Nkp46 antibodies, methods of making and using the same

Abstract

The present application discloses an antibody targeting NKp46, a preparation method and use thereof. Specifically, the present application discloses a new human monoclonal antibody targeting NKp46. The present application also discloses a method for preparing the monoclonal antibody. The monoclonal antibody of the present application can specifically bind to the NKp46 antigen with high specificity, has high affinity and has significant anti-tumor activity.

Description

Technical Field

[0001] This invention belongs to the field of antibodies, specifically relating to an NKp46 antibody, its preparation method, and its application. Background Technology

[0002] Maintaining effective immune surveillance without triggering an autoimmune response requires accurate effector T cell responses. Autoimmune diseases arise when the immune system initiates an immune response against its own antigens. Although the mechanisms involved in initiating and maintaining autoimmune responses are not fully understood, the presence of previously immune-neglected antigens in secondary lymphoid organs may be involved.

[0003] Natural killer (NK) cells are a subpopulation of lymphocytes involved in non-traditional immunity. NK cells provide an effective immune surveillance mechanism, thereby eliminating unwanted cells such as tumor cells or virus-infected cells. NK cell activity is regulated through a complex mechanism involving two types of activation and inhibition signals.

[0004] CD94-NKG2A is an important inhibitory receptor on NK cells that interacts with atypical MHC class I molecules, HLA-E. Some of these receptors have the ability to regulate the threshold for T cell antigen receptor-dependent T cell activation. In rare cases of inhibitory receptor deficiency, these activation isoforms may amplify the function of T cell effectors and contribute to autoimmune pathology. The amino acid sequence of NKG2A varies in mammals, including primates. For example, the human NKG2A protein shares less than 90% homology with that of rhesus monkeys.

[0005] Several distinct NK-specific receptors have been confirmed to play crucial roles in NK cell-mediated recognition and killing of HLA class I-deficient target cells. These receptors (named NKp30, NKp46, and NKp44) are members of the Ig superfamily. Their cross-linking (induced by specific mAbs) leads to strong NK cell activation, resulting in increased intracellular Ca2+. ++ Elevated levels trigger cytotoxicity and lymphokine release. Importantly, monoclonal antibody-mediated activation of NKp30, NKp46, and / or NKp44 leads to activation of NK cell cytotoxicity against a variety of target cells. These findings provide evidence for a central role of these receptors in native cytotoxicity.

[0006] Although research has been conducted on antibodies targeting NKp46, there is still a need to obtain more active and more specific antibodies. Summary of the Invention

[0007] The purpose of this invention is to provide NKp46 antibody, its preparation method and application.

[0008] Specifically, the present invention provides an NKp46 antibody comprising one or more of the heavy chain variable region of the NKp46 antibody, including heavy chain CDR1 (VH-CDR1), heavy chain CDR2 (VH-CDR2), and heavy chain CDR3 (VH-CDR3), and one or more of the light chain variable region of the NKp46 antibody, including light chain CDR1 (VL-CDR1), light chain CDR2 (VL-CDR2), and light chain CDR3 (VL-CDR3).

[0009] In a first aspect of the invention, a heavy chain variable region of an antibody is provided, said heavy chain variable region comprising the following three complementarity-determining regions (CDRs):

[0010] VH-CDR1, as shown in SEQ ID NO:9,

[0011] VH-CDR2 shown in SEQ ID NO:13, and

[0012] VH-CDR3 as shown in SEQ ID NO:17;

[0013] or,

[0014] VH-CDR1 shown in SEQ ID NO:10,

[0015] VH-CDR2 shown in SEQ ID NO:14, and

[0016] VH-CDR3 as shown in SEQ ID NO:18;

[0017] or,

[0018] VH-CDR1 shown in SEQ ID NO:11,

[0019] VH-CDR2 shown in SEQ ID NO:15, and

[0020] VH-CDR3 as shown in SEQ ID NO:19;

[0021] or,

[0022] VH-CDR1 shown in SEQ ID NO:12,

[0023] VH-CDR2 shown in SEQ ID NO:16, and

[0024] VH-CDR3, as shown in SEQ ID NO:20,

[0025] Among them, any of the above-mentioned amino acid sequences also includes a derivative sequence which has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining the binding affinity for NKP46.

[0026] In another preferred embodiment, the heavy chain variable region further includes a human-derived FR region or a mouse-derived FR region.

[0027] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the chimeric antibody is shown in SEQ ID NO:1-4.

[0028] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the humanized antibody is shown in SEQ ID NO:33-40.

[0029] In a second aspect of the invention, a heavy chain of an antibody is provided, said heavy chain having the heavy chain variable region described in the first aspect of the invention.

[0030] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0031] In another preferred embodiment, the heavy chain constant region is of human, mouse, or rabbit origin.

[0032] In a third aspect of the invention, a light chain variable region of an antibody is provided, the light chain variable region comprising the following three complementarity-determining regions (CDRs):

[0033] VL-CDR1 shown in SEQ ID NO:21,

[0034] VL-CDR2 shown in SEQ ID NO:25, and

[0035] VL-CDR3 as shown in SEQ ID NO:29;

[0036] or,

[0037] VL-CDR1 shown in SEQ ID NO:22,

[0038] VL-CDR2 shown in SEQ ID NO:26, and

[0039] VL-CDR3 as shown in SEQ ID NO:30;

[0040] or,

[0041] VL-CDR1 shown in SEQ ID NO:23,

[0042] VL-CDR2 shown in SEQ ID NO:27, and

[0043] VL-CDR3 as shown in SEQ ID NO:31;

[0044] or,

[0045] VL-CDR1 shown in SEQ ID NO:24

[0046] VL-CDR2 shown in SEQ ID NO:28, and

[0047] VL-CDR3, as shown in SEQ ID NO:32,

[0048] Among them, any of the above-mentioned amino acid sequences also includes a derivative sequence which has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining the binding affinity for NKP46.

[0049] In another preferred embodiment, the light chain variable region further includes a human-derived FR region or a mouse-derived FR region.

[0050] In another preferred embodiment, the amino acid sequence of the light chain variable region of the chimeric antibody is shown in SEQ ID NO:5-8.

[0051] In another preferred embodiment, the amino acid sequence of the light chain variable region of the humanized antibody is shown in SEQ ID NO:41-48.

[0052] In a fourth aspect of the invention, a light chain of an antibody is provided, said light chain having the light chain variable region described in the third aspect of the invention.

[0053] In another preferred embodiment, the light chain of the antibody further includes a light chain constant region.

[0054] In another preferred embodiment, the light chain constant region is of human, mouse, or rabbit origin.

[0055] In a fifth aspect of the invention, an antibody is provided, the antibody having:

[0056] (1) The heavy chain variable region described in the first aspect of the present invention; and / or

[0057] (2) The light chain variable region described in the third aspect of the present invention;

[0058] Alternatively, the antibody may have: the heavy chain described in the second aspect of the invention; and / or the light chain described in the fourth aspect of the invention.

[0059] In another preferred embodiment, the antibody further includes a heavy chain constant region and / or a light chain constant region.

[0060] In another preferred embodiment, the heavy chain constant region is of human origin, and / or the light chain constant region is of human origin.

[0061] In another preferred embodiment, the heavy chain constant region is the human antibody heavy chain IgG1 constant region, and the light chain constant region is the human antibody light chain kappa constant region.

[0062] In another preferred embodiment, the antibody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies, or combinations thereof.

[0063] In another preferred embodiment, the CDR region of the humanized antibody contains variations of 1, 2, or 3 amino acids.

[0064] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, or rabbit.

[0065] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0066] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0067] In another preferred embodiment, the antibody is a bispecific antibody or a multispecific antibody.

[0068] In another preferred embodiment, the antibody is a monoclonal antibody.

[0069] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0070] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, and more preferably 10%.

[0071] In another preferred embodiment, the at least one amino acid sequence that has been added, deleted, modified and / or substituted is an amino acid sequence with at least 80% homology.

[0072] In another preferred embodiment, the derived sequence, which has been added, deleted, modified, and / or substituted with at least one amino acid, has NKP46 binding activity.

[0073] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:1; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:5.

[0074] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:2; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:6.

[0075] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:3; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:7.

[0076] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:4; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:8.

[0077] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44.

[0078] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 or SEQ ID NO:48.

[0079] In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2, 3, or 4 in the sequence listing.

[0080] In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO:5 in the sequence listing.

[0081] In another preferred embodiment, the antibody has an affinity KD of (0.5-2)×10⁻⁶ for NKP46. -10 .

[0082] In another preferred embodiment, the antibody has a specific binding effect only on the human NKp46 antigen and does not bind to human NKp44 and NKp30.

[0083] In another preferred embodiment, the antibody can bind to NKP46 on the surface of NK cells and activate the NK cells.

[0084] In another preferred embodiment, the NK cells include NK92 cells or human NK cells.

[0085] In a sixth aspect, the present invention provides a recombinant protein having:

[0086] (i) the heavy chain variable region as described in the first aspect of the invention, the heavy chain as described in the second aspect of the invention, the light chain variable region as described in the third aspect of the invention, the light chain as described in the fourth aspect of the invention, or the antibody as described in the fifth aspect of the invention; and

[0087] (ii) Optional tag sequences to assist in expression and / or purification.

[0088] In another preferred embodiment, the tag sequence includes a 6His tag.

[0089] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0090] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0091] In a seventh aspect, the present invention provides a CAR construct in which the scFv segment of the antigen-binding domain is a binding region specifically binding to NKP46, and the scFv has a heavy chain variable region as described in the first aspect of the present invention and a light chain variable region as described in the third aspect of the present invention.

[0092] In an eighth aspect, the present invention provides a recombinant immune cell expressing an exogenous CAR construct as described in the seventh aspect of the present invention.

[0093] A ninth aspect of the present invention provides an antibody-drug conjugate comprising:

[0094] (a) An antibody portion, said antibody portion being selected from the group consisting of: the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, or the antibody of the fifth aspect of the present invention, or a combination thereof; and

[0095] (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0096] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0097] In a tenth aspect of the present invention, there is provided a use of an active ingredient selected from the group consisting of: the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, or the antibody of the fifth aspect of the present invention, the recombinant protein of the sixth aspect of the present invention, the immune cell of the eighth aspect of the present invention, the antibody-drug conjugate of the ninth aspect of the present invention, or combinations thereof, wherein the active ingredient is used for (a) preparing a detection reagent, detection plate or kit; and / or (b) preparing a medicament for the prevention and / or treatment of cancer and / or diseases related to abnormal expression or function of NKP46.

[0098] In another preferred embodiment, the detection reagent, detection plate, or kit is used for:

[0099] (1) Detect NKP46 in the sample; and / or

[0100] (2) Detection of NKP46 on the surface of immune cells; and / or

[0101] (3) Detect immune cells expressing NKP46.

[0102] In another preferred embodiment, the immune cells are selected from the group consisting of NK cells, T cells, B cells, basophils, and dendritic cells.

[0103] In another preferred embodiment, the drug is used to activate the cytotoxic activity of NK cells.

[0104] In another preferred embodiment, the diseases associated with abnormal NKP46 expression or function are selected from the group consisting of: cancer, autoimmune diseases, metabolic diseases, infectious diseases, or combinations thereof.

[0105] In another preferred embodiment, the cancer includes solid tumors and hematologic malignancies.

[0106] In another preferred embodiment, the metabolic-related diseases include diabetes, diet-induced obesity, and lipoinflammatory disease.

[0107] In another preferred embodiment, the infectious diseases include bacterial and viral infections.

[0108] In an eleventh aspect, the present invention provides a pharmaceutical composition comprising:

[0109] (i) an active ingredient selected from the group consisting of: the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, or the antibody of the fifth aspect of the present invention, the recombinant protein of the sixth aspect of the present invention, the immune cell of the eighth aspect of the present invention, the antibody-drug conjugate of the ninth aspect of the present invention, or combinations thereof; and

[0110] (ii) Pharmaceutically acceptable carriers.

[0111] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0112] In another preferred embodiment, the pharmaceutical composition is an injection.

[0113] A twelfth aspect of the present invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:

[0114] (1) The heavy chain variable region described in the first aspect of the present invention, the heavy chain described in the second aspect of the present invention, the light chain variable region described in the third aspect of the present invention, the light chain described in the fourth aspect of the present invention, or the antibody described in the fifth aspect of the present invention; or

[0115] (2) The recombinant protein described in the sixth aspect of the present invention;

[0116] (3) The CAR construct described in the seventh aspect of the present invention.

[0117] In a thirteenth aspect of the present invention, a carrier is provided, said carrier containing the polynucleotide described in the twelfth aspect of the present invention.

[0118] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0119] In a fourteenth aspect of the present invention, a genetically engineered host cell is provided, said host cell containing the vector described in the thirteenth aspect of the present invention or the polynucleotide described in the twelfth aspect of the present invention integrated into the genome.

[0120] The fifteenth aspect of the present invention provides a method for in vitro detection of NKP46 in samples (including diagnostic or non-diagnostic samples), the method comprising the steps of:

[0121] (1) In vitro, the sample is contacted with the antibody described in the fifth aspect of the present invention;

[0122] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of NKP46 in the sample.

[0123] In a sixteenth aspect, the present invention provides a detection plate comprising: a substrate (support plate) and a test strip, wherein the test strip contains an antibody as described in the fifth aspect of the present invention or an immunoconjugate as described in the ninth aspect of the present invention.

[0124] According to a seventeenth aspect of the present invention, a kit is provided, the kit comprising:

[0125] (1) A first container containing the antibody as described in the fifth aspect of the present invention; and / or

[0126] (2) A second container containing a secondary antibody against the antibody described in the fifth aspect of the present invention;

[0127] Alternatively, the kit may contain the detection plate described in the sixteenth aspect of the present invention.

[0128] An eighteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:

[0129] (a) Culturing the host cells described in the fourteenth aspect of the present invention under suitable expression conditions;

[0130] (b) Isolate a recombinant polypeptide from the culture, wherein the recombinant polypeptide is an antibody as described in the fifth aspect of the present invention or a recombinant protein as described in the sixth aspect of the present invention.

[0131] A nineteenth aspect of the present invention provides a method for activating NK cell cytotoxic activity, the method comprising: administering to a desired subject an antibody as described in the fifth aspect of the present invention, an antibody-drug conjugate of the antibody, or CAR-T cells expressing the antibody, or a combination thereof.

[0132] In another preferred embodiment, the method further includes administering other drugs or treatments to the subject in need for combined treatment.

[0133] In another preferred embodiment, the other drugs or treatment methods include: antitumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.

[0134] In a twentieth aspect of the present invention, a method for preparing a chimeric antibody is provided, comprising the steps of:

[0135] After cloning the nucleotide sequences of the heavy chain variable region described in the first aspect of the present invention and / or the light chain variable region described in the third aspect of the present invention into an expression vector containing the nucleotide sequence of the human antibody constant region, the human-mouse chimeric antibody is expressed by transfecting animal cells.

[0136] In a twenty-first aspect of the present invention, a method for preparing a humanized antibody is provided, comprising the steps of:

[0137] The nucleotide sequence of the CDR region in the heavy chain variable region described in the first aspect of the present invention and / or the light chain variable region described in the third aspect of the present invention is inserted into a nucleotide sequence template containing the FR region of a human antibody, and then cloned into an expression vector containing the constant region of a human antibody, and then expressed as a humanized antibody by transfecting animal cells.

[0138] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0139] Figure 1 The figure shows the antibody titers in mouse serum after cell immunization, measured by ELISA / FACS. The numbers in the figure represent the unimmunized, first-collected, and second-collected immunized serum of mice in Group 1 (Balb / c) and Group 2 (SJL).

[0140] Figure 2 The figure shows the antibody titers in mouse serum after protein immunization, measured by ELISA / FACS. The numbers in the figure represent the unimmunized, first-collected, and second-collected immunized serum of mice in groups 3 (Balb / c) and 4 (SJL).

[0141] Figure 3 The figure shows the antibody titers in mouse serum after DNA immunization, measured by ELISA / FACS. The numbers in the figure represent the unimmunized, first-collected, and second-collected immunized serum of mice in groups 5 (Balb / c) and 6 (SJL).

[0142] Figure 4a The binding reaction between hNKp46 antibody and CHOK1-hNKp46 was detected by FACS.

[0143] Figure 4b The binding reaction between hNKp46 antibody and CHOK1-cNKp46 was detected by FACS.

[0144] Figure 4c The binding reaction of hNKp46 antibody and CHOK1-blank was detected by FACS.

[0145] Figure 5 The activation of Jurkat-NFAT-hNKp46 / hCD3z cells by the hNKp46 antibody was detected using the Jurkat reporter assay.

[0146] Figure 6aThe proportion of CD69-positive NK cells after NKp46 treatment of primary NK cells was detected by flow cytometry (FACS).

[0147] Figure 6b The proportion of CD107a-positive NK cells after NKp46 treatment with primary NK cells was detected by flow cytometry (FACS).

[0148] Figure 7 The secretion of IFNγ stimulated by hNKp46 antibody was detected by ELISA.

[0149] Figure 8 To improve the specificity of hNKp46 antibody detection by ELISA. Detailed Implementation

[0150] Through extensive and in-depth research, the inventors of this invention have obtained a group of murine anti-human NKp46 antibodies with novel amino acid sequences using hybridoma technology. The NKp46 antibodies described in this invention can bind to human NKp46 with high affinity (KD reaches 1.27 × 10⁻⁶). -10 M); and the NKp46 antibody can bind to the extracellular region of the NKp46 protein receptor; the NKp46 antibody can significantly increase the killing effect of NK92 or human NK cells on target cells; based on primary NK cell activity assays, its biological activity is significantly superior to the reference antibody. Furthermore, the NKp46 antibody of this invention has high specificity and lacks cross-reactivity with human NKp44 and NKp30 family protein antigens. The NKp46 antibody of this invention can be used to activate the body's innate immune response to tumors, alone or in combination with anti-PD-1 monoclonal antibodies or other anti-tumor drugs in tumor immunotherapy. The antibody of this invention can be used in the preparation of drugs for treating tumors, autoimmune diseases, etc. This invention was completed based on these findings.

[0151] the term

[0152] NKp46

[0153] Natural killer (NK) cells are a crucial type of lymphocyte in the body, playing a vital role in both innate and adaptive immunity. NK cells possess two types of surface receptors, which can be classified into inhibitory and activating receptors based on their function. These receptors mediate different recognition patterns of NK cells, transmitting different activation and inhibition signals. The CD94 / NKG2 family is a well-studied receptor family, mainly including members such as NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H. Among them, NKG2A is an inhibitory receptor, and its ligand is the non-classical major histocompatibility complex class I molecule—HLA-E. NK cells also express activating receptors, such as CD16a (FcγRIIIA), NKG2D, SLAM family members, and natural cytotoxic receptors (NCRs). Complete activation of NK cells requires the joint participation of different activating receptors. NKp46 is a 46 kDa glycoprotein belonging to the immunoglobulin (Ig) superfamily and is frequently expressed on tumor-infiltrating lymphocytes. Antibody activation of NKp46 not only induces NK cell killing but also triggers the release of cytokines. Therefore, NKp46 is a promising target for anti-tumor therapy. This application provides an antibody against human NKp46 discovered using a hybridoma technology platform.

[0154] Antibody

[0155] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0156] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0157] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0158] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0159] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0160] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be obtained using standard DNA recombination techniques and are all useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as a chimeric antibody having a variable region derived from a mouse monoclonal antibody and a constant region derived from a human immunoglobulin (see, for example, U.S. Patents 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety). A humanized antibody is an antibody molecule derived from a non-human species, having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0161] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0162] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0163] Table A

[0164]

[0165]

[0166] Anti-NKp46 antibodies

[0167] In this invention, the antibody is an anti-NKp46 antibody. This invention provides a highly specific and high-affinity antibody against NKp46, comprising a heavy chain and a light chain, wherein the heavy chain contains a heavy chain variable region (VH) amino acid sequence, and the light chain contains a light chain variable region (VL) amino acid sequence.

[0168] The antibody of the present invention, which binds to human NKp46 protein, has an antigen-binding domain comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region includes three complementarity-determining regions VHCDR1, VHCDR2, and VHCDR3; the light chain variable region includes three complementarity-determining regions VLCDR1, VLCDR2, and VLCDR3; wherein...

[0169] (a) The variable region of the antibody heavy chain comprises VH-CDR1 containing the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; VH-CDR2 containing the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; and VH-CDR3 containing the amino acid sequences shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, wherein any of the above amino acid sequences further comprises a derived sequence that optionally has been added, deleted, modified, and / or substituted with at least one amino acid, and is capable of retaining NKp46 binding affinity.

[0170] (b) The variable region of the antibody light chain comprises VL-CDR1 containing the amino acid sequences shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24; VL-CDR2 containing the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28; and VL-CDR3 containing the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, wherein any of the above amino acid sequences further comprises a derived sequence that optionally has been added, deleted, modified, and / or substituted with at least one amino acid and is capable of retaining NKp46 binding affinity.

[0171] Preferably, the heavy chain variable region (VH) has a complementary determinant region (CDR) selected from the group consisting of:

[0172] VH-CDR1, as shown in SEQ ID NO:9,

[0173] VH-CDR2 shown in SEQ ID NO:13, and

[0174] VH-CDR3 as shown in SEQ ID NO:17;

[0175] or,

[0176] VH-CDR1 shown in SEQ ID NO:10,

[0177] VH-CDR2 shown in SEQ ID NO:14, and

[0178] VH-CDR3 as shown in SEQ ID NO:18;

[0179] or,

[0180] VH-CDR1 shown in SEQ ID NO:11,

[0181] VH-CDR2 shown in SEQ ID NO:15, and

[0182] VH-CDR3 as shown in SEQ ID NO:19;

[0183] or,

[0184] VH-CDR1 shown in SEQ ID NO:12,

[0185] VH-CDR2 shown in SEQ ID NO:16, and

[0186] VH-CDR3 as shown in SEQ ID NO:20.

[0187] Preferably, the light chain variable region (VL) has a complementary determinant region (CDR) selected from the group consisting of:

[0188] VL-CDR1 shown in SEQ ID NO:21,

[0189] VL-CDR2 shown in SEQ ID NO:25, and

[0190] VL-CDR3 as shown in SEQ ID NO:29;

[0191] or,

[0192] VL-CDR1 shown in SEQ ID NO:22,

[0193] VL-CDR2 shown in SEQ ID NO:26, and

[0194] VL-CDR3 as shown in SEQ ID NO:30;

[0195] or,

[0196] VL-CDR1 shown in SEQ ID NO:23,

[0197] VL-CDR2 shown in SEQ ID NO:27, and

[0198] VL-CDR3 as shown in SEQ ID NO:31;

[0199] or,

[0200] VL-CDR1 shown in SEQ ID NO:24

[0201] VL-CDR2 shown in SEQ ID NO:28, and

[0202] VL-CDR3 as shown in SEQ ID NO:32.

[0203] Among them, any of the above amino acid sequences also includes a derivative sequence that has optionally been added, deleted, modified and / or substituted at least one amino acid and is capable of retaining NKp46 binding affinity.

[0204] Among them, any of the above amino acid sequences also includes a derivative sequence that has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining NKG2A binding affinity.

[0205] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or substituting at least one amino acid sequence preferably has at least 80% homology or sequence identity, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the amino acid sequence.

[0206] Methods for determining sequence homology or identity known to those skilled in the art include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied. Math., 48:1073 (1988). Preferred methods for determining identity aim to achieve the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used for identity determination.

[0207] Preferably, the antibody described herein is one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single-chain antibody fragment (scFv), single-domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies. The monoclonal antibody can be developed using various methods and techniques, including hybridoma technology, phage display technology, and single-lymphocyte gene cloning technology. The mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice using hybridoma technology.

[0208] The full-length antibody protein is a conventional full-length antibody protein in the art, comprising a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein, together with the human heavy chain constant region and the human light chain constant region, constitute a fully human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0209] The antibody of the present invention can be a double-chain or single-chain antibody, and can be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

[0210] The antibody derivatives described in this invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0211] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.

[0212] The animal is preferably a mammal, such as a mouse.

[0213] The antibodies of this invention may be chimeric antibodies, humanized antibodies, CDR-grafted and / or modified antibodies that target NKp46 (e.g., human NKp46).

[0214] In the above-described content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0215] In the above-described content of the present invention, more preferably, the number of added, deleted, modified and / or substituted amino acids can be 1-7, more preferably 1-5, more preferably 1-3, and more preferably 1-2.

[0216] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:1-4.

[0217] In another preferred embodiment, the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:5-8.

[0218] In another preferred embodiment, the heavy chain variable region (VH) amino acid sequence and / or the light chain variable region amino acid sequence of the antibody targeting NKp46 are shown in Table 1 below:

[0219] Table 1

[0220] Antibody number VH sequence number VL sequence number 21B2C8 SEQ ID NO:1 SEQ ID NO:5 24H4C4 SEQ ID NO:2 SEQ ID NO:6 29C9B5 SEQ ID NO:3 SEQ ID NO:7 32H2F2 SEQ ID NO:4 SEQ ID NO:8

[0221] In another preferred embodiment, the heavy chain variable region of the humanized antibody contains the amino acid sequence shown in SEQ ID NO:33-40.

[0222] In another preferred embodiment, the light chain variable region of the humanized antibody contains the amino acid sequence shown in SEQ ID NO:41-48.

[0223] In another preferred embodiment, the heavy chain variable region (VH) amino acid sequence and / or the light chain variable region amino acid sequence of the humanized antibody targeting NKp46 are shown in Tables 2 and 3 below:

[0224] Table 2

[0225] Antibody number VH sequence number VL sequence number 21B2C8-VH1+VL1 SEQ ID NO:33 SEQ ID NO:41 21B2C8-VH1+VL2 SEQ ID NO:33 SEQ ID NO:42 21B2C8-VH1+VL3 SEQ ID NO:33 SEQ ID NO:43 21B2C8-VH1+VL4 SEQ ID NO:33 SEQ ID NO:44 21B2C8-VH2+VL1 SEQ ID NO:34 SEQ ID NO:41 21B2C8-VH2+VL2 SEQ ID NO:34 SEQ ID NO:42 21B2C8-VH2+VL3 SEQ ID NO:34 SEQ ID NO:43 21B2C8-VH2+VL4 SEQ ID NO:34 SEQ ID NO:44 21B2C8-VH3+VL1 SEQ ID NO:35 SEQ ID NO:41 21B2C8-VH3+VL2 SEQ ID NO:35 SEQ ID NO:42 21B2C8-VH3+VL3 SEQ ID NO:35 SEQ ID NO:43 21B2C8-VH3+VL4 SEQ ID NO:35 SEQ ID NO:44 21B2C8-VH4+VL1 SEQ ID NO:36 SEQ ID NO:41 21B2C8-VH4+VL2 SEQ ID NO:36 SEQ ID NO:42 21B2C8-VH4+VL3 SEQ ID NO:36 SEQ ID NO:43 21B2C8-VH4+VL4 SEQ ID NO:36 SEQ ID NO:44

[0226] Table 3

[0227]

[0228]

[0229] Recombinant protein

[0230] The present invention also provides a recombinant protein comprising one or more of the heavy chain CDR1 (VH-CDR1), heavy chain CDR2 (VH-CDR2), and heavy chain CDR3 (VH-CDR3) of an NKp46 antibody, and / or one or more of the light chain CDR1 (VL-CDR1), light chain CDR2 (VL-CDR2), and light chain CDR3 (VL-CDR3) of an NKp46 antibody.

[0231] CDR1(VH):

[0232] NYWMY (SEQ ID NO:9)

[0233] SYWMH (SEQ ID NO:10)

[0234] WMH (SEQ ID NO:11)

[0235] SYWMH (SEQ ID NO:12)

[0236] CDR2(VH):

[0237] MIHPNSGSNKYNEKFKS(SEQ ID NO:13)

[0238] MIHPNSGSTKYNEKFKS(SEQ ID NO:14)

[0239] MIHPNSGTTNYNEKFKS(SEQ ID NO:15)

[0240] MIHPHTDITKYNEKFQN(SEQ ID NO:16)

[0241] CDR3(VH):

[0242] EGRGHGNYNTMDY (SEQ ID NO:17)

[0243] QGYGSGYWYFDV(SEQ ID NO:18)

[0244] EGRFDY (SEQ ID NO:19)

[0245] AGRFDY (SEQ ID NO:20)

[0246] The sequences of the light chains CDR1-3 are as follows:

[0247] CDR1(VL):

[0248] KASQDINSYLS (SEQ ID NO:21)

[0249] KASQDINSYLN (SEQ ID NO:22)

[0250] RASQDIGSSLN(SEQ ID NO:23)

[0251] RASQDIGSSLN(SEQ ID NO:24)

[0252] CDR2(VL):

[0253] RANRLVD (SEQ ID NO:25)

[0254] RANRLVD (SEQ ID NO:26)

[0255] ATSSLDS (SEQ ID NO:27)

[0256] ATSNLDS(SEQ ID NO:28)

[0257] CDR3(VL):

[0258] LQYDEFPYT(SEQ ID NO:29)

[0259] LQYDEFPYT(SEQ ID NO:30)

[0260] LQYASSPFT(SEQ ID NO:31)

[0261] LQYASSPFT(SEQ ID NO:32)

[0262] Among them, any of the above amino acid sequences also includes a derivative sequence that has optionally been added, deleted, modified and / or substituted at least one amino acid and is capable of retaining NKp46 binding affinity.

[0263] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or substituting at least one amino acid sequence preferably has at least 80% homology or sequence identity, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the amino acid sequence.

[0264] In another preferred embodiment, the recombinant protein of the present invention comprises a heavy chain variable region of an NKp46 antibody and / or a light chain variable region of an NKp46 antibody, wherein the heavy chain variable region of the antibody contains the amino acid sequences shown in SEQ ID NO: 1-4; the heavy chain variable region of the humanized antibody contains the amino acid sequences shown in SEQ ID NO: 33-40; the light chain variable region of the antibody contains the amino acid sequences shown in SEQ ID NO: 5-8; and the light chain variable region of the humanized antibody contains the amino acid sequences shown in SEQ ID NO: 41-48.

[0265] In another preferred embodiment, the sequence numbers of the recombinant protein and its constituent heavy chain CDR1-3 and light chain CDR1-3 are shown in Table 4:

[0266] Table 4. Sequence numbers of the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3.

[0267]

[0268]

[0269] Among them, any of the above amino acid sequences also includes a derivative sequence that has optionally been added, deleted, modified and / or substituted at least one amino acid and is capable of retaining NKp46 binding affinity.

[0270] Preferably, the recombinant protein further includes an antibody heavy chain constant region and / or an antibody light chain constant region. The antibody heavy chain constant region is conventional in the art, preferably a rat-derived antibody heavy chain constant region or a human-derived antibody heavy chain constant region, and more preferably a human-derived antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat-derived antibody light chain constant region or a human-derived antibody light chain constant region, and more preferably a human-derived antibody light chain constant region.

[0271] The recombinant protein is a conventional protein in the art. Preferably, it is one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single-chain antibody fragment (scFv), single-domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies. The monoclonal antibody can be developed using various methods and techniques, including hybridoma technology, phage display technology, and single-lymphocyte gene cloning technology. The mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice using hybridoma technology.

[0272] The full-length antibody protein is a conventional full-length antibody protein in the art, comprising a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein, together with the human heavy chain constant region and the human light chain constant region, constitute a fully human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0273] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.

[0274] The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, comprising an Fd segment of a light chain variable region, a light chain constant region, and a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab').

[0275] The single-domain antibody is a conventional single-domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

[0276] The single-region antibody described is a conventional single-region antibody in the art, which includes only the heavy chain variable region.

[0277] The recombinant protein is prepared using conventional methods in the art. Preferably, the preparation method involves isolating the protein from a recombinant expression transformant or obtaining it through artificial synthesis of the protein sequence. The preferred method for isolating the protein from the recombinant expression transformant is as follows: cloning a nucleic acid molecule encoding the protein and carrying a point mutation into a recombinant vector; transforming the obtained recombinant vector into a transformant to obtain a recombinant expression transformant; and culturing the obtained recombinant expression transformant to isolate and purify the recombinant protein.

[0278] Nucleic acid

[0279] The present invention also provides a nucleic acid that encodes the heavy chain variable region or light chain variable region of the aforementioned antibody (e.g., an antibody against NKp46) or recombinant protein or antibody against NKp46.

[0280] Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID NO:49-52; and / or, the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID NO:53-56.

[0281] More preferably, the nucleotide sequence encoding the nucleic acid of the heavy chain variable region is shown in SEQ ID NO:49-52; and the nucleotide sequence encoding the nucleic acid of the light chain variable region is shown in SEQ ID NO:53-56.

[0282] The method for preparing the nucleic acid is a conventional method in the art, and preferably includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned protein by artificial full-sequence synthesis.

[0283] Those skilled in the art will understand that the base sequence encoding the amino acid sequence of the aforementioned protein can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homologue. The polynucleotide homologue of this invention can be prepared by substituting, deleting, or adding one or more bases of the gene encoding the protein sequence, while maintaining antibody activity.

[0284] carrier

[0285] The present invention also provides a recombinant expression vector containing the nucleic acid.

[0286] The recombinant expression vector described herein can be obtained by conventional methods in the art, namely, by linking the nucleic acid molecule described in this invention to various expression vectors. The expression vector can be any conventional vector in the art, as long as it can accommodate the aforementioned nucleic acid molecule. Preferably, the vector includes various plasmids, granules, bacteriophages, or viral vectors, etc.

[0287] The present invention also provides a recombinant expression transformant comprising the above-described recombinant expression vector.

[0288] The recombinant expression transformant is prepared using conventional methods in the art, preferably by transforming the recombinant expression vector into host cells. The host cells can be any common host cells in the art, as long as they allow the recombinant expression vector to replicate stably and effectively express the carried nucleic acid. Preferably, the host cells are E. coli TG1 or E. coli BL21 cells (expressing single-chain antibodies or Fab antibodies), or HEK293 or CHO cells (expressing full-length IgG antibodies). Transforming the aforementioned recombinant expression plasmid into host cells yields the preferred recombinant expression transformant of this invention. The transformation method is a conventional method in the art, preferably chemical transformation, heat shock, or electroporation.

[0289] Antibody preparation

[0290] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0291] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0292] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0293] Currently, the DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0294] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0295] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0296] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0297] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0298] The antibodies of this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0299] Antibody-drug conjugates (ADCs)

[0300] The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[0301] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0302] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0303] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0304] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0305] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0306] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0307] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids.

[0308] In this invention, the drug-linker can be used to form an ADC in a simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form an ADC.

[0309] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0310] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0311] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0312] In some implementations, the antibody-drug conjugate (ADC) has the following molecular formula:

[0313]

[0314] in:

[0315] Ab is an antibody.

[0316] LU stands for connector;

[0317] D is a drug;

[0318] Furthermore, the subscript p is a value selected from 1 to 8.

[0319] application

[0320] The present invention also provides the use of the antibodies, antibody-drug conjugates (ADCs), recombinant proteins, and / or immune cells of the present invention, for example, for the preparation of diagnostic agents or pharmaceutical preparations.

[0321] Preferably, the drug is a drug for the prevention and / or treatment of diseases associated with abnormal NKp46 expression or function.

[0322] In this invention, the diseases associated with abnormal NKp46 expression or function are those conventionally associated with abnormal NKp46 expression or function in the art. Preferably, the diseases associated with abnormal NKp46 expression or function are cancer, autoimmune diseases, and inflammatory diseases.

[0323] In this invention, the cancer is a conventional cancer in the art, preferably a cancer that overexpresses NKp46.

[0324] In this invention, the autoimmune disease is a conventional autoimmune disease in the art, preferably hemolytic anemia, pernicious anemia, polyarteritis nodosa, systemic lupus erythematosus, Alzheimer's disease, diabetes, etc.

[0325] The uses of antibodies, ADCs, recombinant proteins, and / or immune cells of this invention include (but are not limited to):

[0326] (i) Diagnosis, prevention, and / or treatment of tumor occurrence, growth, and / or metastasis, especially tumors with high NKp46 expression. The tumors include (but are not limited to): cancers with NKp46 overexpression, etc.

[0327] (ii) Diagnosing, preventing and / or treating autoimmune diseases, including (but not limited to): preferably hemolytic anemia, pernicious anemia, polyarteritis nodosa, systemic lupus erythematosus, Alzheimer's disease, diabetes, etc.

[0328] (iii) Diagnosing, preventing and / or treating inflammatory diseases, including (but not limited to): rheumatoid arthritis, glomerulonephritis, myasthenia gravis, multiple sclerosis, alopecia areata, etc.

[0329] Detection uses and kits

[0330] The antibody or its ADC of the present invention can be used in detection applications, such as for testing samples, thereby providing diagnostic information.

[0331] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include, for example, resected tumor samples, tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.

[0332] The samples used in this invention include fixed or preserved cell or tissue samples.

[0333] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0334] Pharmaceutical Composition

[0335] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein or its ADC or corresponding immune cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0336] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0337] The antibody described in this invention can also be expressed in cells by a nucleotide sequence for cell therapy, such as chimeric antigen receptor T-cell immunotherapy (CAR-T) or NK-cell immunotherapy (CAR-NK).

[0338] The pharmaceutical composition described in this invention is a pharmaceutical composition for the prevention and / or treatment of diseases associated with abnormal NKp46 expression or function.

[0339] The pharmaceutical composition of the present invention can be directly used to bind to NKp46 protein molecules, and therefore can be used for the prevention and treatment of diseases such as tumors.

[0340] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0341] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and 0.01-99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0342] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0343] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0344] This invention provides the use of the above-described pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of diseases associated with abnormal NKp46 expression or function. Preferably, the diseases associated with abnormal NKp46 expression or function are cancer, autoimmune diseases, and inflammatory diseases.

[0345] Methods and compositions for detecting NKp46 protein in samples

[0346] The present invention also provides a method for detecting NKp46 protein in a sample (e.g., detecting NKp46 overexpressing cells), comprising the following steps: contacting the antibody with the sample to be tested in vitro, and detecting whether the antibody and the sample to be tested bind to form an antigen-antibody complex.

[0347] The term "overexpression" is conventional in the art and refers to the overexpression of NKp46 protein in RNA or protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing, and changes in protein degradation), as well as local overexpression and increased functional activity due to changes in protein transport patterns (increased nuclear localization) (e.g., in the case of increased enzymatic hydrolysis of the substrate).

[0348] In this invention, the detection method for whether or not an antigen-antibody complex is formed is a conventional detection method in the art, and preferably a flow cytometry (FACS) assay.

[0349] This invention provides a composition for detecting NKp46 protein in a sample, comprising the above-described antibody, recombinant protein, antibody-drug conjugate, immune cell, or a combination thereof as an active ingredient. Preferably, it further comprises a compound composed of functional fragments of the above-described antibody as an active ingredient.

[0350] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0351] The main advantages of this invention are:

[0352] (1) The NKp46 antibody described in this invention has a high affinity for NKp46 protein (affinity constant KD reaches 1.27 × 10⁻⁶). -10 );

[0353] (2) The NKp46 antibody of the present invention can bind to the extracellular region of the NKp46 protein receptor;

[0354] (3) The NKp46 antibody described in this invention has high specificity;

[0355] (4) The killing experiments mediated by primary isolated NK cells and NK92 cell lines demonstrated that the antibody of the present invention can significantly increase the lysis and killing activity of NK cells from the two cell sources against tumor cells.

[0356] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0357] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available. The room temperatures described in the examples are conventional room temperatures in the art, typically 10-30°C.

[0358] Example 1: Preparation of NKp46 antibody using hybridoma technology

[0359] (I) Preparation of Immunogen A

[0360] The nucleotide sequence containing the full-length amino acid sequence encoding the human NKp46 protein (as shown in SEQ ID NO: 57 in the sequence listing) was cloned into the pLVX vector (purchased from Ingenie Biotech, V044-50), and plasmids were prepared according to established standard molecular biology methods. For specific methods, please refer to Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). CHOK1 cells were infected with the virus and selectively cultured for 2 weeks in F12K medium containing 0.8 μg / mL puromycin and 10% (w / w) fetal bovine serum. Subcloning was performed in 96-well plates using limiting dilution and incubated at 37°C with 5% (v / v) CO2. After approximately 2 weeks, a subset of single clones were amplified into 6-well plates. The amplified clones were screened using flow cytometry with an NKp46 antibody (previously constructed by the applicant). Cell lines with good growth, high fluorescence intensity, and single clones were selected for further expansion and cryopreservation in liquid nitrogen to obtain immunogen A. The specific selection results are shown in Table 5. In Table 5, the percentage of positive cells (%) refers to the percentage of positive cells out of the total number of cells. This resulted in a series of CHOK1 cell lines expressing NKp46 positive expression (Table 5).

[0361] Table 5. Screening of CHOK1-hNKp46 stable cell lines

[0362]

[0363]

[0364] (II) Preparation of Immunogen B

[0365] The full-length amino acid sequence cDNA of NKp46 (as shown in SEQ ID NO:58 in the sequence listing) was cloned into the pCpc vector (prepared by Ruizhi Chemicals) and coated onto a 1.0 μm gold colloidal bullet. Immunization was then performed using the Helios Gene Gun System (Bio-rad, catalog number 165-2431). For details on coating the 1.0 μm gold colloidal bullet and the immunization method, please refer to the Helios Gene Gun instruction manual.

[0366] (III) Preparation of hybridoma cells and antibody screening

[0367] A. Immunogen A (CHOK1-hNKp46) was used to immunize 6-8 week old SJL mice raised under SPF conditions. Immunogen D was cultured in T-75 cell culture flasks to 90% confluence. The culture medium was aspirated, and the cells were washed twice with F12K basal medium (Invitrogen). Then, they were treated with enzyme-free cell dissociation buffer (Invitrogen) at 37°C until the cells could be detached from the culture dish wall. The cells were collected, washed twice with F12K basal medium, and after cell counting, the cells were diluted to 2×10⁻⁶ with phosphate-buffered saline (pH 7.2). 7 Cells per milliliter. Each mouse received an intraperitoneal injection of 0.5 ml of cell suspension for each immunization. The interval between the first and second immunizations was 2 weeks, and subsequent immunizations were spaced 3 weeks apart. Except for the first immunization, blood was collected one week after each immunization, and serum antibody titers and specificities were detected using ELISA and FACS.

[0368] The results are as follows Figure 1 As shown, mouse serum bound to the immunogen to varying degrees, exhibiting an antigen-antibody reaction, with the highest dilution around 10,000. FACS results showed that mouse serum could also specifically bind to human NKp46 on the cell surface, with the highest dilution at 1,000-fold.

[0369] B. Immunogen C (hNKp46-ECD-Fc, purchased from Acro Biosystems) was used for immunization with 6-8 week old Balb / c and SJL mice, which were housed under SPF conditions. The mice were immunized using the HOCK immunization method. The method is shown in Table 6.

[0370] Table 6. HOCK Immunization Procedure

[0371]

[0372]

[0373] The antibody titer and specificity in serum were detected using ELISA and FACS, and the results are as follows: Figure 2 As shown, the serum of mice immunized with human NKp46ECD-Fc showed varying degrees of binding to the immunogen, exhibiting an antigen-antibody reaction. The highest dilution was around 100,000. FACS results showed that the mouse serum could also specifically bind to human NKp46 on the cell surface, with the highest dilution being 1,000-fold.

[0374] C. Immunogen B (pCP-hNKp46) immunization was performed using 6-8 week old Balb / c or SJL mice housed under SPF conditions. All mice were immunized four times via abdominal injection using a Helios gene gun, four injections per dose, with 1.0 microgram of cDNA per injection. The interval between the initial immunization and the first booster immunization was 2 weeks, and subsequent booster immunizations were spaced 3 weeks apart. Blood samples were collected one week after each booster immunization, and serum antibody titers were detected using ELISA or FACS.

[0375] The results are as follows Figure 3 As shown, mouse serum bound to the immunogen to varying degrees, exhibiting an antigen-antibody reaction. The serum antibody titer detected by FACS reached over 1:1000, and the ELISA titer was over 1:100,000.

[0376] Before completing the AC step, each selected mouse was given a final intraperitoneal injection of 100 μg of purified hNKp46-ECD-Fc. The mice were sacrificed 3-4 days later, and spleen cells were collected. NH4OH was added to a final concentration of 1% (w / w) to lyse the erythrocytes in the spleen cells, obtaining a spleen cell suspension. The cells were washed three times by centrifugation at 1000 rpm in DMEM basal medium, and then mixed with mouse myeloma cells SP2 / 0 (purchased from ATCC) at a live cell ratio of 5:1. Cell fusion was performed using a high-efficiency electrofusion method (see METHODS IN ENZYMOLOGY, VOL. 220). The fused cells were diluted in DMEM medium containing 20% ​​fetal bovine serum and 1×HAT (percentages by weight). Then, the cells were diluted 1×10⁻⁶ times. 5 Add 200 μL / well to each well of a 96-well cell culture plate and incubate at 37°C with 5% CO2. The percentages are volume percentages. After 14 days, use ELISA and Acumen (microplate cell assay) to screen for cell confluence plate supernatants. The OD values ​​from the ELISA are then recorded. 450nm Positive clones with a percentage of >1.0 and >15 in Acumen were amplified into 24-well plates and cultured in DMEM (invitrogen) containing 10% (w / w) fetal bovine serum (HT) at 37°C and 5% (v / v) CO2. After 3 days of culture, the culture medium from the 24-well plates was centrifuged, and the supernatant was collected. Antibody subtype analysis was performed on the supernatant, and the binding activity against NKp46-positive cells was determined using FACS.

[0377] Based on the 24-well plate screening results, hybridoma clones that showed positive binding to CHOK1 cells expressing human NKp46 and monkey NKp46 in the FACS experiment, and negative binding to control CHOK1 cells, were selected as candidate clones.

[0378] Hybridoma cells meeting the criteria were selected and subcloned in 96-well plates using limiting dilution. The cells were cultured in DMEM medium (Invitrogen) containing 10% (w / w) FBS at 37°C and 5% (v / v) CO2. Ten days after subcloning, preliminary screening was performed using ELISA and Acumen. Single positive clones were selected and amplified into 24-well plates for further culture. Antigen binding positivity was confirmed by FACS three days later.

[0379] Based on the test results of the 24-well plate samples, the optimal clone was selected and cultured in DMEM medium containing 10% (w / w) FBS (purchased from Invitrogen) at 37°C and 5% (v / v) CO2. The hybridoma cells of this invention were obtained by liquid nitrogen cryopreservation and can be used for subsequent antibody production and purification.

[0380] Example 2: Determination of amino acid sequences in the variable regions of the light and heavy chains

[0381] Total RNA isolation: After testing the supernatants obtained from different subclone cultures in Example 1 for antigen binding (i.e., after verification using the FACS detection method described in Example 4), total RNA was collected by centrifugation at a rate of 1×10⁻⁶. 6 Hybridoma cells were cryopreserved. Sequencing was performed by Baiying Biotechnology, and the amino acid sequence numbers of the heavy chain variable region, light chain variable region, and CDR region of some clones are shown in Table 7.

[0382] Table 7. Amino acid sequence numbers of positive clones

[0383]

[0384] Example 3: Production and purification of chimeric antibodies

[0385] The selected sequences were sent to Bio-Pharmaceutical for the production of chimeric antibodies. The purified NKp46 antibody was analyzed for protein concentration (A280 / 1.4) and purity, and the results are shown in Table 8 below.

[0386] Table 8. Properties of purified antibodies

[0387]

[0388]

[0389] Example 4: Identification of chimeric antibodies

[0390] A. Flow cytometry (FACS) assay for antibody binding to NKp46-expressing cells

[0391] The nucleotide sequence encoding the full-length amino acid sequence of monkey NKp46 was cloned into the pLVX vector (purchased from Clontech) and a plasmid was prepared. The pLVX plasmid was used to infect the CHOK1 cell line to obtain a stable CHOK1 cell line containing monkey NKp46 (here referred to as the CHOK1-cynoNKp46 stable cell line). The CHOK1-mouseNKp46 stable cell line was prepared using the same method. The CHOK1-hNKp46, CHOK1-cynoNKp46, and CHOK1-mouseNKp46 stable cell lines were cultured in T-75 cell culture flasks until 90% confluence. The culture medium was aspirated, and the cells were washed twice with PBS buffer (purchased from Invitrogen). Then, the cells were treated with enzyme-free cell dissociation solution (Versene solution, purchased from Life Technologies) and collected. After washing and centrifuging the cells twice with PBS buffer, cell counting was performed, and the cells were resuspended in FACS buffer (PBS + 2% FBS, the percentages are by mass) to a concentration of 2 × 10⁶ cells / mL. 6 Cells / mL were added at a rate of 100 μL per well to a 96-well FACS plate. The purified NKp46 antibody sample obtained in Example 3 was added at 100 μL per well, and the plate was incubated on ice for 1 hour. Cells were washed twice by centrifugation with FACS buffer, and 100 μL of fluorescent (Alexa 488) labeled secondary antibody (Invitrogen) was added to each well. The plate was incubated on ice for 1 hour. Cells were washed three times by centrifugation with FACS buffer, and then resuspended in 100 μL of PBS buffer. Results were detected and analyzed using FACS (FACS Canto II, BD Biosciences).

[0392] The results are shown in Figure 4. The selected antibody can specifically bind to human NKp46 on the cell surface. Figure 4a ), macaque NKp46 ( Figure 4b ), without binding to parental CHOKI cells ( Figure 4c The IgG control is human IgG, and the data in the figure are the average fluorescence intensity values ​​of the cell population measured by MFI.

[0393] B. Reporter gene experiment

[0394] F(ab')2 human IgG antibody was prepared to a final concentration of 2.2 μg / ml using PBS and added to 100 μl per well of a 96-well plate (corning, 3903). The plate was then incubated overnight at 4°C. The next day, the F(ab')2 human IgG antibody in the 96-well plate was washed twice with PBS, and then diluted with culture medium. Starting from 3.33 nM (2x preparation required), the antibody was diluted eight times in three-fold increments. 50 μl of culture medium containing diluted antibody was added to each well. Finally, 50 μl of a total of 5e4 Jurkat-NFAT-hNKp46 / CD3z-2G3 cells was added to each well. The plate was then incubated at 37°C for 5 h. 100 μl of luciferase detection buffer was added to each well, and the cells were detected using the VARIOSKAN LUX instrument.

[0395] The results are as follows Figure 5 As shown, the selected antibody can specifically recognize hNKp46 on the cell surface, activate the downstream NFAT signaling pathway, and thus lead to an increase in luciferase expression level. The control antibodies hIgG1 and hIgG4 could not activate the downstream NFAT signaling pathway.

[0396] C. Flow cytometry (FACS) assay for the expression of CD107a and CD69 in NK cells.

[0397] Freshly obtained whole blood was diluted 1:1 with PBS to obtain diluted whole blood. Using a sterile pipette, the diluted whole blood was gently spread evenly on the surface of Ficoll (purchased from GE Healthcare). The volume ratio of Ficoll to diluted whole blood was 3:4. Avoid shaking to mix. Centrifuge at 400g at room temperature (20°C) for 30 minutes. The centrifuge tube separated into three layers: the upper layer was plasma, and the middle milky white layer was mononuclear lymphocytes. The middle layer of cells was gently aspirated with a sterile pipette and collected into a new centrifuge tube. The medium was diluted three times with PBS phosphate buffer and centrifuged at 100g at room temperature for 10 minutes, discarding the supernatant. The lymphocytes were resuspended in PBS phosphate buffer to 10 mL. Platelets were removed by repeating the previous steps. Finally, the lymphocytes were resuspended in 10 mL of multi-component RPMI 1640 medium (purchased from Invitrogen) containing 10% fetal bovine serum for later use. This is the peripheral blood mononuclear lymphocyte (PBMC) sample. The percentages mentioned are mass percentages. The primary NK cell was subsequently isolated from the PBMC (purchased from Stemcell).

[0398] Primary NK cells were isolated from PBMCs and added to 96-well plates containing purified NKp46 antibody. The cells were cultured in vitro for 5 days using the following culture medium formulation: RPMI 1640, 10% FBS. Cells were washed twice with PBS buffer, and the collected cells were resuspended in FACS buffer (PBS + 1% BSA, percentages are by mass) to a concentration of 1 × 10⁻⁶. 6 Cells / mL were added at a rate of 100 μL per well to a 96-well FACS plate, along with APC-antiCD107a and PE-antiCD69 (purchased from Biolegend), and incubated at 4°C for 1 hour. Cells were washed three times by centrifugation with FACS buffer, then resuspended in 100 μL of FACS buffer. Results were detected and analyzed using FACS (FACSCantoII, purchased from BD).

[0399] The results are as follows Figure 6a and Figure 6b As shown, compared with control IgG, the selected antibody increased the proportion of CD107a and CD69 positive NK cells.

[0400] D. Detection of changes in IFNγ levels using enzyme-linked immunosorbent assay (ELISA).

[0401] Primary NK cells were isolated from PBMCs and added to a 96-well plate containing immobilized purified NKp46 antibody obtained in Example 3. The plates were cultured in vitro for 4-5 days using the following culture medium: RPMI 1640, 5% FBS, 10 ng / ml IL-2, and 20 ng / ml IL-12. The supernatant was collected, and the cytokine IFNγ release level was detected using an ELISA (from R&D).

[0402] The results are as follows Figure 7 As shown, IFNγ levels increase over time, and NKp46 antibody can significantly increase the level of IFNγ released by NK cells.

[0403] Example 5: Determination of NKp46 antibody affinity constant

[0404] Affinity constants were determined using an Octet red96 instrument (purchased from Fortiebio). Specific procedures and methods followed the instrument manual and the manufacturer's detailed instructions. Specifically: the Anti-hIgG Fc sensor (purchased from Fortiebio) captured 5 μg / mL of the target antibody, then the hNKp46-his protein was serially diluted two-fold to an initial concentration of 100 nM and incubated at 30°C for 5 minutes. The binding and dissociation of NKp46-his protein with the antibody were detected by measuring changes in the interference wavelength using the Octet instrument. The dissociation constant and binding constant were obtained by fitting using User Software, and the affinity constant is the ratio of the dissociation constant to the binding constant. The results are shown in Table 9:

[0405] Table 9. Affinity constants of NKp46 antibody to NKp46-his

[0406] Ab ID KD(M) kon(1 / Ms) kdis(1 / s) Full R^2 32H2F2 1.27E-10 3.04E+05 3.87E-05 0.9917 21B2CB 5.19E-10 2.44E+05 1.27E-04 0.9816 29C9B5 5.45E-10 2.50E+05 1.37E-04 0.9921 24H4C4 5.24E-09 2.71E+05 1.42E-03 0.9838 tab1 5.50E-09 1.17E+05 6.46E-04 0.983 tab4 3.38E-09 1.72E+05 5.81E-04 0.9872

[0407] Example 6: ELISA detection of the binding of NKp46 antibody to NKp46, NKp44 and NKp30

[0408] His-tagged NKp46, NKp44, and NKp30 proteins (purchased from AcroBiosystems and Sinocare, respectively) were diluted to 1 μg / ml with PBS and added to 100 μl / well of ELISA microplates. The plates were incubated overnight at 4°C. After blocking with ELISA blocking buffer (PBS phosphate buffer containing 1% BSA, pH 7.4, percentages are by weight) at 37°C for two hours, the test lead antibody obtained in Example 3, diluted to 10 μg / ml with PBS, was added and incubated at 37°C for 1 hour. Goat anti-human IgG Fc horseradish peroxidase secondary antibody (purchased from Sigma, product number A0170) was added and incubated at room temperature for 30 minutes. Then, 100 μl / well of TMB chromogenic buffer was added and incubated at room temperature for 15 minutes. Finally, 50 μl of 1N hydrochloric acid was added to terminate the chromogenic reaction, and the OD was read using an ELISA reader. 450nm reading.

[0409] The results are as follows Figure 8 As shown, the NKp46 antibody selectively binds to the NKp46 protein but does not bind to the NKp44 or NKp30 proteins.

[0410] Example 7: Humanization of NKp46 Antibody

[0411] The bioactivity characteristics of the NKp46 antibody sequences in Examples 3-6 were analyzed, and the 21B2CB and 24H4C4 sequences were selected for humanization. A common method for antibody humanization is complementarity-determining region (CDR) transplantation. The CDR region is involved in the antigen-antibody binding interaction. The CDR region of the non-human antibody is transplanted into the human framework. During transplantation, back mutation sites are usually designed based on changes in affinity, etc., to preserve the best affinity and function while maximizing humanization.

[0412] Preliminary analysis was performed on the antibody variable region (VH or VL), framework region, and complementarity-determining region (CDR). Based on database information, the corresponding sequences of CDRs, HV loops, and FRs for 21B2CB and 24H4C4 were further confirmed. A structural model of the murine antibody was obtained through modeling, and residues within the framework region were identified. Human VH and VL transplantation recipients were selected, and the CDR regions of the murine antibody were directly transplanted into the human framework region recipients, labeled as VH1 and VL1. The transplanted VH1 / VL1 and murine antibody VH / VL sequences were compared, and the amino acid residue distribution at key sites, i.e., reversion mutation sites, was analyzed. Based on the degree of impact of the reversion mutation on the transplanted antibody structure, one or more reversion mutations were performed on the transplanted sequence to obtain humanized VHs and VLs, namely VH1 / 2 / 3 / 4 and VL1 / 2 / 3 / 4.

[0413] DNA sequences of all VHs and VLs associated with the 21B2CB and 24H4C4 sequences were synthesized, namely the four VH sequences and four VL sequences of 21B2CB and the four VH sequences and four VL sequences of 24H4C4. These were combined to form 16 different VH-VL antibody sequences, and the pcDNA3.4 expression plasmid was constructed. After verification by EcoRI and HindIII restriction enzyme digestion, the plasmid was transfected into HEK293 cells to express 16 antibodies. The antibody expression supernatant was collected, and the affinity of the antibodies for the His-tagged NKp46 antigen was detected by Biacore 8K. The results are shown in Tables 10 and 11. Based on the affinity ranking results and the number of reversion mutations, three antibodies were selected for expression and purification.

[0414] Table 10 Affinity constants of 21B2CB-related antibody expression supernatant with His-hNKp46

[0415]

[0416]

[0417] Table 11 Affinity constants of 21B2CB-related antibody expression supernatant with His-hNKp46

[0418]

[0419]

[0420] The purified lead antibody was further tested using a Biacore 8K assay to detect the affinity of the humanized antibody for his-hNKp46. The experimental results are shown in Table 12. The humanized antibodies all retained good affinity for his-hNKp46. Based on the results of the detection of the number of reversed mutations and the affinity constant, the combination of 21B2C8-VH3+VL2 and 24H4C4-VH1+VL3 was selected as the highest priority sequence for further development.

[0421] Table 12 Affinity constants of humanized antibodies to His-hNKp46

[0422]

[0423]

[0424] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Shanghai Huaiyue Biotechnology Co., Ltd. <120> NKp46 antibody, its preparation method and application <130> P2021-1205 <160> 58 <170> SIPOSequenceListing 1.0 <210> 1 <211> 122 <212> PRT <213> Mouse (Mus musculus) <400> 1 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Met Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Asn Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Arg Gly His Gly Asn Tyr Asn Thr Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 2 <211> 121 <212> PRT <213> Mouse (Mus musculus) <400> 2 Gln Val Gln Leu Gln Gln Pro Gly Thr Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Tyr Gly Ser Gly Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Thr Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 3 <211> 110 <212> PRT <213> Mouse (Mus musculus) <400> 3 Gln Val Gln Val Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Trp Met His Trp Val 20 25 30 Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile His Pro 35 40 45 Asn Ser Gly Thr Thr Asn Tyr Asn Glu Lys Phe Lys Ser Lys Ala Thr 50 55 60 Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser 65 70 75 80 Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Gly Arg 85 90 95 Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 100 105 110 <210> 4 <211> 115 <212> PRT <213> Mouse (Mus musculus) <400> 4 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro His Thr Asp Ile Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Gln Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ala Gly Arg Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 <210> 5 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 5 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr 65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 6 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 6 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr 65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 7 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 1>Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Phe 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 8 <211> 107 <212> PRT <213> mice (Mus musculus) <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Leu Gln Glu Glu Pro Asn Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Asn Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 5 <212> PRT <213> Mouse (Mus musculus) <400> 9 Asn Tyr Trp Met Tyr 1 5 <210> 10 <211> 5 <212> PRT <213> Mouse (Mus musculus) <400> 10 Ser Tyr Trp Met His 1 5 <210> 11 <211> 3 <212> PRT <213> Mouse (Mus musculus) <400> 11 Trp Met His 1 <210> 12 <211> 5 <212> PRT <213> Mouse (Mus musculus) <400> 12 Ser Tyr Trp Met His 1 5 <210> 13 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 13 Met Ile His Pro Asn Ser Gly Ser Asn Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser <210> 14 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 14 Met Ile His Pro Asn Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser <210> 15 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 15 Met Ile His Pro Asn Ser Gly Thr Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser <210> 16 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 16 Met Ile His Pro His Thr Asp Ile Thr Lys Tyr Asn Glu Lys Phe Gln 1 5 10 15 Asn <210> 17 <211> 13 <212> PRT <213> Mouse (Mus musculus) <400> 17 Glu Gly Arg Gly His Gly Asn Tyr Asn Thr Met Asp Tyr 1 5 10 <210> 18 <211> 12 <212> PRT <213> Mouse (Mus musculus) <400> 18 Gln Gly Tyr Gly Ser Gly Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 19 <211> 6 <212> PRT <213> Mouse (Mus musculus) <400> 19 Glu Gly Arg Phe Asp Tyr 1 5 <210> 20 <211> 6 <212> PRT <213> Mouse (Mus musculus) <400> 20 Ala Gly Arg Phe Asp Tyr 1 5 <210> twenty one <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> twenty one Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser 1 5 10 <210> twenty two <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> twenty two Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Asn 1 5 10 <210> twenty three <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> twenty three Arg Ala Ser Gln Asp Ile Gly Ser Ser Leu Asn 1 5 10 <210> twenty four <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> twenty four Arg Ala Ser Gln Asp Ile Gly Ser Ser Leu Asn 1 5 10 <210> 25 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 25 Arg Ala Asn Arg Leu Val Asp 1 5 <210> 26 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 26 Arg Ala Asn Arg Leu Val Asp 1 5 <210> 27 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 27 Ala Thr Ser Ser Leu Asp Ser 1 5 <210> 28 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 28 Ala Thr Ser Asn Leu Asp Ser 1 5 <210> 29 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 29 Leu Gln Tyr Asp Glu Phe Pro Tyr Thr 1 5 <210> 30 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 30 Leu Gln Tyr Asp Glu Phe Pro Tyr Thr 1 5 <210> 31 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 31 Leu Gln Tyr Ala Ser Ser Pro Phe Thr 1 5 <210> 32 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 32 Leu Gln Tyr Ala Ser Ser Pro Phe Thr 1 5 <210> 33 <211> 122 <212> PRT <213> Artificial Sequence <400> 33 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Asn Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Arg Gly His Gly Asn Tyr Asn Thr Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 34 <211> 122 <212> PRT <213> Artificial Sequence <400> 34 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Asn Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Arg Gly His Gly Asn Tyr Asn Thr Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 35 <211> 122 <212> PRT <213> Artificial Sequence <400> 35 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Asn Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Arg Gly His Gly Asn Tyr Asn Thr Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 36 <211> 122 <212> PRT <213> Artificial Sequence <400> 36 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Met Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Asn Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Arg Gly His Gly Asn Tyr Asn Thr Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 37 <211> 121 <212> PRT <213> Artificial Sequence <400> 37 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Tyr Gly Ser Gly Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 121 <212> PRT <213> Artificial Sequence <400> 38 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Tyr Gly Ser Gly Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 39 <211> 121 <212> PRT <213> Artificial Sequence <400> 39 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Tyr Gly Ser Gly Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 40 <211> 121 <212> PRT <213> Artificial Sequence <400> 40 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Tyr Gly Ser Gly Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 41 <211> 107 <212> PRT <213> Artificial Sequence <400> 41 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 42 <211> 107 <212> PRT <213> Artificial Sequence <400> 42 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 43 <211> 107 <212> PRT <213> Artificial Sequence <400> 43 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 44 <211> 107 <212> PRT <213> Artificial Sequence <400> 44 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr 65 70 75 80 Glu Asp Met Ala Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 45 <211> 107 <212> PRT <213> Artificial Sequence <400> 45 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 46 <211> 107 <212> PRT <213> Artificial Sequence <400> 46 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 47 <211> 107 <212> PRT <213> Artificial Sequence <400> 47 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 48 <211> 107 <212> PRT <213> Artificial Sequence <400> 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Tyr Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Tyr 65 70 75 80 Glu Asp Ile Ala Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 49 <211> 366 <212> DNA <213> Artificial Sequence <400> 49 caggtgcagc tgcagcagcc aggcgccgag gtggtgaaac ccggcgcttc cgtgatgctg 60 agctgcaaag ccagcggcta caccttcacc aactactgga tgtactgggt gaaacagagg 120 cctggacagg gcctggagtg gattggaatg atccacccca actccggcag caacaaatac 180 aacgaaaaat tcaaaagcaa ggccaccctg accgtggaca agagcagcag cacagcctac 240 atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcac cagagaaggc 300 agaggacatg gcaactacaa caccatggac tattggggcc agggaaccag cgtgaccgtg 360 agcagc 366 <210> 50 <211> 363 <212> DNA <213> Artificial Sequence <400> 50 caggtgcagc tgcagcagcc aggcacagag ctggtgaagc ccggcgccag cgtgaaactg 60 agctgcaaag ccagcggcta caccttcaca agctactgga tgcactgggt gaagcagagg 120 cctggccagg gactggagtg gatcgggatg atccacccca acagcggaag caccaaatac 180 aatgagaagt tcaaaagcaa agccaccctg accgtggaca aaagcagcag cacagcctac 240 atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc cagacagggc 300 tacggaagcg gatactggta tttcgacgtg tggggaacag ggacaacagt gacagtgagc 360 agc 363 <210> 51 <211> 330 <212> DNA <213> Artificial Sequence <400> 51 caggtgcagg tgcagcagag cggcgccgag ctggtgaagc ccggagctag tgtgaagctg 60 agctgcaaag ccagcggcta ctggatgcac tgggtgaagc agaggcctgg ccagggactg 120 gagtggatcg gcatgatcca tcccaactcc gggaccacca actataacga gaaattcaag 180 agcaaagcta cactgaccgt ggacaaaagc agcagcacag cctacatgca gctgagcagc 240 ctgaccagcg aggacagcgc cgtgtactac tgcgccagag agggcagatt cgactactgg 300 ggacagggca ccaccctgac cgtgagcagc 330 <210> 52 <211> 345 <212> DNA <213> Artificial Sequence <400> 52 caggtgcagc tgcagcagcc aggcgccgag ctggtgaagc ccggagcttc tgtgaagctg 60 agctgcaaag ccagcggcta cacattcaca tcctactgga tgcactgggt gaagcagagg 120 cctggacagg gcctggagtg gattgggatg atccaccccc acaccgacat caccaagtac 180 aacgaaaaat tccagaacaa agccaccctg accgtggaca agagcagcag cacagcctac 240 atgcagctgt ccagcctgac cagcgaagat tctgctgtgt actactgcgc cggagccggc 300 agattcgact actggggaca gggcaccacc ctgaccgtga gcagc 345 <210> 53 <211> 321 <212> DNA <213> Artificial Sequence <400> 53 gacattaaga tgacccagag ccccagcagc atgtacgcca gcctgggaga gagagtgacc 60 atcacctgca aggccagcca ggacatcaac agctatctga gctggtttca gcagaagccc 120 ggcaagagcc ccaaaaccct gatctaccgc gccaacagac tggtggacgg cgtgcccagc 180 agattcagcg gcagcggcag cggacaggac tactccctga ctatcagcag cctggaatac 240 gaagacatgg gaatctacta ctgcctgcag tacgacgagt tcccatacac cttcggaggc 300 gggacaaagc tggagatcaa a 321 <210> 54 <211> 321 <212> DNA <213> Artificial Sequence <400> 54 gacattaaga tgacccagag ccccagcagc atgtacgcca gcctgggaga gagagtgacc 60 atcacctgca aggccagcca ggacatcaac agctatctga actggtttca gcagaagccc 120 ggcaagagcc ccaaaaccct gatctaccgc gccaacagac tggtggacgg agtgcccagc 180 agttcagcg gcagcggcag cggacaggac tactccctga ctatcagcag cctggaatat 300. gaggacatgg gcatctatta ctgcctgcag tacgacgaat ttccatatac cttcggcggc ggcaccaagc tggagatcaa a <210> 55 <211> 321 <212> DNA <213> Artificial Sequence <400> 55 60. gacattcaga tgacccagag ccccagcagc ctgagcgcca gcctgggaga gagagtgagc ctgacctgca gagccagcca ggacatcggc agcagcctga actggctgca gcaggaacct 180. gacggcacca tcaaaagact gatctacgcc accagcagcc tggacagcgg agtgccaaaa agttcagcg gaagcagaag cggcagcgac tacagcctga ccatcagcag cctggaagc gaggacttcg tggactacta ctgcctgcag tacgcctcta gccccttcac cttcggcgcc 300 ggcaccaaac tggagctgaa a <210> 56 <211> 345 <212> DNA <213> Artificial Sequence <400> 56 caggtgcagc tgcagcagcc aggcgccgag ctggtgaagc ccggagcttc tgtgaagctg agctgcaaag ccagcggcta cacattcaca tcctactgga tgcactggt gaagcagagg 120 cctggacagg gcctggagtg gattgggatg atccaccccc acaccgacat caccaagtac 180 aacgaaaaat tccagaaca agccaccctg accgtggaca agagcagcag cacagcctac 240 atgcagctgt ccagcctgac cagcgaagat tctgctgtgt actactgcgc cggagccggc 300 agatcgact actggggaca gggcaccacc ctgaccgtga gcagc 345 <210> 57 <211> 304 <212> PRT <213> Homo sapiens <400> 57 Met Ser Ser Thr Leu Pro Ala Leu Leu Cys Val Gly Leu Cys Leu Ser 1 5 10 15 Gln Arg Is Only Gln Gln Thr Leu Pro Lys Pro Phe With Trp 20 25 30 Ala Glu Pro His Phe Met Val Pro Lys Glu Lys Gln Val Thr Ile Cys 35 40 45 Cys Gln Gly And Tyr Gly Only Val Glu Tyr Gly Leu His Phe Glu Gly 50 55 60 Ser Leu Phe Ala Val Asp Arg Pro Lys Pro Glu Arg Ile Asn Lys 65 70 75 80 Val Lys Phe Tyr Ile Pro Asp Met Asn Ser Arg Met Ala Gly Gln Tyr 85 90 95 Ser Cys Ile Tyr Arg Val Gly Glu Leu Trp Ser Glu Pro Ser Asn Leu 100 105 110 Leu Asp Leu Val Val Thr Glu Met Tyr Asp Thr Pro Thr Leu Ser Val 115 120 125 His Pro Gly Pro Glu Val Ile Ser Gly Glu Lys Val Thr Phe Tyr Cys 130 135 140 Arg Leu Asp Thr Ala Thr Ser Met Phe Leu Leu Leu Lys Glu Gly Arg 145 150 155 160 Ser Ser His Val Gln Arg Gly Tyr Gly Lys Val Gln Ala Glu Phe Pro 165 170 175 Leu Gly Pro Val Thr Thr Ala His Arg Gly Thr Tyr Arg Cys Phe Gly 180 185 190 Ser Tyr Asn Asn His Ala Trp Ser Phe Pro Ser Glu Pro Val Lys Leu 195 200 205 Leu Val Thr Gly Asp Ile Glu Asn Thr Ser Leu Ala Pro Glu Asp Pro 210 215 220 Thr Phe Pro Ala Asp Thr Trp Gly Thr Tyr Leu Leu Thr Thr Glu Thr 225 230 235 240 Gly Leu Gln Lys Asp His Ala Leu Trp Asp His Thr Ala Gln Asn Leu 245 250 255 Leu Arg Met Gly Leu Ala Phe Leu Val Leu Val Ala Leu Val Trp Phe 260 265 270 Leu Val Glu Asp Trp Leu Ser Arg Lys Arg Thr Arg Glu Arg Ala Ser 275 280 285 Arg Ala Ser Thr Trp Glu Gly Arg Arg Leu Asn Thr Gln Thr Leu 290,295,300 <210> 58 <211> 915 <212> DNA <213> Homo sapiens <400> 58 atgtcttcca cactccctgc cctgctctgc gtcgggctgt gtctgagtca gaggatcagc 60 gccagcagc agactccc aaaaccgttc atctggccg agccccattt catggttcca 120 aaggaaaagc aagtgaccat ctgttgccag ggaattatg gggctgttga attackagctg 180 cactttgaag gaagcctttt tgccgtggac agaccaaac cccctgagcg gattacaaa 240 gtcaattct acatcccgga catgaactcc cgcatggcag ggcaattct ctgcatctat 300 cgggttgggg agctctggtc agagcccagc aacttgctgg atctggtggt aacagaaatg 360 tatgacacac ccaccctctc ggttcatcct ggacccgaag tgatctcggg agaaaggtg 420 accttctact gccgtctaga cactgcaaca agcatgttct tactgctcaa ggaggaga 480 tccagccacg tacagcgcgg atacgggaag gtccaggcgg agttccccct gggccctgtg 540 accacagcc acagaggac ataccgatgt tttggctcct ataacaacca tgcctggtct 600 ttccccagtg agccagtgaa gctcctggtc acaggcgaca ttgagaacac cagccttgca 660 cctgaagacc ccacctttcc tgcagacact tggggcacct accttttaac cacagagacg 720 ggactccaga aagaccatgc cctctgggat cacactgccc agaatctcct tcggatgggc 780 ctggcctttc tagtcctggt ggctctagtg tggttcctgg ttgaagaactg gctcagcagg 840 aagagacta gagagcgagc cagcagagct tccacttggg aaggcaggag aaggtgaac 900 acacagactc tttga 915

Claims

1. An antibody or antigen-binding fragment that binds NKp46, characterized in that, The antibody or antigen-binding fragment has the following three complementary determinant CDRs for the heavy chain variable region and three complementary determinant CDRs for the light chain variable region: VH-CDR1 shown in SEQ ID NO: 9, VH-CDR2 shown in SEQ ID NO: 13, VH-CDR3 shown in SEQ ID NO: 17, VL-CDR1 shown in SEQ ID NO: 21, VL-CDR2 shown in SEQ ID NO: 25, and VL-CDR3 as shown in SEQ ID NO:

29.

2. An antibody that binds NKp46, characterized in that, The antibody has the following characteristics: (1) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1; and (2) The amino acid sequence is shown in the light chain variable region of SEQ ID NO:

5.

3. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the antibody or antigen-binding fragment that binds to NKp46 as described in claim 1; and (ii) Optional tag sequences to assist in expression and / or purification.

4. An antibody conjugate, characterized in that, The antibody conjugate contains: (a) An antibody portion comprising an antibody or antigen-binding fragment that binds to NKp46 as described in claim 1; and (b) A coupling portion conjugated to the antibody portion, the coupling portion being selected from the group consisting of detectable markers, radionuclides, or combinations thereof.

5. A pharmaceutical composition, characterized by, The pharmaceutical composition contains: (i) An active ingredient selected from the group consisting of: an antibody or antigen-binding fragment that binds NKp46 as described in claim 1, or an antibody as described in claim 2, an antibody conjugate as described in claim 4, or a combination thereof; as well as (ii) Pharmaceutically acceptable carriers.

6. A method for the non-diagnostic in vitro detection of NKp46 in a sample, characterized in that, The method includes the following steps: (1) In vitro, the sample is contacted with the antibody as described in claim 2; (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of NKp46 in the sample.

7. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-6. The polynucleotides encode polypeptides selected from the following group: (1) The antibody or antigen-binding fragment that binds to NKp46 as described in claim 1; or (2) The recombinant protein as described in claim 3.

8. A vector, characterized in that, The carrier contains the polynucleotide as described in claim 7.

9. A genetically engineered host cell, characterized in that, The host cell contains the vector as described in claim 8 or the genome is integrated with the polynucleotide as described in claim 7.

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