Nkp80 antibodies and uses thereof

Abstract

The present application relates to the field of biotechnology, in particular, the present application relates to NKp80 antibody and its application. The present application screens a mouse anti-NKp80 monoclonal antibody, the monoclonal antibody has higher binding activity with human or cynomolgus monkey NKP80 protein extracellular segment, can target binding high expression NKp80 cell, also can combine human primary T cell and NK cell. In addition, the double specific binding molecule prepared by using the anti-NKp80 antibody can also specifically target and combine human NKp80 protein and cynomolgus monkey NKp80 protein, and the double specific binding molecule can target and combine high expression NKp80 and B7H6 cell, thereby being used for treating or preventing diseases, such as tumor (for example, colorectal cancer and the like).

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to NKp80 antibodies and their applications. More specifically, this invention relates to antibodies or antigen-binding fragments thereof capable of specifically recognizing NKp80, bispecific binding molecules, nucleic acid molecules, expression vectors, recombinant cells, compositions, drugs, and kits for detecting NKp80. Background Technology

[0002] T cells and natural killer cells (NK cells) are two crucial types of immune cells in the anti-tumor immune response, playing a vital role in the direct killing of tumors. NK cells are important members of the innate immune system; unlike T cells, NK cells do not express antigen-specific receptors. NK cells possess a broad spectrum of tumor-killing capabilities and also play a significant role in enhancing antibody and T cell responses. Currently, NK cell-based tumor immunotherapy takes various forms and employs diverse methods.

[0003] NKp80 protein is a C-type lectin-like receptor, normally expressed on the surface of peripheral blood NK cells and NKT cells, serving as a surface marker of activated NK cells. NKp80 is usually expressed on the cell surface in a dimer form, lacking charged amino acids in its transmembrane region and lacking the conventional ITAM activation motif intracellularly (Alessandro Moretta et al., 2001). Studies have found that NKp80 is also expressed on the surface of a small group of CD8 T cells with an effector memory phenotype, which can promote T cell responses and thus enhance immune responses (Sabrina Kuttruff et al., 2009). AICL protein is a ligand of NKp80, normally expressed on the surface of myeloid cells. Myeloid cells upregulate AICL expression upon stimulation by Toll-like receptors. Therefore, during NK cell-monocyte interaction, NKp80 stimulates myeloid cells to release pro-inflammatory cytokines, playing a crucial role in the initiation and maintenance of inflammation (Stefan Welte et al., 2006). Therefore, NKp80 molecules can serve as a promising potential target for cancer treatment.

[0004] Bispecific antibodies are antibodies that can simultaneously and specifically bind to two antigen sites. Bispecific antibodies used in cancer therapy can be classified into three categories based on their mechanism of action: retargeting effector cells; immunomodulation; and dual binding to tumor cell receptors. Antibodies for retargeting function constitute the majority, and the two currently marketed antibodies for cancer therapy are based on T cell retargeting. Due to their characteristics, bispecific antibodies can also be effectively used in other therapeutic systems, such as dual immunomodulation or targeting two molecules on the same cell membrane.

[0005] Currently, there are relatively few publicly available NKp80 monoclonal and biclonal antibodies, and the variety needs to be further expanded, while the specificity needs to be improved. Summary of the Invention

[0006] This application is based on the inventor's discoveries and understanding of the following facts and problems:

[0007] NKp80 is present on the surface of peripheral blood NK cells and NKT cells, serving as a surface marker for activated NK cells. During NK cell-monocyte interaction, NKp80 stimulates myeloid cells to release pro-inflammatory cytokines, which plays a crucial role in the initiation and maintenance of inflammation. NKp80 molecules could therefore serve as a promising potential therapeutic target for tumors.

[0008] The inventors of this application have successfully screened a mouse-derived anti-NKp80 monoclonal antibody. This monoclonal antibody has high binding activity to the extracellular domain of human or cynomolgus monkey NKp80 protein, and can target and bind to cells that highly express NKp80, as well as human primary T cells and NK cells (cells that express NKp80 protein on their surface).

[0009] Furthermore, the bispecific binding molecule prepared using the anti-NKp80 antibody can also specifically target and bind to human NKp80 protein and cynomolgus monkey NKp80 protein. Moreover, the bispecific binding molecule can target and bind to cells that highly express NKp80 and B7H6, thereby treating or preventing diseases such as tumors (e.g., colorectal cancer). For example, a bispecific antibody containing NKp80 scFv targeting B7H6 can promote the specific killing of B7H6 by PBMCs. + Tumor cells.

[0010] In a first aspect, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises:

[0011] The heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:1, 2, and 3, or amino acid sequences having at least 80% identity with SEQ ID NO:1, 2, and 3, respectively; and / or

[0012] The light chain variable regions CDR1, CDR2, and CDR3 sequences are shown as in SEQ ID NO:4, 5, and 6, or amino acid sequences having at least 80% identity with 4, 5, and 6, respectively. The antibody or antigen-binding fragment according to embodiments of the present invention can specifically bind to the human or cynomolgus monkey NKp80 protein with high affinity, and also provides a basis for the development of NKp80-based redirected multispecific antibodies.

[0013] According to embodiments of the present invention, the antibody or antigen-binding fragment may further include at least one of the following additional technical features:

[0014] According to an embodiment of the present invention, the antibody or antigen-binding fragment is a murine antibody or antigen-binding fragment.

[0015] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises:

[0016] The heavy chain variable region CDR1 sequence shown in SEQ ID NO:1, the heavy chain variable region CDR2 shown in SEQ ID NO:2, the heavy chain variable region CDR3 shown in SEQ ID NO:3, the light chain variable region CDR1 shown in SEQ ID NO:4, the light chain variable region CDR2 shown in SEQ ID NO:5, and the light chain variable region CDR3 shown in SEQ ID NO:6.

[0017] HCDR1 amino acid sequence: DYYMN SEQ ID NO:1

[0018] HCDR2 amino acid sequence: DIHPNNGGTSYNQKFKG SEQ ID NO:2

[0019] HCDR3 amino acid sequence: DYGGTYTNWAQNYFDY SEQ ID NO:3

[0020] LCDR1 amino acid sequence: RASQSVSTPSYSYMH SEQ ID NO:4

[0021] LCDR2 amino acid sequence: YASNLES SEQ ID NO:5

[0022] LCDR3 amino acid sequence: QHSWEIPWT SEQ ID NO:6

[0023] According to an embodiment of the present invention, the antibody or antigen-binding fragment includes at least one of a heavy chain FR region and a light chain FR region.

[0024] According to an embodiment of the present invention, at least a portion of at least one of the heavy chain FR region and the light chain FR region is derived from at least one of primate-derived antibodies and murine antibodies or mutants thereof.

[0025] According to an embodiment of the present invention, the light chain variable region of the antibody or antigen-binding fragment further comprises a light chain FR region of a mouse κ chain or a mouse κ chain variant, or a light chain FR region of a mouse λ chain or a mouse λ chain variant; wherein, the antibody heavy chain variable region further comprises a heavy chain FR region of mouse IgG1 or a variant thereof, or a heavy chain FR region of IgG2 or a variant thereof, or a heavy chain FR region of IgG3 or a variant thereof.

[0026] According to embodiments of the present invention, the antibody or antigen-binding fragment includes a heavy chain variable region as shown in SEQ ID NO:7; and / or

[0027] As shown in SEQ ID NO:8, the light chain variable region.

[0028] SEQ ID NO:7:

[0029] EVQLQQSGPELVNPGASVKISCKTSGYTFTDYYMNWVKQSHGKSLEWIGDIHPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARDYGGTYTNWAQNYFDYWGQGTTLTVSS

[0030] SEQ ID NO:8

[0031] DIVQTQSPASLAVSLGQRATISCRASQSVSTPSYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPWTFGGGTKLEIK

[0032] According to an embodiment of the present invention, the antibody or antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a primate antibody and a murine antibody or a mutant thereof.

[0033] According to an embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from mouse IgG antibodies or their mutants, or human IgG antibodies or their mutants.

[0034] According to an embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from mouse IgG1 antibody or its mutant or human IgG1 antibody or its mutant.

[0035] According to an embodiment of the present invention, the antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:9 and a light chain of the amino acid sequence shown in SEQ ID NO:10.

[0036] SEQ ID NO:9

[0037] EVQLQQSGPELVNPGASVKISCKTSGYTFTDYYMNWVKQSHGKSLEWIGDIHPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARDYGGTYTNWAQNYFD YWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0038] SEQ ID NO:10

[0039] DIVQTQSPASLAVSLGQRATISCRASQSVSTPSYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPWTFGGGTK LEIKAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0040] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody.

[0041] According to an embodiment of the present invention, the monoclonal antibody includes at least one of full-length antibody, Fv antibody, single-chain antibody, Fab antibody, single-domain antibody, and minimum recognition unit.

[0042] A second aspect of the present invention provides a multispecific binding molecule. According to an embodiment of the present invention, the multispecific binding molecule comprises at least:

[0043] A first binding region, the first binding region comprising the antibody or antigen binding fragment described in the first aspect; and

[0044] The second binding region is capable of specifically binding to target cell surface antigens.

[0045] The bispecific binding molecules of the present invention not only specifically bind to NKp80, but also specifically bind to one or more other antigens. The multispecific molecules of the present invention include at least bispecific, trispecific, and tetraspecific molecules. Based on their multispecificity, the binding molecules of the present invention can target NK cells and T cells to other antigens, eliminating cells producing such antigens through NK cell or T cell-mediated cell killing or phagocytosis. A bispecific binding molecule of one embodiment of the present invention can specifically bind to human or cynomolgus monkey NKp80 and B7H6 proteins, and can be used in scientific research or to effectively kill B7H6-positive tumor cells.

[0046] Those skilled in the art will understand that the binding activity of the second binding region is not particularly limited and may also have other binding activities, as long as the bispecific antibody has the antibody or antigen-binding fragment described in the first aspect, and both the antibody or antigen-binding fragment and the second binding region can effectively function. Furthermore, the antibody or antigen-binding fragment described in this invention can be used to prepare more specific antibodies, such as trispecific, tetraspecific, and pentaspecific antibodies. Based on the multispecificity of these antibodies, the antibody or antigen-binding fragment of this invention can target the surface of cells that highly express multiple antigens, eliminating cells producing these antigens through cell-killing action.

[0047] According to an embodiment of the present invention, the bispecific binding molecule may further include at least one of the following additional technical features:

[0048] According to an embodiment of the present invention, the multispecific binding molecule is a bispecific binding molecule.

[0049] According to an embodiment of the present invention, the bispecific binding molecule includes a symmetrical bispecific binding molecule or an asymmetrical bispecific binding molecule.

[0050] According to an embodiment of the present invention, the bispecific binding molecule is a symmetrical bispecific binding molecule.

[0051] According to an embodiment of the present invention, the first binding region comprises peptide chain 1 and peptide chain 2, wherein peptide chain 1 includes heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 1, 2, and 3, respectively.

[0052] The peptide chain 2 includes light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:4, 5, and 6, respectively.

[0053] According to an embodiment of the present invention, peptide chain 1 includes the heavy chain variable region shown in SEQ ID NO:7, and peptide chain 2 includes the light chain variable region shown in SEQ ID NO:8.

[0054] According to an embodiment of the present invention, the first binding region further includes a first linker peptide, wherein the N-terminus of the first linker peptide is connected to the C-terminus of the peptide chain 2, and the C-terminus of the first linker peptide is connected to the N-terminus of the peptide chain 1; or the N-terminus of the first linker peptide is connected to the C-terminus of the peptide chain 1, and the C-terminus of the first linker peptide is connected to the N-terminus of the peptide chain 2.

[0055] According to an embodiment of the present invention, the first linker peptide has an amino acid sequence (GGS)n, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Those skilled in the art will understand that conventional linkers, such as conventional flexible or rigid amino acid fragments, can be used.

[0056] According to an embodiment of the present invention, the first linker peptide has the amino acid sequence shown in SEQ ID NO:13.

[0057] According to an embodiment of the present invention, the first binding region comprises a single-chain antibody having an amino acid sequence as shown in SEQ ID NO:11.

[0058] According to an embodiment of the present invention, the first binding region further includes a first heavy chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate antibody, and a mouse antibody or a mutant thereof.

[0059] According to an embodiment of the present invention, the first heavy chain constant region is derived from a human IgG antibody or a mutant thereof.

[0060] According to an embodiment of the present invention, the first heavy chain constant region has the amino acid sequence shown in SEQ ID NO:28.

[0061] According to an embodiment of the present invention, the first binding region has an amino acid sequence as shown in SEQ ID NO:14.

[0062] According to an embodiment of the present invention, the second binding region has B7H6 binding activity.

[0063] According to an embodiment of the present invention, the second binding region includes at least one of a full-length antibody, an Fv antibody, a single-chain antibody, a Fab antibody, a single-domain antibody, and a minimum recognition unit having B7H6 binding activity.

[0064] According to an embodiment of the present invention, the second binding region includes an anti-B7H6 single-chain antibody.

[0065] According to an embodiment of the present invention, the anti-B7H6 single-chain antibody includes a light chain variable region and a heavy chain variable region of the anti-B7H6 antibody.

[0066] According to an embodiment of the present invention, the heavy chain variable regions CDR1, CDR2, and CDR3 sequences of the anti-B7H6 antibody are shown in SEQ ID NO:30-32.

[0067] According to an embodiment of the present invention, the light chain variable regions CDR1, CDR2, and CDR3 sequences of the anti-B7H6 antibody are shown in SEQ ID NO:33-35.

[0068] According to an embodiment of the present invention, the heavy chain variable region of the anti-B7H6 antibody has the amino acid sequence shown in SEQ ID NO:36, and the light chain variable region of the anti-B7H6 antibody has the amino acid sequence shown in SEQ ID NO:37.

[0069] According to an embodiment of the present invention, the anti-B7H6 single-chain antibody further comprises a second linker peptide that links the heavy chain variable region and the light chain variable region of the anti-B7H6 antibody. According to an embodiment of the present invention, the second linker peptide has an amino acid sequence (GGS)n, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and more preferably 7. Those skilled in the art will understand that conventional linkers, such as conventional flexible or rigid amino acid fragments, can be used.

[0070] According to an embodiment of the present invention, the second linker peptide has the amino acid sequence shown in SEQ ID NO:13.

[0071] Optionally, the anti-B7H6 single-chain antibody has an amino acid sequence as shown in SEQ ID NO:12.

[0072] According to an embodiment of the present invention, the bispecific antibody provided by the present invention includes a first antigen-binding region and a second binding region, wherein the first antigen-binding region contains an antibody targeting NKp80 as described in the first aspect of the present invention, the antibody or antigen-binding fragment being an anti-NKp80 single-chain antibody, see SEQ ID NO:11, and the second binding region contains an antibody targeting another target, including an anti-B7H6 single-chain antibody, see SEQ ID NO:12.

[0073] According to an embodiment of the present invention, the second binding region further includes a second heavy chain constant region, at least a portion of which is derived from at least one of a primate antibody and a murine antibody or a mutant thereof.

[0074] According to an embodiment of the present invention, the second heavy chain constant region is derived from a human IgG antibody or a mutant thereof.

[0075] According to an embodiment of the present invention, the second heavy chain constant region has the amino acid sequence shown in SEQ ID NO:29.

[0076] According to an embodiment of the present invention, the N-terminus of the second heavy chain constant region is connected to the C-terminus of the anti-B7H6 single-chain antibody.

[0077] According to an embodiment of the present invention, the second binding region has the amino acid sequence shown in SEQ ID NO:15.

[0078] According to an embodiment of the present invention, the first heavy chain constant region and the second heavy chain constant region are connected by a knot-into-hole structure.

[0079] A third aspect of the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect or the multispecific binding molecule described in the second aspect.

[0080] According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect of the present invention. The heavy chain coding sequence of the NKp80 antibody (chimeric antibody 53G7) is SEQ ID NO: 16, and the light chain coding sequence is SEQ ID NO: 17.

[0081] It should be noted that those skilled in the art will understand that the nucleic acids mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases herein, the complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this invention include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0082] A fourth aspect of the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the third aspect.

[0083] The expression vector may include optional control sequences operatively linked to the nucleic acid molecule. The control sequences are one or more control sequences that direct the expression of the nucleic acid molecule in a host. The expression vector proposed in this invention can efficiently and massively express the antibody or antigen-binding fragment in suitable host cells, thereby effectively enabling its use for tumor-specific treatment or prevention.

[0084] In this article, "operably ligated" refers to ligating a foreign gene to a vector so that the control elements within the vector, such as transcriptional and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. When ligating the aforementioned nucleic acid molecules to a vector, the nucleic acid molecules can be directly or indirectly linked to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. These control elements can be directly derived from the vector itself or be exogenous, i.e., not derived from the vector itself. Those skilled in the art will understand that nucleic acid molecules used to encode antibody or antigen-binding fragments can be independently inserted into different vectors, but commonly they are inserted into the same vector. Commonly used vectors include plasmids, bacteriophages, etc.

[0085] A fifth aspect of the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect; or

[0086] It is capable of expressing the antibody or antigen-binding fragment described in the first aspect or the multispecific binding molecule described in the second aspect.

[0087] According to an embodiment of the present invention, the recombinant cells are obtained by introducing the expression vector described in the fourth aspect into host cells.

[0088] According to some embodiments of the present invention, the antibody or antigen-binding fragment or the bispecific binding molecule can be effectively obtained in vitro in large quantities through recombinant cells.

[0089] According to some specific embodiments of the present invention, the recombinant cells are not particularly limited, and either prokaryotic cells or eukaryotic cells can be used.

[0090] According to an embodiment of the present invention, the recombinant cells are eukaryotic cells.

[0091] According to an embodiment of the present invention, the recombinant cell is a mammalian cell. According to some specific embodiments of the present invention, when the cell is a eukaryotic cell, such as a mammalian cell, the expression efficiency of the recombinant antibody is higher.

[0092] A sixth aspect of the present invention provides a composition. According to an embodiment of the present invention, the composition comprises at least one of the antibody or antigen-binding fragment of the first aspect, the multispecific binding molecule of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, or the recombinant cell of the fifth aspect.

[0093] The compositions of the present invention can also be administered in combination with each other or in combination with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Therefore, the compositions may also contain a chemotherapeutic agent. The antibodies or antigen-binding fragments or multispecific binding molecules of the present invention may also be combined with a second therapeutic agent. It should be noted that the compositions include combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the present invention. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0094] A seventh aspect of the present invention provides a medicament. According to an embodiment of the present invention, the medicament comprises at least one of the antibody or antigen-binding fragment of the first aspect, the multispecific binding molecule of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cell of the fifth aspect, or the composition of the sixth aspect.

[0095] According to embodiments of the present invention, the drug may further include a pharmaceutically acceptable carrier.

[0096] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0097] The effective amount of the antibody or antigen-binding fragment or the multispecific binding molecule described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0098] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0099] The medicament of this invention contains a safe and effective amount of the active ingredient of this invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched with the route of administration, wherein the route of administration may be oral, nasal, intradermal, subcutaneous, intramuscular, intravenous, or intraperitoneal. The dosage forms of the medicament of this invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, it is prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The medicament is preferably manufactured under sterile conditions. The antibody or antigen-binding fragment can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0100] The eighth aspect of this invention provides the use of the antibody or antigen-binding fragment described in the first aspect, the multispecific binding molecule described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, and the composition described in the sixth aspect in the preparation of a medicament for the prevention and / or treatment of cancer.

[0101] The cancers include at least one of the following: colorectal cancer, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

[0102] A ninth aspect of the present invention provides a kit for detecting NKp80. According to an embodiment of the present invention, the kit contains at least one of the antibody or antigen-binding fragment described in the first aspect, the multispecific binding molecule described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, and the recombinant cells described in the fifth aspect.

[0103] As previously described, the antibody or antigen-binding fragments of some specific embodiments of the present invention can effectively bind to human or cynomolgus monkey NKp80 protein. The bispecific binding molecule can bind to both human or cynomolgus monkey NKp80 protein and B7H6 protein. Therefore, kits containing the antibody or antigen-binding fragments can effectively perform qualitative or quantitative detection of human or cynomolgus monkey NKp80 protein, and kits containing the bispecific binding molecule can effectively perform qualitative or quantitative detection of human or cynomolgus monkey NKp80 protein and B7H6 protein. The kits provided by the present invention can be used, for example, in immunoblotting, immunoprecipitation, and other kits involving the detection of human or cynomolgus monkey NKp80 and antibody-specific binding properties. These kits may contain any one or more of the following: antagonist, anti-NKp80 antibody, or pharmaceutical reference material; protein purification column; immunoglobulin affinity purification buffer; cell assay diluent; instructions or literature, etc. Anti-NKp80 antibodies can be used for different types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, they can be tested on the serum or blood of a subject to detect related diseases. They can also be used in scientific research to detect human or cynomolgus monkey NKp80 protein, or human or cynomolgus monkey NKp80 protein and B7H6 protein in a sample.

[0104] The tenth aspect of this invention provides the use of the antibody or antigen-binding fragment described in the first aspect, the multispecific binding molecule described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, and the recombinant cells described in the fifth aspect in the preparation of a kit for detecting NKp80.

[0105] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0106] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0107] Figure 1 The flow cytometry results of 293T cells overexpressing human NKp80 and monkey NKp80 are shown.

[0108] Figure 2A The image shows the ELISA results of chimeric NKp80 antibody (53G7) binding to human NKp80;

[0109] Figure 2B The image shows the ELISA results of chimeric NKp80 antibody (53G7) binding to cynomolgus monkey NKp80;

[0110] Figure 3A The image shows the results of chimeric NKp80 antibody (53G7) binding to 293T cells overexpressing human NKp80;

[0111] Figure 3B The image shows the results of chimeric NKp80 antibody (53G7) binding to 293T cells overexpressing cynomolgus monkey NKp80;

[0112] Figure 4A This demonstrates the binding of the chimeric NKp80 antibody (53G7) to CD3 in human PBMCs. + Result image of cells;

[0113] Figure 4B This demonstrates the binding of the chimeric NKp80 antibody (53G7) to CD56 in human PBMCs. + Result image of cells;

[0114] Figure 5 The figure shows the results of the in vitro cytotoxicity experiment of HCT-15 cells by the NKp80×B7H6 bispecific binding protein and PBMC. Detailed Implementation

[0115] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0116] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0117] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0118] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0119] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0120] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0121] The antibody or antigen-binding fragments described in this invention are typically prepared by biosynthetic methods. Based on the nucleotide sequence described in this invention, those skilled in the art can readily obtain the encoding nucleic acid of this invention using various known methods. These methods include, but are not limited to, PCR, artificial DNA synthesis, etc., and specific methods can be found in J. Sambrook, *Molecular Cloning: A Laboratory Manual*. As one embodiment of this invention, the encoding nucleic acid sequence of this invention can be constructed by synthesizing nucleotide sequences in segments and then performing overlap extension PCR. The antibody or antigen fragments are numbered and defined using the Kabat numbering system.

[0122] the term

[0123] The term "antibody" is used in the broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind antigens (e.g., Fab', F'(ab)2, Fv, single-chain antibodies, biantibodies, Fab), and recombinant peptides containing the foregoing, provided they exhibit the desired biological activity. An "immunoglobulin" or "tetrameric antibody" is a tetrameric glycoprotein (a tetrapeptide chain structure linked by interchain disulfide bonds) composed of two heavy chains and two light chains, each containing a variable region and a constant region. The antigen-binding portion can be produced using recombinant DNA technology or through enzymatic or chemical cleavage of intact antibodies. The antigenicity of immunoglobulins varies depending on the amino acid composition and sequence of the constant region of the heavy chain. Immunoglobulins can be classified into five classes: IgM, IgD, IgG, IgA, and IgE. The same class of immunoglobulins can also be subdivided based on their amino acid composition, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin light chains are classified into κ chains or λ chains based on their constant regions. In this invention, the variable region of the antibody heavy chain may further include a heavy chain constant region, which contains human or mouse IgG1, IgG2, IgG3, IgG4, or variants thereof. In this invention, the variable region of the antibody light chain may further include a light chain constant region, which contains human or mouse κ, λ chains, or variants thereof.

[0124] The "antigen-binding fragment" described in this invention refers to Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments (ScFv fragments) that bind to human NKp80, all possessing antigen-binding activity; these fragments contain one or more CDR regions selected from SEQ ID NO:1 to SEQ ID NO:6 of the antibody described in this invention. The Fv fragment contains variable regions of the antibody heavy chain and light chain, but no constant regions, and is the smallest antibody fragment possessing all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. Two antibody variable regions can also be linked into a single polypeptide chain using different linkers, referred to as a single-chain antibody or single-chain Fv.

[0125] Antibody fragments or antigen-binding portions particularly include Fab, Fab', F(ab')2, Fv, domain antibodies (dAb), complementarity-determining region (CDR) fragments, CDR-transplanted antibodies, single-chain antibodies (scFv), single-chain antibody fragments, chimeric antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, linear antibodies; chelated recombinant antibodies, tribody or bibody antibodies, intracellular antibodies, nanobodies, small modular immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion proteins, camelified antibodies, VHH-containing antibodies or their variants or derivatives, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer a specific antigen bound to the polypeptide, such as one, two, three, four, five or six CDR sequences, provided that the antibody retains the desired biological activity.

[0126] As used herein, the "heavy chain variable region" refers to a region of an antibody molecule containing at least one complementarity-determining region (CDR) of the antibody heavy chain variable domain. The heavy chain variable region may contain one, two, or three CDRs of the antibody heavy chain. The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is termed the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is termed the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR3.

[0127] The terms "complementarity-determining region" or "CDR" or "CDR sequence" refer to the amino acid sequence in an antibody responsible for antigen binding. For example, these typically include amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health). of Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., amino acid residues near 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable region, and amino acid residues near 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)).

[0128] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by murine antibodies. The variable region gene is cloned from mouse hybridoma cells, and then the constant region gene of a human antibody is cloned as needed. The mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic expression system. In a preferred embodiment of the invention, the light chain of the NKp80 chimeric antibody further comprises the Fc region of a human κ, λ chain, or a variant thereof. The heavy chain of the NKp80 chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof.

[0129] In this article, "monoclonal antibody" refers to an antibody with a single antigen-binding site.

[0130] In this article, "dual antibody" refers to an antibody with two different antigen-binding sites.

[0131] In this article, the term "single-domain antibody" refers to a naturally occurring heavy chain antibody found in camel / alpaca blood that lacks the light chain and consists only of the heavier H chain. The amino-terminal (N-terminus) amino acid sequence of the peptide chain varies considerably and is called the variable region (V region), while the carboxyl-terminus (C-terminus) is relatively stable with minimal variation and is called the constant region (C region). The V region of the H chain is called VH. Within the variable region, certain areas exhibit even greater variability in amino acid composition and sequence, termed the hypervariable region (HVR). The hypervariable region is the site of antigen-antibody binding and is therefore also called the complementarity-determining region (CDR). The heavy chain variable region has three CDRs. CDR1 and CDR3 are slightly longer than those in humans, and CDR3 protrudes outward in the tertiary structure. Therefore, it is speculated that single-domain antibodies have higher antigen-binding specificity and affinity than traditional antibodies.

[0132] The variable domains of single-domain antibodies exhibit the same general structure as a relatively conserved framework region (FR) linked by three hypervariable regions or CDRs. From the N-terminus to the C-terminus, the heavy chain contains domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignment of each domain conforms to the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991; or Chothia and Lesk, Journal of Molecular Biology, 196:901-917, 1987; Chothia et al., Nature, 342:878-883, 1989).

[0133] The hypervariable region of an antibody refers to the CDR amino acid residues responsible for antigen binding. The hypervariable region contains amino acid residues from the CDR, such as 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain (as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and / or residues from the hypervariable ring, such as 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain (as described in Chothia et al., Journal of Molecular Biology 196:901-917 (1987)).

[0134] In this document, the term "mutant" or "variant" may refer to a molecule obtained by mutating one or more nucleotides or amino acids into any naturally occurring or engineered molecule.

[0135] In this invention, the term "mouse antibody" refers to a monoclonal antibody against human NKp80 prepared according to the knowledge and skills in the art. Preparation involves injecting the test subject with an antigen, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. In a preferred embodiment of the invention, the mouse NKp80 antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a mouse IgG1, IgG2, IgG3 or a variant thereof.

[0136] Monoclonal antibodies are antibodies obtained from a substantially homogeneous group of antibodies, where all antibodies in the mixture have a single amino acid sequence derived from a single clone. Monoclonal antibodies are typically highly specific and target a single antigenic site or epitope. In contrast, polyclonal antibody formulations usually consist of a mixture of antibodies with different amino acid sequences targeting the same or different determinants (epitaxes). In addition to their specificity, monoclonal antibodies have the advantage of being synthesized in homologous cultures, free from contamination by other immunoglobulins with different specificities and characteristics.

[0137] As used herein, the term "hinge" refers to the region between the CH1 and CH2 regions of the antibody heavy chain. This region includes interchain disulfide bonds, is rich in proline, does not form α-helices, and readily extends and twists to a certain extent, which facilitates complementary binding between the antibody's antigen-binding site and the antigenic epitope. In the heavy chain single-domain antibody of this invention, the variable region of the heavy chain is connected to the Fc region via the hinge region.

[0138] As used herein, the term "Fc region" refers to a protein containing the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin, excluding the heavy chain variable region and heavy chain constant region 1 (CH1). In this invention, an Fc fragment means that it includes not only the native amino acid sequence but also its mutant sequence. The immunoglobulin Fc region can be derived from humans or animals, such as cattle, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs.

[0139] As used herein, the term "constant region" refers to the C-terminus of a polypeptide chain, specifically the 3 / 4 or 4 / 5 segment of the H chain and the 1 / 2 segment of the L chain. The number, type, sequence, conformation, and sugar content of amino acids in this segment are relatively stable; hence, this segment is called the constant region, or C-region. The C-regions of the H and L chains are denoted by CH and CL, respectively. The CH lengths vary among different types of immunoglobulins. IgG, IgA, and IgD have three CHs: CH1, CH2, and CH3; IgM and IgE have four CHs: CH1, CH2, CH3, and CH4. Each heavy chain consists of a variable region (VH) and first, second, third, and fourth (optionally) constant regions (CH1, CH2, CH3, and CH4, respectively). Typically, natural intact antibodies are Y-shaped, with the stem of the Y-structure composed of the second and third constant regions of two heavy chains linked by disulfide bonds. Each arm of the “Y” structure includes the heavy chain VH and CH1 (VH-CH1) and the light chain (VL-CL).

[0140] In this paper, the term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, see, for example, Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm by Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm by Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method by Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; and the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997)). And the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266: 460-480 (1996)); or GAP, BESTFIT, BLASTAltschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0141] Without substantially affecting antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention to obtain variants of the antibody or its functional fragments. These are all considered to be included within the scope of protection of the present invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences of the present invention can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. Sequence identity described in the present invention can be measured using sequence analysis software, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are shown in N-terminus to C-terminus arrangement.

[0142] As previously described, the monoclonal antibodies of the present invention may be full-length antibodies or may contain only their functional fragments (e.g., Fab, F(ab')2, or scFv fragments), or may be modified to affect function. The present invention includes anti-B7H6 antibodies with modified glycosylation patterns. In some applications, modification to remove undesirable glycosylation sites may be useful, or to antibodies lacking a fucose moiety on the oligosaccharide chain to, for example, enhance antibody-dependent cytotoxicity (ADCC) function. In other applications, galactosylation modification may be performed to alter complement-dependent cytotoxicity (CDC).

[0143] In this paper, the term "full-length antibody" refers to a tetrapeptide chain structure composed of two identical light chains and two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE). Immunoglobulins of the same class can also be divided into different subclasses based on their amino acid composition, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin light chains are classified into κ chains or λ chains based on their constant regions.

[0144] As used herein, the term "functional fragment" specifically refers to antibody fragments such as CDR transplantation antibodies, Fab, Fab', F(ab')2, Fv, or scFv, nanobodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol ("PEGylated, PEGylated") (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol), which have B7H6 binding activity. Preferably, the functional fragment will consist of or contain a portion of the heavy chain variable region or light chain variable region of its source antibody, the portion of which is sufficient to retain the same binding specificity and sufficient affinity as its source antibody, preferably at least 1 / 100 of the affinity for B7H6, and more preferably at least 1 / 10. This functional fragment will contain at least 3 amino acids, preferably 5, 10, 15, 25, 50, and 100 consecutive amino acids from the antibody sequence from which it is derived.

[0145] In this paper, the term "CDR-transplanted antibody" refers to the transplantation of the CDR of a monoclonal antibody from one species into the variable region of an antibody from another species. For example, the CDR of a murine monoclonal antibody can be transplanted into the variable region of a human antibody to replace the human antibody's CDR, thereby giving the human antibody the antigen-binding specificity of the murine monoclonal antibody while reducing its heterologous nature.

[0146] In this article, the term "Fab antibody" or "Fab" generally refers to an antibody containing only Fab molecules, which consist of the VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond.

[0147] In this paper, the term “nanobody” (single-domain antibody or VHH antibody), which was originally described as an antigen-binding immunoglobulin (variable) domain of “heavy chain antibody” (i.e., “antibody lacking light chains”) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)), contains only the heavy chain variable region (VH) and the conventional CH2 and CH3 regions, through which it specifically binds to the antigen.

[0148] In this article, the term "Fv antibody" generally refers to an antibody composed only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds. It is the smallest functional fragment of an antibody that retains the complete antigen-binding site.

[0149] In this paper, the term "single-chain antibody" or "scFv" refers to a fragment consisting of the variable regions of the antibody heavy chain and light chain linked by a short peptide.

[0150] In this paper, the "knob into hole structure" refers to the formation of a button (hole) mutation in the CH3 region of the constant region of the antibody heavy chain, facilitating heavy chain interlocking and the formation of a heterodimer. For example, in this application, this is achieved by mutating the amino acids in the CH3 domain of the constant region of the human IgG1 heavy chain (T366S, L368A, Y407V, Y349C mutations in one chain, i.e., "hole"; and T366W, S354C mutations in the other chain, i.e., "knob"). The amino acid numbers here are based on the Kabat numbering system. For example, "T366S" means that the T amino acid at position 366 according to the Kabat numbering system is replaced by an S amino acid.

[0151] As used herein, the term "nucleic acid" is used interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form, encompassing nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, said nucleic acids being synthetic, naturally occurring or unnatural, having similar binding properties to a reference nucleic acid, and being metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including one or more such nucleic acid molecules in a single or separate vector, and one or more such nucleic acid molecules present at one or more locations in a host cell. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences.

[0152] As used herein, the term "vector" refers to a delivery system that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) into and express that genetic element, for example, to produce a protein, RNA, or DNA encoded by that genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. Vectors may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into the cell, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors. In some embodiments, the vector (e.g., expression vector) provided by the present invention contains a nucleic acid sequence encoding a fusion protein as described in the present invention, at least one promoter operatively linked to the nucleic acid sequence (e.g., SV40, CMV, EF1α), and at least one selection marker.

[0153] As used herein, the term "host cell" refers to a cell into which exogenous polynucleotides and / or vectors can be introduced or have been introduced. The host cell contains the vector, which can be introduced into mammalian cells to construct host cells, which are then used to express the antibodies or antigen-binding fragments provided in this invention. The corresponding antibodies or fusion proteins can be obtained by culturing the host cells. Suitable mammalian cells include CHO cells, etc.

[0154] As used herein, the term "composition" refers to a form in which the biological activity of the active ingredient is permitted and which does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition will be applied.

[0155] As used herein, the term “treatment” means the temporary or permanent, partial or complete elimination, reduction, suppression or improvement of the clinical symptoms, manifestations or progression of an event, disease or condition.

[0156] As used herein, the term "diagnosis" refers to the identification, revelation, ascertainment, and / or definition of the location of a pathological state, disease, or condition. In some specific embodiments, the pharmaceutical compositions of the present invention, when administered to a subject or exposed to a sample from a subject, aid in the diagnosis of cancer, tumor formation, or condition.

[0157] In many embodiments, the terms “subject” and “patient” are used interchangeably, regardless of whether the subject has received or is currently receiving any form of treatment. As used herein, the terms “subject” or “patient” refer to a mammalian subject or patient. Unless otherwise indicated, the terms “patient” or “subject” are used interchangeably herein. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, poultry, goats, and sheep. In some embodiments, the subject is a human. In some embodiments, the subject is a person suspected of having cancer, an autoimmune disease or condition, and / or an infection. “Exogenous” means a substance produced outside an organism, cell, or human body, depending on the circumstances. “Endogenous” means a substance produced inside a cell, organism, or human body, depending on the circumstances.

[0158] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0159] The sequence information involved in this invention is shown in Table 1 below:

[0160] Table 1:

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167] Example 1: Construction of 293T cells overexpressing human and cynomolgus monkey NKp80

[0168] HEK293T cells were seeded in T150 culture flasks and cultured in DMEM complete medium. After overnight culture, endotoxin-free psPAX2, pMD2.G, and pCDH-CMV-NKp80-IRES-puro vectors (pCDH-CMV-MCS-IRES-puro vectors with the coding sequence of human NKp80 protein (SEQ ID NO:20) (SEQ ID NO:21) or cynomolgus monkey NKp80 protein (SEQ ID NO:22) (SEQ ID NO:23) inserted between the multiple cloning sites) were added to 1.5 mL of Opti-MEM (Gibco, catalog number 31985070) medium at a ratio of 7:3:10, followed by 100 μL of P3000 transfection reagent. Then, 100 μL of Lipofectamine 3000 (thermo, catalog number L3000008) transfection reagent was added to 1.5 mL of Opti-MEM medium and mixed thoroughly. Mix DNA dilution buffer and liposome dilution buffer at a 1:1 volume ratio, incubate at room temperature for 5-10 min, then add to 293T cells. After culturing for 48 hours, harvest the viral supernatant. Centrifuge the viral supernatant at 2000g for 10 min at 4°C, collect the supernatant, filter through a 0.45μm filter, add PEG8000 solution (Shanghai Sangon Biotech), mix thoroughly, and incubate overnight at 4°C. Centrifuge at 2200g for 90 min. A white precipitate appears at the bottom of the centrifuge tube; resuspend the virus in sterile PBS buffer.

[0169] Polybrene (Sigma, TR-1003) (final concentration 8 μg / ml) was added to DMEM medium, mixed well, and then an appropriate amount of virus solution was added. 2E5 293T cells were placed in 24-well plates, virus-containing medium was added, and the plates were incubated for 8 hours before being replaced with fresh culture. After 48 hours, the expression level of NKp80 on the surface of 293T cells was detected by flow cytometry. Once a positive cell population appeared, a limiting dilution was performed (digestion and dilution to a density of 4 cells / ml), and the cells were seeded into 96-well plates at a density of 200 μl per well. After culturing for 2 weeks until the cells clearly formed single cell clusters, the expression level of NKp80 on the surface of each clone was detected by flow cytometry. All positive cells were considered 293T cells overexpressing human NKp80 or cynomolgus monkey NKp80. The flow cytometry results for expression identification are shown below. Figure 1 .

[0170] Example 2: Preparation of extracellular protein of NKp80 in humans and cynomolgus monkeys

[0171] The amino acid sequence of the extracellular segment of human NKp80 is shown in SEQ ID NO:24, and the nucleotide sequence is shown in SEQ ID NO:25. The amino acid sequence of the extracellular segment of cynomolgus monkey NKp80 is shown in SEQ ID NO:26, and the nucleotide sequence is shown in SEQ ID NO:27.

[0172] Human or cynomolgus monkey NKp80 extracellular protein was prepared by transiently transfecting ExpiCHO-S cells (Gibco, catalog number A29127) with a pcDNA3.4 vector (histidine-tagged) carrying the nucleic acid sequence of the extracellular segment of human or cynomolgus monkey NKp80. The day before transfection, the ExpiCHO-S cells were adjusted to a cell density of (3-4) × 10⁻⁶ cells. 6 Incubate overnight at 37°C, 8% CO2, with shaking at 120 rpm, using a 1 / ml incubator. On the day of transfection, cells grew to 7 × 10⁹ cells / ml. 6 -1×10 7 When the cell viability is greater than 95%, prepare for transfection by diluting the cells to 6 × 10⁶ / ml using fresh, pre-warmed ExpiCHO medium (Gibco, catalog number A2910002). 6 / ml, then take a plasmid containing the extracellular coding sequence of human or cynomolgus monkey NKp80 and transfect it into ExpiCHO-S cells using ExpiFectamine CHO transfection reagent (Gibco, catalog number A29129). Incubate at 37°C, 8% CO2, and 120 rpm with shaking. 18-22 h post-transfection, immediately add ExpiFectamine CHO Enhancer and ExpiCHO Feed to the transfected cells, mix well, and incubate at 32°C, 5% CO2, and 120 rpm with shaking. On day 5 post-transfection, add another 8 ml of ExpiCHO Feed to the cells, mix well, and continue culturing. Observe cell count and cell viability changes daily. Harvest cells by centrifugation when cell viability drops below 80% or after 10-14 days of culture. The expression supernatant was filtered through a 0.22 μm filter membrane. Histidine-tagged antibodies were captured from the expression supernatant using a HisTrap excel affinity chromatography column (Cytiva, catalog number 17371206). After equilibrating the column with phosphate buffer at pH 7.4, the supernatant was passed through the affinity chromatography column and eluted with phosphate elution buffer containing 500 mM imidazole. Finally, the supernatant was concentrated and replaced with PBS buffer. The purified human and cynomolgus monkey NKp80 extracellular fragment proteins were identified by SDS-PAGE and found to have a purity of over 90%. The human and cynomolgus monkey NKp80 extracellular fragment proteins were finally obtained.

[0173] Example 3: Preparation of anti-human NKp80 hybridoma monoclonal antibody

[0174] Anti-human NKp80 monoclonal antibodies were generated by immunizing mice. Balb / c mice, female, 6 weeks old (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), were used as the immunogenic antigen, which was the extracellular segment of human NKp80 protein prepared in Example 2. The first immunization was performed intraperitoneally with the antigen emulsified with Freund's complete adjuvant (Sigma, F5881). Subsequent immunizations were performed intraperitoneally with the antigen mixed with the Ribi adjuvant system (Sigma, S6322).

[0175] Mice with high antibody titers in their serum were selected for spleen cell fusion. 72 hours prior to fusion, the selected mice were immunized via intraperitoneal injection. Using an optimized PEG-mediated fusion procedure, spleen lymphocytes were fused with myeloma Sp2 / 0 cells (ATCC, CRL-8287) to obtain hybridoma cells. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1×HAT, and 1×OPI) and aliquoted into 96-well cell culture plates, incubated at 37°C and 5% CO2. On day 5 post-fusion, 50 μL of HAT complete medium was added to each well. From day 7 to 8 post-fusion, the medium was completely replaced with HT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1×HT, and 1×OPI), 200 μL per well, depending on cell growth density.

[0176] On days 10-11 post-fusion, flow cytometry was performed to detect cell conjugation based on cell growth density. Positive wells were replaced with new medium, and the cells were promptly expanded to 24-well plates according to density. Cell lines transferred to 24-well plates were retested and then preserved for the first subcloning. Cells showing positive results in the first subcloning were preserved and subjected to a second subcloning. Cells showing positive results in the second subcloning were preserved and subjected to protein expression. Antibodies were further prepared using serum-free cell culture and purified by protein G affinity chromatography for subsequent functional activity assays.

[0177] Example 4 Hybridoma Sequencing

[0178] By combining the mouse-derived 53G7 antibody hybridomas selected in the experiment, the total number of candidate hybridoma cells was cultured to 10. 6Cells were collected by centrifugation at 800 rpm for 10 minutes, and total RNA was extracted using the Trizol kit (Invitrogen). Using the total RNA as a template, a cDNA library was synthesized by reverse transcription (Invitrogen), and the corresponding variable region nucleic acid sequence of hybridoma cells was amplified by PCR using the cDNA as a template. The primer sequences used in the PCR amplification reaction were complementary to the first frame region or signal peptide region and constant region of the antibody variable region (Larrick, JW, et al., (1990) Scand. J. Immunol., 32, 121128 and Coloma, JJ et al., (1991) BioTechniques, 11, 152156). In a 50 μl reaction system, add 2 μL of cDNA, 5 μL of 10×PCR buffer, 2 μL (5 μM) of upstream and downstream primers, 2 μL of dNTPs, 1 μL of Taq enzyme (Takara, Ex Taq), and 38 μL of H2O. Perform pre-denaturation at 95℃ for 5 min, followed by temperature cycling for PCR amplification. The reaction conditions were: 94℃ denaturation for 30 s, 58℃ annealing for 45 s, 72℃ extension for 50 s, for a total of 32 cycles, followed by a final extension at 72℃ for 7 min. Sequencing of the amplified products yielded the heavy and light chain variable region sequences of the mouse monoclonal antibody.

[0179] Example 5: Transient expression of anti-human NKp80 antibody

[0180] The heavy chain variable region sequence of anti-human NKp80 antibody 53G7 is shown in SEQ ID NO:7, and the light chain variable region sequence is shown in SEQ ID NO:8. Nucleic acid sequences encoding the heavy chain amino acid sequence (SEQ ID NO:9) (SEQ ID NO:16) and the light chain amino acid sequence (SEQ ID NO:10) (SEQ ID NO:17) were recombined into the pTT5 plasmid using molecular biology techniques.

[0181] NKp80 antibody was prepared by transiently transfecting ExpiCHO-S cells (Gibco, catalog number A29127) with a pTT5 vector carrying the light and heavy chains of anti-human NKp80 antibody. The day before transfection, the ExpiCHO-S cells were adjusted to a cell density of (3-4) × 10⁻⁶ cells. 6 Incubate overnight at 37°C, 8% CO2, with shaking at 120 rpm. On the day of transfection, cells grew to 7 × 10⁹ / ml. 6 -1×10 7 When the cell count is 95% or higher, prepare for transfection by diluting the cells to 6 × 10⁶ / ml using fresh, pre-warmed ExpiCHO medium (Gibco, catalog number A2910002). 6 / ml, then take plasmids containing anti-human NKp80 light and heavy chains at a molar ratio of 2:1 and transfect them into ExpiCHO-S cells using ExpiFectamine CHO transfection reagent (Gibco, catalog number A29129). Incubate at 37℃, 8% CO2, and 120 rpm with shaking. 18-22 h post-transfection, immediately add ExpiFectamine CHO Enhancer and ExpiCHO Feed to the transfected cells, mix well, and incubate at 32℃, 5% CO2, and 120 rpm with shaking. On day 5 post-transfection, add another 8 ml of ExpiCHO Feed to the cells, mix well, and continue culturing. Observe cell count and cell viability changes daily. Harvest cells by centrifugation when cell viability drops below 80% or after 10-14 days of culture. The expression supernatant was filtered through a 0.22 μm filter membrane. Antibodies with Fc domains were captured from the expression supernatant using a Mabselectprism A affinity chromatography column (Cytiva, catalog number 17549854). After equilibrating the column with phosphate buffer at pH 7.2, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100 mM citric acid, pH 2.7). Finally, the elution was concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE and its purity was above 95%, thus obtaining the 53G7 antibody.

[0182] Example 6: NKp80 antibody ELISA binding assay

[0183] ELISA assays were used to detect the binding properties of chimeric NKp80 antibody 53G7. Human and cynomolgus monkey NKp80 extracellular fragment protein was diluted to 2 μg / ml with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), and 100 μl was added to each well of an ELISA plate and incubated overnight at 4°C. The plate was then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μl of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate and incubated at room temperature for 2 h. The plate was then washed three more times with PBST. A gradient concentration of anti-NKp80 antibody 53G7 was added to each well and incubated at room temperature for 1 h. The plate was then washed three more times with PBST. Add 100 μl of HRP-goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-036-097) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash three times with PBST, add TMB to each well, and incubate at room temperature in the dark for 2-5 minutes. Terminate the reaction with 2M sulfuric acid in each well. Finally, read the OD450 value using a microplate reader.

[0184] The results are as follows Figure 2A and 2B As shown, Figure 2AThis indicates that the 53G7 antibody of the present invention can bind to human NKp80. Figure 2B This indicates that the 53G7 antibody of the present invention can bind to cynomolgus monkey NKp80, hIgG1 as a control.

[0185] Example 7: NKp80 antibody flow cytometry combined experiment

[0186] (1) NKp80 antibody binds to 293T cells overexpressing human and cynomolgus monkey NKp80 protein.

[0187] 293T cells overexpressing human NKp80 or 293T cells overexpressing cynomolgus monkey NKp80 were diluted with PBS to a concentration of 2 × 10⁻⁶. 6 / mL, add 100μL / tube to a 1.5ml EP tube, add 10μL / tube of goat serum, and block at 4℃ for 30min. Add gradient concentrations of NKp80 antibody and incubate at 4℃ for 30min. Add 1mL of PBS to the EP tube, centrifuge at 3500rpm×5min at 4℃, discard the supernatant, and wash once with PBS. After centrifugation, discard the supernatant, resuspend the cells in 100μL / tube of PBS, add 0.1μL / tube of Alexa647-labeled goat anti-mouse antibody secondary antibody (Jackson ImmunoResearch, 109-606-170), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200μL / tube of PBS and analyze using flow cytometry.

[0188] Streaming results as follows Figure 3A and 3B ,in Figure 3A The results showed that the 53G7 antibody could bind to 293T cells overexpressing human NKp80. Figure 3B The results showed that the 53G7 antibody could bind to 293T cells overexpressing cynomolgus monkey NKp80, indicating that the 53G7 antibody can bind to the NKp80 protein on the cell surface.

[0189] (2) NKp80 antibody binds to human PBMCs

[0190] Human peripheral blood mononuclear cells (PBMCs) were adjusted to a cell density of 5 × 10⁻⁶. 7Add 100 μL of NKp80 antibody to each 1.5 mL EP tube, along with a gradient concentration, and incubate at 4 °C for 30 min. Add 1 mL of PBS to the EP tube, centrifuge at 3500 rpm for 5 min at 4 °C, discard the supernatant, and wash once with PBS. Dilute anti-human CD45 antibody (biolegend, B356107), anti-human CD3 antibody (biolegend, B375999), anti-human CD56 antibody (biolegend, B371788), and goat anti-mouse antibody secondary antibody (Jackson ImmunoResearch, 109-606-170) with PBS, adding 100 μL to each well, mix well, and incubate at 4 °C for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μL of PBS per tube and analyze using flow cytometry.

[0191] Figure 4A The results showed that the 53G7 antibody could bind to CD3 in PBMCs. + T cells, Figure 4B The results showed that the 53G7 antibody could bind to CD56 in PBMCs. + NK cells indicate that the 53G7 antibody can bind to human primary T cells and NK cells.

[0192] Example 8: Bispecific antibody targeting B7H6 containing NKp80 single-chain antibody (scFv)

[0193] (1) Preparation of a bispecific antibody targeting B7H6 containing NKp80 scFv

[0194] A bispecific antibody was designed, containing two monovalent units. One monovalent unit is an anti-NKp80 scFv-Fc form, with variable region amino acids derived from the sequence in Example 4 of this invention. The other monovalent unit is an anti-B7H6 scFv-Fc form (sequence from CN 116284397A). This bispecific antibody is named NKp80×B7H6. This bispecific antibody contains two polypeptide chains: a heavy chain containing the NKp80 single-chain antibody scFv (SEQ ID NO: 14) and a heavy chain containing the B7H6 single-chain antibody scFv (SEQ ID NO: 15). Due to the molecule's unique asymmetric structure, different amino acid mutations were introduced into the constant regions of the two chains to reduce the formation of homodimers. Simultaneously, to prevent cross-linking activation caused by the Fcγ receptor, a (L234A / L235A) mutation was also introduced into the constant region of the heavy chain.

[0195] The bispecific antibody recombinant protein was prepared by one-step affinity purification using the method described in Example 5 and in combination with the plasmid ratio (1:1 or other ratios). Table 2 below lists the information for the bispecific antibody NKp80×B7H6.

[0196] Table 2

[0197] amino acid sequence SEQ ID NO: 14 SEQ ID NO: 15 nucleotide sequence SEQ ID NO: 18 SEQ ID NO: 19

[0198] (2) Cell killing experiment

[0199] After digestion, HCT-15 colorectal cancer cells were counted, and the cell density was adjusted to 2 × 10⁻⁶. 5 / ml. Turn on the RTCA instrument (Agilent), select the experimental mode, and fill in the cell and drug information. Enter the schedule to set the experimental steps. Add 50μl of fresh culture medium (89% RPMI 1640 medium + 10% fetal bovine serum + 1% penicillin-streptomycin) to the plate, place it in the instrument, close it, and click "Start" for step one. After completion, remove the plate, add 100μl of cell suspension, incubate at room temperature for 15-30 minutes to prevent edge effects, place it in the instrument, and click "Start". Once growth reaches the logarithmic growth phase, pause the process and add 50μl of PBMC (4×10⁻⁶ cells / ml). 6 Add the sample ( / ml) and a gradient concentration of bispecific antibody, click Start, and analyze after a period of time. Figure 5 The results showed that as the concentration of NKp80×B7H6 bispecific antibody increased, the killing efficiency of PBMCs against colorectal cancer HCT-15 cells gradually increased, indicating that NKp80×B7H6 can effectively promote the specific killing of B7H6 cells by PBMCs. + Tumor cells.

[0200] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0201] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that binds to NKp80, characterized in that, include: The heavy chain variable region CDR1 sequence shown in SEQ ID NO:1, the heavy chain variable region CDR2 shown in SEQ ID NO:2, the heavy chain variable region CDR3 shown in SEQ ID NO:3, the light chain variable region CDR1 shown in SEQ ID NO:4, the light chain variable region CDR2 shown in SEQ ID NO:5, and the light chain variable region CDR3 shown in SEQ ID NO:

6.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, include: Heavy chain FR region and light chain FR region.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The heavy chain FR region and the light chain FR region are derived from at least one of primate-derived antibodies and murine antibodies.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, include: As shown in SEQ ID NO:7, the heavy chain variable region; and The light chain variable region as shown in SEQ ID NO:

8.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that, The antibody or its antigen-binding fragment contains a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region and the light chain constant region are derived from at least one of a primate-derived antibody and a murine antibody.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, Both the light chain constant region and the heavy chain constant region are derived from mouse IgG antibodies or human IgG antibodies.

7. The antibody or its antigen-binding fragment according to claim 6, characterized in that, Both the light chain constant region and the heavy chain constant region are derived from mouse IgG1 antibody or human IgG1 antibody.

8. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The antibody or its antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:9 and a light chain of the amino acid sequence shown in SEQ ID NO:

10.

9. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a monoclonal antibody.

10. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The monoclonal antibody is a full-length antibody.

11. A bispecific antibody, characterized in that, At least including: A first binding region, wherein the first binding region comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10; and The second binding region specifically binds to the target cell surface antigen B7H6.

12. The bispecific antibody according to claim 11, characterized in that, The bispecific antibody includes symmetrical bispecific antibodies or asymmetrical bispecific antibodies.

13. The bispecific antibody according to claim 11, characterized in that, The bispecific antibody is a symmetrical bispecific antibody.

14. The bispecific antibody according to claim 11, characterized in that, The first binding region comprises peptide chain 1 and peptide chain 2, wherein peptide chain 1 includes heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 1, 2, and 3, respectively. The peptide chain 2 includes light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:4, 5, and 6, respectively.

15. The bispecific antibody according to claim 14, characterized in that, The peptide chain 1 includes the heavy chain variable region shown in SEQ ID NO:7, and the peptide chain 2 includes the light chain variable region shown in SEQ ID NO:

8.

16. The bispecific antibody according to claim 14, characterized in that, The first binding region further includes a first linker peptide, wherein the N-terminus of the first linker peptide is connected to the C-terminus of the peptide chain 2, and the C-terminus of the first linker peptide is connected to the N-terminus of the peptide chain 1; or the N-terminus of the first linker peptide is connected to the C-terminus of the peptide chain 1, and the C-terminus of the first linker peptide is connected to the N-terminus of the peptide chain 2.

17. The bispecific antibody according to claim 16, characterized in that, The first linker peptide has an amino acid sequence (GGS)n, where n is an integer greater than or equal to 1.

18. The bispecific antibody according to claim 17, characterized in that, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

19. The bispecific antibody according to claim 17, characterized in that, The first linker peptide has the amino acid sequence shown in SEQ ID NO:

13.

20. The bispecific antibody according to claim 11, characterized in that, The first binding region includes a single-chain antibody having an amino acid sequence as shown in SEQ ID NO:

11.

21. The bispecific antibody according to claim 20, characterized in that, The first binding region further includes a first heavy chain constant region, which is derived from at least one of a primate-derived antibody and a murine antibody.

22. The bispecific antibody according to claim 21, characterized in that, The first heavy chain constant region is derived from human IgG antibody.

23. The bispecific antibody according to claim 21, characterized in that, The first heavy chain constant region has the amino acid sequence shown in SEQ ID NO:

28.

24. The bispecific antibody according to claim 11, characterized in that, The first binding region has an amino acid sequence as shown in SEQ ID NO:

14.

25. The bispecific antibody according to claim 11, characterized in that, The second binding region includes at least one of a full-length antibody, an Fv fragment, a single-chain antibody, and a Fab antibody that has B7H6 binding activity.

26. The bispecific antibody according to claim 25, characterized in that, The second binding region includes an anti-B7H6 single-chain antibody.

27. The bispecific antibody according to claim 26, characterized in that, The anti-B7H6 single-chain antibody includes a light chain variable region and a heavy chain variable region.

28. The bispecific antibody according to claim 27, characterized in that, The heavy chain variable regions CDR1, CDR2, and CDR3 sequences of the anti-B7H6 antibody are shown in SEQ ID NO:30-32.

29. The bispecific antibody according to claim 27, characterized in that, The light chain variable regions CDR1, CDR2, and CDR3 sequences of the anti-B7H6 antibody are shown in SEQ ID NO:33-35.

30. The bispecific antibody according to claim 27, characterized in that, The heavy chain variable region of the anti-B7H6 antibody has the amino acid sequence shown in SEQ ID NO:36, and the light chain variable region of the anti-B7H6 antibody has the amino acid sequence shown in SEQ ID NO:

37.

31. The bispecific antibody according to claim 27, characterized in that, The anti-B7H6 single-chain antibody further includes a second linker peptide that links the heavy chain variable region and the light chain variable region of the anti-B7H6 antibody.

32. The bispecific antibody according to claim 31, characterized in that, The second linker peptide has an amino acid sequence (GGS)n, where n is an integer greater than or equal to 1.

33. The bispecific antibody according to claim 32, characterized in that, The n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

34. The bispecific antibody according to claim 32, characterized in that, The second linker peptide has the amino acid sequence shown in SEQ ID NO:

13.

35. The bispecific antibody according to claim 26, characterized in that, The anti-B7H6 single-chain antibody has the amino acid sequence shown in SEQ ID NO:

12.

36. The bispecific antibody according to claim 11, characterized in that, The second binding region further includes a second heavy chain constant region, which is derived from at least one of a primate-derived antibody and a murine antibody.

37. The bispecific antibody according to claim 36, characterized in that, The second heavy chain constant region is derived from human IgG antibody.

38. The bispecific antibody according to claim 36, characterized in that, The second heavy chain constant region has the amino acid sequence shown in SEQ ID NO:

29.

39. The bispecific antibody according to claim 26 or 36, characterized in that, The N-terminus of the second heavy chain constant region is connected to the C-terminus of the anti-B7H6 single-chain antibody.

40. The bispecific antibody according to claim 11, characterized in that, The second binding region has the amino acid sequence shown in SEQ ID NO:

15.

41. The bispecific antibody according to claim 21 or 36, characterized in that, The first heavy chain constant region and the second heavy chain constant region are connected by a knot-into-hole structure.

42. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10, or the bispecific antibody as described in any one of claims 11 to 41.

43. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 42.

44. A recombinant cell, characterized in that, The recombinant cell is a mammalian cell, and the recombinant cell carries the nucleic acid molecule of claim 42 or the expression vector of claim 43.

45. The recombinant cell according to claim 44, characterized in that, The recombinant cells are obtained by introducing the expression vector of claim 43 into host cells.

46. ​​A composition, characterized in that, It includes at least one of the antibodies or antigen-binding fragments thereof as described in any one of claims 1 to 10, the bispecific antibodies as described in any one of claims 11 to 41, the nucleic acid molecule as described in claim 42, the expression vector as described in claim 43, or the recombinant cells as described in claims 44 or 45.

47. A drug, characterized in that, It includes at least one of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10, the bispecific antibody as described in any one of claims 11 to 41, the nucleic acid molecule as described in claim 42, the expression vector as described in claim 43, the recombinant cell as described in claim 44 or 45, or the composition as described in claim 46.

48. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the bispecific antibody according to any one of claims 11 to 41, the nucleic acid molecule according to claim 42, the expression vector according to claim 43, the recombinant cell according to claim 44 or 45, or the composition according to claim 46 in the preparation of a medicament for the prevention and / or treatment of cancer. The cancer in question is colorectal cancer.

49. A kit for detecting NKp80, characterized in that, The kit contains at least one of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10, the bispecific antibody as described in any one of claims 11 to 41, the nucleic acid molecule as described in claim 42, and the expression vector as described in claim 43.

50. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the bispecific antibody according to any one of claims 11 to 41, the nucleic acid molecule according to claim 42, the expression vector according to claim 43, and the recombinant cells according to claim 44 or 45 in the preparation of a kit for detecting NKp80.

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