, 10E10 and 3B9 of the present invention can bind to human and cynomolgus monkey NKp46.

[0169] Example 3: Flow cerebrospinal fluid binding experiments of mouse-derived NKp46 antibodies

[0170] Flow cytometry experiments were performed to detect the binding characteristics of the three NKp46 antibodies (10D10 antibody, 10E10 antibody, and 3B9 antibody) obtained in Example 1. Mouse-derived NKp46 antibody was added to human peripheral blood mononuclear cells, and the signal intensity after antibody addition was used to determine the binding characteristics of the antibody and PBMC cells. The specific steps are as follows:

[0171] 2 × 10 human peripheral blood mononuclear cells (PBMCs) in PBS6 The cells were diluted to 100 μL / tube and added to 1.5 mL EP tubes. 10 μL / tube of goat serum was added and blocked for 30 minutes at 4°C. NKp46 antibody was added at different concentrations and incubated for 30 minutes at 4°C. 1 mL of PBS was added to the EP tubes, and the tubes were centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was completely removed and the tubes were washed once with PBS. After centrifugation, the supernatant was completely removed and the cells were resuspended in 100 μL / tube of PBS. 0.1 μL / tube of Alexa-647-labeled goat anti-mouse secondary antibody (Biolegend) and 0.5 μL / tube of FITC-labeled CD3 antibody were added and incubated for 30 minutes at 4°C in the dark. After two washes with PBS and centrifugation, the supernatant was completely removed. The cells were resuspended in 200 μL / tube of PBS and analyzed by flow cytometry. The results are shown in Figure 2.

[0172] As can be seen from the results, the murine-derived NKp46 antibodies of the present invention, 10D10, 10E10 and 3B9, are able to bind to human PBMC cells.

[0173] Example 4: ELISA binding experiments of human-mouse chimeric NKp46 antibodies

[0174] 1. The inventors designed human-mouse chimeric NKp46 antibodies using the heavy chain variable regions and light chain variable regions of the three NKp46 antibodies (10D10 antibody, 10E10 antibody, and 3B9 antibody) obtained in Example 1. The human-mouse chimeric 10D10 antibody has a heavy chain shown in the amino acid sequence of SEQ ID NO:31 and a light chain shown in the amino acid sequence of SEQ ID NO:32. The human-mouse chimeric 10E10 antibody has a heavy chain shown in the amino acid sequence of SEQ ID NO:35 and a light chain shown in the amino acid sequence of SEQ ID NO:36. The human-mouse chimeric 3B9 antibody has a heavy chain shown in the amino acid sequence of SEQ ID NO:39 and a light chain shown in the amino acid sequence of SEQ ID NO:40. Next, human-mouse chimeric 10D10 antibody (also called 10D10-hIgG1LALA), human-mouse chimeric 10E10 antibody (also called 10E10-hIgG1LALA), and human-mouse chimeric 3B9 antibody (also called 3B9-hIgG1LALA) are prepared by conventional molecular biology techniques.

[0175] The specific preparation steps were to transiently transfect ExpiCHO-S cells (Gibco, item A29127) with pcDNA3.4 vectors (synthesized at Jinsirui, Nanjing) encoding the nucleotide sequences of the heavy and light chain amino acid sequences of the above antibodies. The day before transfection, ExpiCHO-S cells were grown to a cell density of (3-4) x 10 6 The cells were adjusted to a concentration of 7 × 10 cells / mL and cultured overnight at 37°C with 8% CO2 and 95 rpm. 6 ~1×10 7 Grow cells to 6 × 10 cells / mL and prepare for transfection when viability is greater than 95%. Discard cells using fresh, prewarmed ExpiCHO medium (Gibco, item A2910002). 6 The pcDNA3.4 plasmids carrying the heavy and light chains (1:1 ratio of light to heavy chain plasmids) were diluted to cells / mL and transfected into ExpiCHO-S cells using ExpiFectamine CHO transfection reagent (Gibco, item A29129). The cells were incubated at 37°C in 8% CO2 The cells were cultured at 32°C with 5% CO2 and shaking at 95 rpm. 18-22 hours after transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were mixed uniformly, and then the transfected cells were immediately added and mixed uniformly. Culture was continued at 32°C with 5% CO2 and shaking at 95 rpm. Five days after transfection, 8 mL of ExpiCHO Feed was added to the cells, mixed uniformly, and culture was continued. Changes in cell number and cell viability were monitored daily, and cells were harvested by centrifugation when cell viability fell below 80% or after 9 days of culture. The expression supernatant was filtered through a 0.45 μm filter, and the Fc domain-containing antibody was captured from the expression supernatant using a protein A affinity chromatography column (purchased from Suzhou Nawei). After equilibrating the chromatography column with phosphate buffer at pH 7.2, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100 mM citric acid, pH 2.7). Finally, the column was concentrated and replaced with PBS buffer. The purified antibody was identified as having a purity of 95% or more by SDS-PAGE, and it was determined that the above antibody was finally obtained.

[0176] 2. ELISA experiments were used to detect the binding properties of the three human-mouse chimeric NKp46 antibodies (i.e., human-mouse chimeric 10D10 antibody, human-mouse chimeric 10E10 antibody, and human-mouse chimeric 3B9 antibody). NKp46 extracellular domain His-tag fusion protein (NKp46-His) was coated on a 96-well plate, and the signal intensity after adding the antibody was used to determine the binding properties of the antibody to NKp46. The specific steps are as follows: Human NKp46-His fusion protein and cynomolgus monkey-His fusion protein (see Example 2 for the specific origin) were each diluted to 1 μg / mL in PBS buffer, added to a 96-well plate at a volume of 100 μL / well, and left to stand overnight at 4° C. The PBS buffer in the 96-well plate was removed, and the plate was washed six times with PBST (PBS containing 0.1% Tween 20, pH 7.2) buffer. 200 μL / well of PBS / 10% BSA was then added, and the plate was incubated at 37° C. for 2 hours for blocking. After removing the blocking solution, the plate was washed six times with PBST. Then, 100 μL / well of the NKp46 antibody to be measured, the positive control antibody (hlgG1 LALA), and the control antibody (IN0321; the heavy chain amino acid sequence is shown in SEQ ID NO:62 and the light chain amino acid sequence is shown in SEQ ID NO:63) diluted to the appropriate concentration in PBST / 0.05% BSA were added and incubated at 37°C for 1 hour. After removing the reaction mixture and washing the plate six times with PBST, 100 μL / well of HRP (horseradish peroxidase)-conjugated anti-human secondary antibody was added and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes. To stop the reaction, 80 μL / well of 4M sulfuric acid was added. The absorbance at 450 mm was read using a microplate reader, and the results are shown in Figure 3.

[0177] As can be seen from the results, the human-mouse chimeric NKp46 antibodies 10D10-hIgG1LALA, 10E10-hIgG1LALA and 3B9-hIgG1LALA of the present invention can bind to human and cynomolgus monkey NKp46, and their binding ability is similar to that of the NKp46 antibody in the Innate Pharma patent (IN0321, derived from US20190367609A1).

[0178] Example 5: Flow cetocemometry binding experiments of human-mouse chimeric NKp46 antibodies

[0179] Flow cytometry experiments were performed to detect the binding characteristics of the two human-mouse chimeric NKp46 antibodies (i.e., human-mouse chimeric 10D10 antibody and human-mouse chimeric 3B9 antibody) prepared in Example 4. The human-mouse chimeric NKp46 antibodies were added to human and cynomolgus monkey peripheral blood mononuclear cells, and the signal intensity after antibody addition was used to determine the binding characteristics of the antibodies to PBMC cells. The specific steps are as follows:

[0180] 2 × 10 human and cynomolgus monkey peripheral blood mononuclear cells (PBMCs) in PBS 6 The cells were diluted to 1 mL / mL and added to 1.5 mL EP tubes at a volume of 100 μL per tube. 10 μL of goat serum was added per tube and blocked at 4°C for 30 minutes. Different concentration gradients of NKp46 antibody and a positive control antibody (hlgG1 LALA) were added and incubated at 4°C for 30 minutes. 1 mL of PBS was added to the EP tubes, and the cells were centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was completely removed and the cells were washed once with PBS. After centrifugation, the supernatant was completely removed and the cells were resuspended in 100 μL of PBS. 0.1 μL per tube of Alexa-647-labeled rat anti-human IgG-Fc secondary antibody (Biolegend) and 0.5 μL per tube of BV510-labeled CD16 antibody were added and incubated at 4°C for 30 minutes in the dark. The cells were washed twice with PBS, centrifuged, and the supernatant was completely removed. The cells were resuspended in 200 μL / tube of PBS and detected using a flow cytometer. The results are shown in FIG.

[0181] As can be seen from the results, the human-mouse chimeric NKp46 antibodies 10D10-hIgG1LALA and 3B9-hIgG1LALA of the present invention can bind to human and cynomolgus monkey PBMC cells.

[0182] Example 6: Mouse antibody humanization experiment

[0183] Based on the light chain variable region and heavy chain variable region sequences of the mouse 10D10 antibody obtained in Example 1, a humanization template was selected that best matched the non-CDR regions. The mouse antibody CDR regions were then grafted onto the selected humanization template, replacing the CDR regions of the human template. Based on the three-dimensional structure of the mouse antibody, buried residues, residues that directly interact with the CDR regions, and residues that significantly affect the conformation of the VL and VH domains were backmutated to obtain a humanized antibody (also referred to as humanized 10D10 antibody, h10D10-hIgG1LALA; its heavy chain amino acid sequence is set forth in SEQ ID NO:29 and its light chain amino acid sequence is set forth in SEQ ID NO:30). A humanized 10D10 antibody (specifically, see Step 1 in Example 4; the only difference is the antibody sequence) was prepared using conventional molecular biology techniques.

[0184] Example 7: ELISA binding experiments of humanized NKp46 antibodies

[0185] An ELISA experiment was conducted to detect the binding properties of the humanized 10D10 antibody prepared in Example 6. NKp46 extracellular domain His-tag fusion protein (NKp46-His) was coated onto a 96-well plate, and the signal intensity after antibody addition was used to determine the binding properties of the antibody to NKp46. The specific steps are as follows:

[0186] Human NKp46-His fusion protein and cynomolgus monkey-His fusion protein (see Example 2 for the specific origin) were each diluted to 1 μg / mL in PBS buffer, added to a 96-well plate at a volume of 100 μL / well, and left to stand overnight at 4° C. The PBS buffer in the 96-well plate was removed, and the plate was washed six times with PBST (PBS containing 0.1% Tween 20, pH 7.2) buffer. 200 μL / well of PBS / 10% BSA was then added, and the plate was incubated at 37° C. for 2 hours for blocking. After removing the blocking solution and washing the plate six times with PBST, 100 μL / well of the NKp46 antibody to be measured, the positive control antibody (hlgG1 LALA), control antibody 1 (IN0321; heavy chain amino acid sequence shown in SEQ ID NO:64, light chain amino acid sequence shown in SEQ ID NO:65), and control antibody 2 (Jeru4602; heavy chain amino acid sequence shown in SEQ ID NO:62, light chain amino acid sequence shown in SEQ ID NO:63) diluted to the appropriate concentration in PBST / 0.05% BSA were added and incubated at 37°C for 1 hour. After removing the reaction mixture and washing the plate six times with PBST, 100 μL / well of horseradish peroxidase (HRP)-conjugated anti-human secondary antibody was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL of TMB (tetramethylbenzidine) was added per well and incubated at room temperature for 3 minutes. To stop the reaction, 80 μL of 4 M sulfuric acid was added per well. The absorbance at 450 mm was read using a microplate reader. The results are shown in Figure 5.

[0187] As can be seen from the results, the humanized NKp46 antibody h10D10-hIgG1LALA of the present invention can bind to human and cynomolgus monkey NKp46, and its binding ability is similar to that of the NKp46 antibody in the Innate Pharma patent (IN0321, derived from US20190367609A1), while another NKp46 antibody, Jeru4602 (derived from US10716864), shows significant differences in its binding ability to human and monkey NKp46.

[0188] Example 8: ELISA binding experiments of NKp46 (mouse-derived 10D10) x MICA bispecific antibodies

[0189] 1. Mouse-derived 10D10xMICA bispecific antibody (abbreviated as 10D10xMICA), mouse-derived 10E10xMICA bispecific antibody (abbreviated as 10E10xMICA), and mouse-derived 3B9xMICA bispecific antibody (abbreviated as 3B9xMICA) were prepared. The structural formulas of the bispecific antibodies are shown in Figure 6A. The mouse-derived 10D10xMICA bispecific antibody has a first peptide segment (mouse-derived 10D10 ScFv-Fc) shown in the amino acid sequence of SEQ ID NO:48, a second peptide segment (MICA antibody heavy chain) shown in the amino acid sequence of SEQ ID NO:53, and a second peptide segment (MICA antibody light chain) shown in the amino acid sequence of SEQ ID NO:54. The mouse-derived 10E10xMICA bispecific antibody has a first peptide segment (mouse-derived 10E10 ScFv-Fc) shown in the amino acid sequence of SEQ ID NO:58, a second peptide segment (MICA antibody light chain) shown in the amino acid sequence of SEQ ID NO:55, and a third peptide segment (MICA antibody light chain) shown in the amino acid sequence of SEQ ID NO:56. The murine 3B9xMICA bispecific antibody has a first peptide segment (murine 3B9 ScFv-Fc) represented by the amino acid sequence of SEQ ID NO:60, a second peptide segment (MICA antibody heavy chain) represented by the amino acid sequence of SEQ ID NO:53, and a second peptide segment (MICA antibody light chain) represented by the amino acid sequence of SEQ ID NO:54. Next, the murine 10D10xMICA bispecific antibody (specifically see Step 1 of Example 4, the only difference being the antibody sequences) was prepared by conventional molecular biology techniques.

[0190] 2. ELISA experiments were performed to detect the binding characteristics of the mouse 10D10 x MICA bispecific antibody prepared above. NKp46 extracellular domain His-tagged fusion protein (NKp46-His) and MICA extracellular domain His-tagged fusion protein (MICA-His) were coated onto a 96-well plate, and the signal intensity after antibody loading was used to determine the binding characteristics of the antibody to NKp46 and MICA. The specific steps are as follows:

[0191] Human NKp46-His fusion protein (see Example 2 for the source) and human MICA-His fusion protein (purchased from Acro) were diluted to 1 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL / well and incubated overnight at 4°C. The PBS buffer was removed from the 96-well plate, and the plate was washed six times with PBST (pH 7.2, 0.1% Tween 20). 200 μL / well of PBS / 10% BSA was added and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed six times with PBST. Then, 100 μL / well of the target bispecific antibody or positive control antibody (hlgG1 LALA) diluted to the appropriate concentration in PBST / 0.05% BSA was added and incubated at 37°C for 1 hour. After removing the reaction mixture and washing the plate six times with PBST, 100 μL / well of HRP (horseradish peroxidase)-conjugated anti-human secondary antibody was added in PBST / 0.05% BSA and incubated at 37°C for 1 h. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 min. 80 μL / well of 4M sulfuric acid was added to stop the reaction. The absorbance at 450 mm was read using a microplate reader, and the results are shown in Figure 6B and Figure 6C.

[0192] As can be seen from the results, the NKp46 (mouse-derived 10D10) x MICA bispecific antibody of the present invention can bind to both NKp46 and MICA.

[0193] Example 9: Bridging ELISA experiments of NKp46 (mouse-derived 10D10) x MICA bispecific antibodies

[0194] The binding characteristics of the mouse-derived 10D10 x MICA bispecific antibody bridge NKp46 prepared in Step 1 of Example 8 to MICA were detected by ELISA. NKp46 extracellular domain His-tagged fusion protein (NKp46-His) was coated onto a 96-well plate. After antibody addition, detection was performed using a biotin-labeled MICA extracellular domain α3 domain Fc fusion protein (Biotin-MICAα3-Fc). The signal intensity was used to determine the binding characteristics of the bispecific antibody bridge NKp46 and MICA. The specific steps are as follows:

[0195] Human NKp46-His fusion protein (see Example 2 for the specific origin) was diluted to 1 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL / well and incubated overnight at 4°C. The PBS buffer was removed from the 96-well plate, and the plate was washed six times with PBST (pH 7.2, PBS containing 0.1% Tween 20). 200 μL / well of PBS / 10% BSA was added and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed six times with PBST. Then, 100 μL / well of the target bispecific antibody or positive control antibody (hlgG1 LALA) diluted to the appropriate concentration in PBST / 0.05% BSA was added and incubated at 37°C for 1 hour. After removing the reaction mixture and washing the plate six times with PBST, 100 μL / well of Biotin-MICAα3-Fc was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 h. 100 μL / well of HRP (horseradish peroxidase)-labeled Avidin was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 h. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 min. To stop the reaction, 80 μL / well of 4M sulfuric acid was added. The absorbance at 450 nm was read using a microplate reader, and the results are shown in Figure 7.

[0196] As can be seen from the results, the NKp46 (mouse-derived 10D10) x MICA bispecific antibody of the present invention can bridge to both NKp46 and MICA.

[0197] Example 10: NKp46 (murine-derived 10D10, 10E10, 3B9) x MICA bispecific antibodies enhance tumor cell killing by PBMCs

[0198] The effect of bispecific antibodies on the killing of A375 cells by PBMCs was examined by constructing a reaction system of the tumor cells + PBMCs + different concentrations of bispecific antibodies. The specific experimental procedures are as follows: (1) Add complete RPMI-1640 medium to a 96-well RTCA plate at a volume of 50 μL / well to perform instrument calibration; (2) 2 × 10 A375 cells in complete RPMI-1640 medium 5 The cells were diluted to 1 mL / mL and added to the RTCA plate obtained in step (1) at a volume of 50 μL / well. The cell count was then detected for 24 hours using the xCELLigence RTCA MP instrument under conditions of 37°C and 5% CO2. (3) NKp46 × MICA bispecific antibody (mouse-derived 10D10 × MICA bispecific antibody, mouse-derived 10E10 × MICA bispecific antibody, or mouse-derived 3B9 × MICA bispecific antibody) or a positive control antibody (hlgG1 LALA) was diluted in complete RPMI-1640 medium to a concentration gradient series and added to the RTCA plate obtained in step (2), with an addition volume of 20 μL / well. (4) 1.25 × 10 PBMCs (purchased from Saikasa Biosciences) in complete RPMI-1640 medium. 6 The mixture was diluted to 80 μL / mL and added to the RTCA plate obtained in step (3), with the volume added being 80 μL / well. (5) The reaction system obtained in step (4) was subjected to cell count detection at 37°C and 5% CO2 for 48 hours using the xCELLigence RTCA MP instrument, and the results are shown in Figure 8.

[0199] As can be seen from the results, the NKp46 (murine-derived 10D10, 10E10, 3B9) x MICA bispecific antibodies of the present invention can promote tumor cell killing by PBMCs.

[0200] Example 11: ELISA binding experiments of NKp46 (humanized 10D10) x MICA bispecific antibodies

[0201] 1. A humanized 10D10×MICA bispecific antibody (abbreviated as h10D10×MICA) was prepared. The structural formula of the bispecific antibody is shown in Figure 9A. The humanized 10D10 (the origin of which is specifically described in Example 2) × MICA bispecific antibody has a first peptide segment (humanized h10D10 ScFv-Fc) represented by the amino acid sequence of SEQ ID NO:46, a second peptide segment (MICA antibody heavy chain) represented by the amino acid sequence of SEQ ID NO:53, and a second peptide segment (MICA antibody light chain) represented by the amino acid sequence of SEQ ID NO:54. The murine-derived 10D10×MICA bispecific antibody (see Step 1 of Example 4 for details; the only difference is the antibody sequence) was then prepared using conventional molecular biology techniques.

[0202] 2. ELISA experiments were performed to detect the binding properties of the humanized 10D10 x MICA bispecific antibody prepared in Step 1 of this example. NKp46 extracellular domain His-tagged fusion protein (NKp46-His) and MICA extracellular domain His-tagged fusion protein (MICA-His) were coated onto a 96-well plate, and the signal intensity after antibody addition was used to determine the binding properties of the antibody to NKp46 and MICA. The specific steps are as follows:

[0203] Human NKp46-His fusion protein and human MICA-His fusion protein (purchased from Acro) were diluted to 1 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL per well and incubated overnight at 4°C. The PBS buffer was removed from the 96-well plate, and the plate was washed six times with PBST (pH 7.2, 0.1% Tween 20). 200 μL of PBS / 10% BSA was added per well and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed six times with PBST. Then, 100 μL of the target bispecific antibody or positive control antibody (hlgG1 LALA) diluted to the appropriate concentration in PBST / 0.05% BSA was added per well and incubated at 37°C for 1 hour. The reaction mixture was removed, and the plate was washed six times with PBST. Then, 100 μL / well of HRP (horseradish peroxidase)-conjugated anti-human secondary antibody was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 h. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added, and the plate was incubated at room temperature for 3 min. To stop the reaction, 80 μL / well of 4M sulfuric acid was added. The absorbance at 450 mm was read using a microplate reader, and the results are shown in Figure 9.

[0204] As can be seen from the results, the NKp46 (humanized 10D10) x MICA bispecific antibody of the present invention can bind to both NKp46 and MICA.

[0205] Example 12: NKp46 (humanized 10D10) x MICA bispecific antibody enhances tumor cell killing by PBMCs

[0206] The effect of bispecific antibodies on the killing of A375 cells by PBMCs was examined by constructing a reaction system of the tumor cells + PBMCs + different concentrations of bispecific antibodies. The specific experimental procedures are as follows: (1) Add complete RPMI-1640 medium to a 96-well RTCA plate at a volume of 50 μL / well to perform instrument calibration; (2) 2 × 10 A375 cells in complete RPMI-1640 medium 5 The cells were diluted to 1 mL / mL and added to the RTCA plate obtained in step (1) at a volume of 50 μL / well. The cell count was then detected for 24 hours using the xCELLigence RTCA MP instrument under conditions of 37°C and 5% CO2. (3) The NKp46 x MICA bispecific antibody or positive control antibody (hlgG1 LALA) prepared in Example 11 was diluted in complete RPMI-1640 medium to a concentration gradient series and added to the RTCA plate obtained in step (2), with an addition volume of 20 μL / well; (4) 1.25 × 10 PBMCs (purchased from Saikasa Biosciences) in complete RPMI-1640 medium. 6 The mixture was diluted to 80 μL / mL and added to the RTCA plate obtained in step (3), with the volume added being 80 μL / well. (5) The reaction system obtained in step (4) was subjected to cell count detection at 37°C and 5% CO2 for 48 hours using the xCELLigence RTCA MP instrument, and the results are shown in Figure 10.

[0207] As can be seen from the results, the NKp46 (humanized 10D10) x MICA bispecific antibody of the present invention can enhance tumor cell killing by PBMCs.

[0208] Therefore, as can be seen from the experimental results of the above examples, all of the antibodies obtained in the present invention can bind to human and cynomolgus monkey NKp46 recombinant proteins, can bind to human and cynomolgus monkey peripheral blood mononuclear cells, and can promote anti-cancer activity in human PBMCs.

[0209] In the description herein, a statement referring to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples" means that a particular feature, structure, material, or characteristic described with reference to that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, general references to such terms do not necessarily refer to the same embodiment or example. In addition, a particular feature, structure, material, or characteristic described may be incorporated in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art can combine and combine the various embodiments or examples described herein and the features of the various embodiments or examples without mutual contradiction.

[0210] Although the embodiments of the present invention have been shown and described, the above embodiments are illustrative and should not be construed as limiting the present invention. Those skilled in the art will understand that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment, Heavy chain variable region CDRs having the amino acid sequences of SEQ ID NOs: 1-3, 7-9 and 13-15 or conservatively modified forms thereof; and An antibody or antigen-binding fragment comprising at least one CDR selected from the light chain variable region CDRs having the amino acid sequence of SEQ ID NOs: 4-6, 10-12, and 16-18, or conservatively modified forms thereof.

2. The antibody or antigen-binding fragment comprises: the heavy chain variable region CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; or the heavy chain variable region CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; or 2. The antibody or antigen-binding fragment of claim 1, comprising the heavy chain variable region CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively.

3. The antibody or antigen-binding fragment comprises: the light chain variable region CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; or the light chain variable region CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively; or 2. The antibody or antigen-binding fragment of claim 1, comprising the light chain variable region CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively.

4. The antibody or antigen-binding fragment comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable region CDR1, CDR2, CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; or heavy chain variable region CDR1, CDR2, CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and light chain variable region CDR1, CDR2, CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively; or The antibody or antigen-binding fragment of claim 1, characterized in that it comprises heavy chain variable region CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively, and light chain variable region CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively.

5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment comprises a heavy chain framework region and / or a light chain framework region.

6. 6. The antibody or antigen-binding fragment according to claim 5, wherein at least a portion of the heavy chain framework region and / or the light chain framework region is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody, a bovine-derived antibody, an equine-derived antibody, a cow-derived antibody, a porcine-derived antibody, a sheep-derived antibody, a goat-derived antibody, a dog-derived antibody, a cat-derived antibody, a rabbit-derived antibody, a camel-derived antibody, a donkey-derived antibody, a deer-derived antibody, a marten-derived antibody, a chicken-derived antibody, a duck-derived antibody, a goose-derived antibody, a turkey-derived antibody, a gamecock-derived antibody, or a variant thereof, preferably at least one of a mouse-derived antibody, a human-derived antibody, and a primate-derived antibody.

7. The antibody or antigen-binding fragment comprises: A heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25, or an amino acid sequence having at least 90% homology thereto, and / or The antibody or antigen-binding fragment of any one of claims 1 to 4, comprising a light chain variable region of an amino acid sequence set forth in any one of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26, or an amino acid sequence having at least 90% identity thereto.

8. The antibody or antigen-binding fragment comprises: A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19 or an amino acid sequence having at least 90% homology thereto, and a light chain variable region having the amino acid sequence set forth in any one of SEQ ID NO: 20 or an amino acid sequence having at least 90% homology thereto; or A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90% homology thereto, and a light chain variable region having the amino acid sequence set forth in any one of SEQ ID NO: 22 or an amino acid sequence having at least 90% homology thereto; or A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90% homology thereto, and a light chain variable region having the amino acid sequence set forth in any one of SEQ ID NO: 24 or an amino acid sequence having at least 90% homology thereto; or 5. The antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 90% identity thereto, and a light chain variable region having the amino acid sequence shown in any one of SEQ ID NO: 26 or an amino acid sequence having at least 90% identity thereto.

9. the antibody or antigen-binding fragment further comprises a constant region; The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the constant region comprises at least one of a heavy chain constant region and a light chain constant region.

10. 10. The antibody or antigen-binding fragment of claim 9, wherein at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody, a bovine-derived antibody, an equine-derived antibody, a dairy cow-derived antibody, a porcine-derived antibody, a sheep-derived antibody, a goat-derived antibody, a dog-derived antibody, a cat-derived antibody, a rabbit-derived antibody, a camel-derived antibody, a donkey-derived antibody, a deer-derived antibody, a marten-derived antibody, a chicken-derived antibody, a duck-derived antibody, a goose-derived antibody, a turkey-derived antibody, a gamecock-derived antibody, or a mutant thereof.

11. the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; or 10. The antibody or antigen-binding fragment of claim 9, wherein the light chain constant region is selected from a kappa or lambda light chain constant region.

12. The antibody or antigen-binding fragment of claim 9, wherein the light chain constant region and the heavy chain constant region are both derived from a mouse-derived antibody or a mutant thereof, or a human-derived antibody or a mutant thereof.

13. the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and / or The antibody or antigen-binding fragment of claim 9, wherein the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

14. The heavy chain constant region and / or comprising a heavy chain constant region as set forth in SEQ ID NO: 27 or an amino acid sequence having at least 80% identity thereto, or a heavy chain constant region as set forth in SEQ ID NO: 28 or an amino acid sequence having at least 80% identity thereto; 10. The antibody or antigen-binding fragment of claim 9, wherein the light chain constant region comprises or is the light chain constant region set forth in SEQ ID NO: 55 or an amino acid sequence having at least 80% identity thereto, or wherein the light chain constant region comprises or is the light chain constant region set forth in SEQ ID NO: 56 or an amino acid sequence having at least 80% identity thereto.

15. The antibody or antigen-binding fragment comprises: A heavy chain having an amino acid sequence set forth in any one of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41, or an amino acid sequence having at least 90% homology thereto, and / or 5. The antibody or antigen-binding fragment of claim 1, comprising a light chain having an amino acid sequence set forth in any one of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, or SEQ ID NO: 42, or an amino acid sequence having at least 90% identity thereto.

16. The antibody or antigen-binding fragment comprises: a heavy chain having the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 80% identity thereto, and a light chain having the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 80% identity thereto; or a heavy chain having the amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence having at least 80% identity thereto, and a light chain having the amino acid sequence shown in SEQ ID NO: 32 or an amino acid sequence having at least 80% identity thereto; or a heavy chain having the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having at least 80% identity thereto, and a light chain having the amino acid sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 80% identity thereto; or a heavy chain having the amino acid sequence set forth in SEQ ID NO: 35 or an amino acid sequence having at least 80% identity thereto, and a light chain having the amino acid sequence set forth in SEQ ID NO: 36 or an amino acid sequence having at least 80% identity thereto; or a heavy chain having the amino acid sequence shown in SEQ ID NO: 37 or an amino acid sequence having at least 80% identity thereto, and a light chain having the amino acid sequence shown in SEQ ID NO: 38 or an amino acid sequence having at least 80% identity thereto; or a heavy chain having the amino acid sequence shown in SEQ ID NO: 39 or an amino acid sequence having at least 80% identity thereto, and a light chain having the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 80% identity thereto; or a heavy chain having the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 80% identity thereto, and a light chain having the amino acid sequence shown in SEQ ID NO: 42 or an amino acid sequence having at least 80% identity thereto; Optionally, the antibody is a polyclonal antibody, a full-length monoclonal antibody, a Fab antibody, a Fab' antibody, a F(ab') antibody, or a Fab' antibody. ２ at least one selected from an antibody, an Fv antibody, a single-chain antibody, a single-domain antibody, and a minimal recognition unit; or The antigen-binding fragment is F(ab') ２ fragment, Fab' fragment, Fab fragment, F(ab) ２ The antibody or antigen-binding fragment according to any one of claims 1 to 4, comprising at least one selected from the group consisting of an Fv fragment, an scFv fragment, an scFv-Fc fusion protein, an scFv-Fv fusion protein, and a minimum recognition unit.

17. A recombinant protein comprising: An antibody or antigen-binding fragment according to any one of claims 1 to 16, and A recombinant protein characterized by comprising at least one of any biologically active protein or fragment thereof, or a biologically active polypeptide or fragment thereof.

18. The recombinant protein of claim 17, wherein the biologically active protein or fragment thereof comprises at least one selected from a protein tag, a protein toxin or fragment thereof, a tumor necrosis factor or fragment thereof, an interferon or fragment thereof, a biological response modifier or fragment thereof, and an Fc fragment.

19. A multispecific antibody, a first binding region comprising the antibody or antigen-binding fragment of any one of claims 1 to 16; and a second binding region having binding activity to the first molecule.

20. The multispecific antibody of claim 19, characterized in that the multispecific antibody comprises at least one selected from a bispecific antibody, a trispecific antibody, and a tetraspecific antibody, and is preferably a bispecific antibody.

21. 20. The multispecific antibody of claim 19, wherein the first molecule comprises at least one selected from a tumor-associated antigen, a virus, a bacterium, an endotoxin, a cytokine, and a cytokine receptor, and is preferably a tumor antigen.

22. 20. The multispecific antibody of claim 19, wherein the first molecule comprises at least one selected from B7H6, MICA, MICB, and PDL1, and preferably MICA.

23. the first binding region comprises a first heavy chain variable region and a first light chain variable region; the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first light chain variable region; or The multispecific antibody according to any one of claims 19 to 22, wherein the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region.

24. the first binding region further comprises a first connecting peptide; the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the first light chain variable region; or The multispecific antibody of claim 23, wherein the C-terminus of the first light chain variable region is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the first heavy chain variable region.

25. the first heavy chain variable region is a heavy chain variable region defined by the antibody or antigen-binding fragment of any one of claims 1 to 16; and / or The multispecific antibody of claim 23, wherein the first light chain variable region is a light chain variable region defined by an antibody or antigen-binding fragment of any one of claims 1 to 16.

26. the first binding region further comprises a first Fc fragment; the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the first light chain variable region, and the C-terminus of the first light chain variable region is connected to the N-terminus of the first Fc fragment; or The multispecific antibody of claim 24, wherein the C-terminus of the first light chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the first heavy chain variable region, and the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first Fc fragment.

27. The first Fc fragment has an amino acid sequence similar to that of the Fc fragment of human-derived wild-type IgG1, 27. The multispecific antibody of claim 26, comprising at least one of the mutation sites C220S, L234A, L235A, T366W and S354C.

28. The multispecific antibody of claim 27, wherein the first Fc fragment has the amino acid sequence shown in SEQ ID NO:

43.

29. the first binding region further comprises a second connecting peptide; Optionally, the N-terminus of the second connecting peptide is connected to the C-terminus of the first light chain variable region and the C-terminus of the second connecting peptide is connected to the N-terminus of the first Fc fragment; or The multispecific antibody of claim 26, wherein the N-terminus of the second connecting peptide is connected to the C-terminus of the first heavy chain variable region, and the C-terminus of the second connecting peptide is connected to the N-terminus of the first Fc fragment.

30. The amino acid sequence of the first connecting peptide and / or the second connecting peptide is (GGGGS) ｎ 30. The multispecific antibody of claim 29, wherein n is an integer of 1 or more, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

31. the first connecting peptide has the amino acid sequence set forth in SEQ ID NO: 44, and / or 31. The multispecific antibody of claim 30, wherein the second connecting peptide is GGGGS.

32. The multispecific antibody of claim 19, wherein the first binding region comprises an amino acid sequence shown in any one of SEQ ID NOs: 45, 47, 57 and 59.

33. The multispecific antibody of claim 19, wherein the first binding region has an amino acid sequence shown in any one of SEQ ID NOs: 46, 48, 58 and 60.

34. the second binding region comprises a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide being connected by an interchain disulfide bond; the first polypeptide comprises a second heavy chain variable region, a CH1 region, and a second Fc fragment, the C-terminus of the second heavy chain variable region being connected to the N-terminus of the CH1 region, and the C-terminus of the CH1 region being connected to the N-terminus of the second Fc fragment; The multispecific antibody of claim 19, wherein the second polypeptide comprises a second light chain variable region and a CL region, and the C-terminus of the second light chain variable region is connected to the N-terminus of the CL region.

35. the CH1 region is selected from the CH1 region of a wild-type IgG1 of human, primate or mouse origin, and / or The second Fc fragment has an amino acid sequence similar to that of the Fc fragment of human-derived wild-type IgG1, 35. The multispecific antibody of claim 34, comprising at least one of the following mutation sites: L234A, L235A, Y349C, T366S, L368A and Y407V.

36. The multispecific antibody of claim 34, wherein the second Fc fragment has the amino acid sequence shown in SEQ ID NO:

49.

37. The multispecific antibody of claim 34, wherein the CL region is a wild-type CL region of human, primate or mouse origin, preferably a wild-type CL region of human origin.

38. The multispecific antibody of claim 34, wherein the second heavy chain variable region has the amino acid sequence shown in SEQ ID NO:

51.

39. The multispecific antibody of claim 34, wherein the second light chain variable region has the amino acid sequence shown in SEQ ID NO:

52.

40. The multispecific antibody of claim 34, wherein the first polypeptide has the amino acid sequence shown in SEQ ID NO:

53.

41. The multispecific antibody of claim 34, wherein the second polypeptide has the amino acid sequence shown in SEQ ID NO:

54.

42. The multispecific antibody of claim 19, wherein the first binding region and the second binding region are connected by a knob-into-hole structure.

43. 43. A nucleic acid molecule, characterized in that said nucleic acid molecule encodes the antibody or antigen-binding fragment of any one of claims 1 to 16, the recombinant protein of any one of claims 17 to 18 or the multispecific antibody of any one of claims 19 to 42.

44. An expression vector carrying the nucleic acid molecule of claim 43.

45. A recombinant cell comprising: Carrying a nucleic acid molecule according to claim 43 or an expression vector according to claim 44, or A recombinant cell characterized in that it contains an expression of an antibody or antigen-binding fragment according to any one of claims 1 to 16, a recombinant protein according to any one of claims 17 to 18 or a multispecific antibody according to any one of claims 19 to 42.

46. 1. A pharmaceutical composition comprising: An antibody or antigen-binding fragment according to any one of claims 1 to 16, a recombinant protein according to any one of claims 17 to 18, a multispecific antibody according to any one of claims 19 to 42, a nucleic acid molecule according to claim 43, an expression vector according to claim 44 or a recombinant cell according to claim 45, and A pharmaceutical composition characterized by optionally containing a pharmaceutically acceptable adjuvant.

47. A conjugate comprising: An antibody or antigen-binding fragment according to any one of claims 1 to 16, a recombinant protein according to any one of claims 17 to 18 or a multispecific antibody according to any one of claims 19 to 42, and A conjugate comprising a conjugation moiety attached to said antibody or antigen-binding fragment, recombinant protein or multispecific antibody.

48. 48. The conjugate of claim 47, wherein the conjugation moiety comprises at least one selected from a vector, a drug, a toxin, a cytokine, a protein tag, a modification, and a chemotherapeutic agent.

49. A kit comprising:

46. ​​A kit comprising an antibody or antigen-binding fragment according to any one of claims 1 to 16, a recombinant protein according to any one of claims 17 to 18, a multispecific antibody according to any one of claims 19 to 42, a nucleic acid molecule according to claim 43, an expression vector according to claim 44 or a recombinant cell according to claim 45.

50. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 16, a recombinant protein according to any one of claims 17 to 18, a multispecific antibody according to any one of claims 19 to 42, a pharmaceutical composition according to claim 46 or a conjugate according to any one of claims 47 to 48 in the preparation of a medicament for the prevention and / or treatment of an NKp46-mediated disease.

51. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 16, a recombinant protein according to any one of claims 17 to 18, a multispecific antibody according to any one of claims 19 to 42, a pharmaceutical composition according to claim 46 or a conjugate according to any one of claims 47 to 48 in the prevention and / or treatment of NKp46-mediated diseases or in the detection of NKp46.

52. An antibody or antigen-binding fragment according to any one of claims 1 to 16, a recombinant protein according to any one of claims 17 to 18, a multispecific antibody according to any one of claims 19 to 42, a pharmaceutical composition according to claim 46 or a conjugate according to any one of claims 47 to 48 for preventing and / or treating a disease mediated by NKp46 or for detecting NKp46.

53. The use according to any one of claims 50 to 52, characterized in that the NKp46-mediated disease comprises at least one selected from tumors or cancers, diseases associated with transplant rejection, autoimmune diseases and infectious diseases.

54. 54. The use of claim 53, wherein the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

55. A method for detecting NKp46, comprising: contacting a sample to be detected with the antibody or antigen-binding fragment of any one of claims 1 to 16, the recombinant protein of any one of claims 17 to 18, the multispecific antibody of any one of claims 19 to 42, the conjugate of any one of claims 47 to 48, or the kit of claim 49 to form an immune complex; determining whether or not the sample to be detected contains NKp46, or determining the amount of NKp46 contained, based on the signal of the immune complex.

56. 1. A method for treating or preventing cancer or tumors, comprising:

47. A method for treating or preventing cancer or tumors, comprising administering to a subject a pharmaceutically acceptable amount of the antibody or antigen-binding fragment of any one of claims 1 to 16, the recombinant protein of any one of claims 17 to 18, the multispecific antibody of any one of claims 19 to 42, the pharmaceutical composition of claim 46, or the conjugate of any one of claims 47 to 48.

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