Anti-NKp46 nano antibody and application thereof

Abstract

The invention belongs to the technical field of antibodies, and particularly relates to an anti-NKp46 nano antibody and application thereof. The nano antibody provided by the invention can be efficiently combined with human and cynomolgus monkey NKp46, promotes NK cell activation, accurately regulates NK cell activity, constructs an NKCE (Natural Killer Cell Treatment) platform targeting NKp46, and becomes a key for research and development of a disease therapy aiming at a specific disease target, so that an effective scheme is provided for disease treatment.

Description

Technical Field

[0001] The present application belongs to the field of antibody technology, and specifically relates to an anti-NKp46 nanobody and its application. Background Art

[0002] NK is an important component of the innate immune system. It can kill cancer and virus-infected cells without relying on previous antigen stimulation, and plays a key antiviral and anti-tumor role. NK cells express a variety of activating receptors on their surface, such as NKp46, NKp30, NKG2D, etc. The ligands of these activating receptors (such as B7H6, MICA / B, etc.) are upregulated in cases of cell malignant transformation (tumor), autoimmune inflammation, infectious inflammation, etc. NK cells recognize and kill abnormal cells by binding to their ligands.

[0003] NKp46 is a member of the immunoglobulin superfamily with two external C2-type Ig-like domains. Its transmembrane (TM) domain transmits activation signals by binding to the transmembrane domain of CD3ζ or FcRγ. NK cells play an important role in the occurrence and development of tumors, autoimmune diseases, transplant rejection, and inflammation. As a key activating receptor for NK cell-mediated immune responses, NKp46 is not only involved in tumor immune surveillance, but also plays a core role in autoimmune diseases, transplant rejection, and inflammatory responses. Given its unique function in regulating immune responses, regulating NKp46 activity has become a key direction for new immunotherapy and drug development, especially in enhancing anti-tumor immune responses and inhibiting excessive immune responses, showing significant clinical application potential.

[0004] At present, Innate Pharma has made significant progress in the research and development of drugs based on NKp46 antibodies. IPH6101 / SAR443579 has demonstrated excellent anti-tumor activity in preclinical models and has entered the Phase II clinical trial stage for the indications of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, and high-risk myelodysplasia.

[0005] However, among the NKp46 antibody patents currently published worldwide, some antibodies have the problem of being unable to bind to monkey NKp46 and having weak NK cell activation ability. Therefore, the development of new nano-antibodies targeting NKp46, which can accurately regulate NK cell activity, and the construction of an NK cell therapy platform (NKCE) targeting NKp46, has become the key to the development of disease therapies, thereby providing an effective solution for disease treatment. Summary of the invention

[0006] Therefore, the present application develops a novel nano-antibody targeting NKp46, which can precisely regulate the activity of NK cells, and constructs an NK cell therapy platform (NKCE) targeting NKp46, and the technical scheme thereof is as follows:

[0007] 1. A nanobody, characterized in that the nanobody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3), wherein:

[0008] CDR-H1 has an amino acid sequence of SEQ ID NO 2: GRTFSNYA,

[0009] CDR-H2 has an amino acid sequence of SEQ ID NO 3: ISRSGDT,

[0010] CDR-H3 has an amino acid sequence of SEQ ID NO 4: AAGPSLYATNIFTAGRWYDY.

[0011] 2. The Nanobody according to item 1, characterized in that the Nanobody comprises an antibody heavy chain variable region VHH, wherein:

[0012] VHH has the sequence SEQ ID NO: 1:

[0013] The amino acid sequence of EVQLVESGGGLVQAGASLTLSCLVPGRTFSNYAMGWFRQAPGKDRDVVAAISRSGDTFYIDSVKGRFTISRDDAKNTVYLQMNSLKPEDKALYYCAAGPS LYATNIFTAGRWYDYWGQGTQVTVSS, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 1.

[0014] 3. The Nanobody according to claim 1, characterized in that the Nanobody comprises a heavy chain, wherein:

[0015] The heavy chain amino acid sequence has the sequence SEQ ID NO: 5:

[0016] EVQLVESGGGLVQAGASLTLSCLVPGRTFSNYAMGWFRQAPGKDRDVVAAISRSGDTFYIDSVKGRFTISRDDAKNTVYLQMNSLKPEDKALYYCAAGPSLYATNIFTAGRWYDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC The amino acid sequence of LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, or the same as SEQ ID NO:5 has an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.

[0017] 4. A nucleic acid molecule comprising a Nanobody encoding any one of items 1-3.

[0018] 5. A vector comprising the nucleic acid described in item 4.

[0019] 6. A host cell comprising the nucleic acid described in Item 4 or the vector described in Item 5.

[0020] 7. A pharmaceutical composition, wherein the pharmaceutical composition comprises:

[0021] The Nanobody described in any one of Items 1-3, or the antibody expressed by the nucleic acid described in Item 4, or the antibody prepared by the vector described in Item 5, or the antibody produced by the host cell described in Item 6, and a pharmaceutically acceptable carrier.

[0022] 8. The pharmaceutical composition according to item 7, wherein the pharmaceutical composition is used to treat or prevent NKp46-mediated related diseases.

[0023] 9. The pharmaceutical composition according to item 8, wherein the NKp46-mediated related diseases are selected from one or more of breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, esophageal cancer, pancreatic cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, ovarian cancer, kidney cancer, thyroid cancer, lymphoma, leukemia, endometrial cancer, melanoma, osteosarcoma, follicular lymphoma, relapsed / refractory multiple myeloma, relapsed / refractory light chain amyloidosis, rheumatoid arthritis, and systemic lupus erythematosus.

[0024] 10. A product or kit for detecting NKp46, comprising the Nanobody or antigen-binding fragment thereof according to any one of items 1 to 3.

[0025] 11. A conjugate, wherein the conjugate comprises:

[0026] The Nanobody described in any one of Items 1-3, or the antibody expressed by the nucleic acid described in Item 4, or the antibody prepared by the vector described in Item 5, or the antibody produced by the host cell described in Item 6.

[0027] 12. The conjugate according to item 11, wherein the conjugate further comprises a drug, a cytokine, a radionuclide or an enzyme linked to the antibody.

[0028] 13. A method for treating or preventing NKp46-mediated related diseases, comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of items 7 to 9 to a user in need of treatment or prevention.

[0029] 14. The method according to item 3, wherein the NKp46-mediated related diseases are selected from one or more of breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, esophageal cancer, pancreatic cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, ovarian cancer, kidney cancer, thyroid cancer, lymphoma, leukemia, endometrial cancer, melanoma, osteosarcoma, follicular lymphoma, relapsed / refractory multiple myeloma, relapsed / refractory light chain amyloidosis, rheumatoid arthritis, and systemic lupus erythematosus.

[0030] 15. Use of the Nanobody or antigen-binding fragment thereof described in any one of items 1 to 3 in the preparation of a product or kit for detecting NKp46.

[0031] 16. Use of the Nanobody according to any one of items 1 to 3 in the preparation of a medicament for treating or preventing NKp46-mediated related diseases.

[0032] 17. The use according to item 16, wherein the NKp46-mediated related diseases are selected from one or more of breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, esophageal cancer, pancreatic cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, ovarian cancer, kidney cancer, thyroid cancer, lymphoma, leukemia, endometrial cancer, melanoma, osteosarcoma, follicular lymphoma, relapsed / refractory multiple myeloma, relapsed / refractory light chain amyloidosis, rheumatoid arthritis, and systemic lupus erythematosus.

[0033] Beneficial effects: The present application provides a NKp46 nanobody or an antigen-binding fragment thereof, which can efficiently bind to human and crab-eating macaque NKp46, promote NK cell activation, accurately regulate NK cell activity, and construct an NK cell therapy platform (NKCE) targeting NKp46; based on the excellent biological properties and strong drugability of the NKp46 nanobody of the present application, it can further promote the treatment of diseases such as follicular lymphoma, relapsed / refractory multiple myeloma (RRMM) and relapsed / refractory light-chain amyloidosis (RRLCA). BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings are used to better understand the present application and do not constitute an improper limitation on the present application.

[0035] Figure 1 This is the ELISA binding curve of NKp46-1 antibody and recombinant human NKp46-His;

[0036] Figure 2 This is the ELISA binding curve of NKp46-1 antibody and recombinant monkey NKp46-his;

[0037] Figure 3 This is the binding curve of NKp46-1 antibody and cells expressing human NKp46;

[0038] Figure 4 Detection of Jurkat-NKp46 activation mediated by 1 antibody for NKp46-1. DETAILED DESCRIPTION

[0039] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0040] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0041] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0042] Generally speaking, the terms used in this specification have the following meanings.

[0043] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or complete immunoglobulins, and can be derived from natural sources or recombinant sources. Antibodies can also be polymers of immunoglobulin molecules. The terms "heavy chain" ("CH"), "light chain" ("CL"), "heavy chain constant region", "light chain constant region", "light chain variable region" ("VL"), "heavy chain variable region" ("VH"), "framework region" ("Framework Region, FR"), and "complementarity determining region" ("CDR") are components of antibodies, and different types of antibodies can be formed through different combinations. For example, conventional IgG antibodies are tetramers composed of two light chains and two heavy chains. Among them, VL and VH are composed of different CDRs and FRs, CDRs contain residues that contact the antigen, determine the antigen specificity of VL and VH, and FRs are used to maintain the variable region structure and determine the position of the CDR loop. Usually, the heavy chain variable region and the heavy chain constant region constitute a complete heavy chain, and the light chain variable region and the light chain constant region constitute a complete light chain.

[0044] In this article, the term "nanoantibody" is used interchangeably with "heavy chain variable region single domain antibody" or "VHH" (variable domain of heavy chain of heavy-chain antibody), that is, a single domain antibody composed only of the variable region of the antibody heavy chain. Because its relative molecular size is at the nanometer level, it is called a nanoantibody. The variable region of the heavy chain antibody of camelids is very similar to the structure of the variable region of the heavy chain of human antibodies, containing three complementary determining regions (CDRs) and a total of four framework regions (FRs) on both sides of each CDR. Nanoantibodies can bind tightly to antigens like normal antibodies, but they are not as easy to adhere to each other and aggregate into clumps as single-chain antibodies. Compared with traditional antibodies, nanoantibodies have the advantages of small relative molecular mass, high affinity, high stability, good solubility, low immunogenicity, strong penetration, and easy transformation and engineering transformation.

[0045] Herein, an "antigen-binding fragment" of a VHH refers to a protein fragment that contains only a portion of an intact VHH, generally including the antigen-binding site of the intact VHH, and thus retains the ability to bind to an antigen.

[0046] As used herein, "fused nanobody" refers to a new fusion molecule formed by combining with other structures, such as genetic engineering technology or chemical reaction. The other structures are selected from, for example, molecules, enzymes, antimicrobial peptides or developing substances that can prolong the half-life of VHH. In the new fusion molecule, the nanobody is directed to bind to its target antigen, and the part fused with the nanobody can play a corresponding function. When used for clinical treatment, it is often necessary for the drug to stay in the body for a long enough time, but the blood clearance rate of the nanobody is very fast, which is not conducive to its function. Therefore, by fusing the nanobody with molecules with a longer life span (such as albumin, antibody Fc end, etc.), the existence time of the nanobody in the blood can be increased, that is, its half-life can be extended, thereby achieving a better therapeutic effect.

[0047] As used herein, the term "HcAb" refers to a functional heavy chain antibody, which is known to be naturally produced by camelids and sharks. Compared to conventional IgG antibodies, HcAb does not contain a light chain polypeptide and is formed as a dimer by two heavy chains lacking the first constant region (CH1). The heavy chain variable region of a single HcAb can constitute VHH.

[0048] As used herein, the term "phagemid" is a vector derived from a filamentous phage, and its basic components mainly include the replication origin, selection marker, and intergenic region (IG region) of the plasmid, and usually also contain the packaging sequence and replication origin of the negative and positive chains, the gene of the phage coat protein, the restriction endonuclease recognition site, the promoter, and the DNA fragment encoding the signal peptide. In addition, the phagemid can also contain a molecular tag to facilitate the screening of the library based on the phagemid. The phagemid cannot independently complete the assembly of the progeny phage particles, and the other structures and functional proteins required to complete its life cycle are provided by the helper phage. The helper phage is a filamentous phage mutant with extremely low efficiency of self-DNA replication. Therefore, when the phage formed by the helper phage and the phagemid package co-infects the host bacteria, a large number of phages containing phagemids can be packaged, while only a small amount of helper phages can be packaged.

[0049] As used in this article, "plasmid" refers to a DNA molecule other than chromosomes (or nucleoids) in organisms such as bacteria, yeast and actinomycetes, which exists in the cytoplasm or nucleus and has the ability to replicate autonomously, so that it can maintain a constant copy number in daughter cells and express the genetic information it carries.

[0050] The term "vector" refers to a vector by which a polynucleotide sequence (e.g., a foreign gene) can be introduced into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phage vectors, viral vectors, etc. Among them, a "viral vector" is a vector transformed from a viral genome, which introduces foreign genes into host cells through viral infection.

[0051] As used herein, the term "specific binding" refers to a binding affinity (equilibrium dissociation constant KD for antigen-antibody binding) of at least 10 -6 In some aspects, the antibody is present in an amount of at least about 10 -7 M, preferably 10 -8 M, 10 - 9 M, 10 -10 M, 10 -11 M or 10 -12 Affinity binding of M.

[0052] In this application, the terms "polynucleotide" or "nucleic acid", "nucleic acid molecule" are used interchangeably and include but are not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (micro RNA), genomic DNA, synthetic DNA, synthetic RNA and / or tRNA.

[0053] As used herein, the "identity" of a sequence refers to the degree of similarity between amino acid sequences or between nucleotide sequences as determined by sequence alignment software, such as BLAST.

[0054] In this specification, "conjugate" refers to an antibody or antigen-binding fragment thereof connected to another agent such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, etc. The connection can be a covalent bond or a non-covalent interaction, such as by electrostatic forces. A variety of linkers known in the art can be used to form a conjugate. Alternatively, the conjugate can be provided in the form of a fusion protein expressed by a polynucleotide encoding the conjugate.

[0055] In this specification, the NKCE molecule is mainly composed of a nanobody targeting NKp46 on the surface of NK cells and a specific antibody targeting an antigen on the surface of the tumor cell membrane. The specific antibody targeting an antigen on the surface of the tumor cell membrane can be in the form of Fab, scFv, nanobody or other forms. The antibodies targeting NK receptors and targeting tumor antigens are connected with a suitable flexible chain (Linker), and then connected to the Fc region or other proteins (including peptides); or directly connected to different positions of the Fc region or other proteins (including peptides) to form bispecific or multispecific antibodies. Linker can be selected from (G4S / G3S)n, 218s, HMA or other forms. The NKCE molecule can retain the Fc region, or lack the Fc region; have other proteins (including peptides) such as HAS, or none.

[0056] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0057] Antibody

[0058] The present application discloses an anti-NKp46 nanobody, characterized in that the nanobody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3).

[0059] In a specific embodiment of the present application, CDR-H1 has an amino acid sequence of SEQ ID NO: 2: GRTFSNYA, CDR-H2 has an amino acid sequence of SEQ ID NO: 3: ISRSGDT, and CDR-H3 has an amino acid sequence of SEQ ID NO: 4: AAGPSLYATNIFTAGRWYDY.

[0060] In a specific embodiment of the present application, the antibody comprises a heavy chain variable region VHH, wherein: VHH has the sequence

[0061] SEQ ID NO: 1:

[0062] The amino acid sequence of EVQLVESGGGLVQAGASLTLSCLVPGRTFSNYAMGWFRQAPGKDRDVVAAISRSGDTFYIDSVKGRFTISRDDAKNTVYLQMNSLKPEDKALYYCA AGPS LYATNIFTAGRWYDYWGQGTQVTVSS, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 1.

[0063] In a specific embodiment of the present application, the antibody comprises a heavy chain, wherein the amino acid sequence of the heavy chain has the sequence SEQ ID NO: 5:

[0064] EVQLVESGGGLVQAGASLTLSCLVPGRTFSNYAMGWFRQAPGKDRDVVAAISRSGDTFYIDSVKGRFTISRDDAKNTVYLQMNSLKPEDKALYYCAAGPSLYATNIFTAGRWYDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC The amino acid sequence of LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, or the same as SEQ ID NO:5 has an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.

[0065] In a specific embodiment of the present application, the antibody is a camel-derived antibody.

[0066] In a specific embodiment of the present application, the antibody is a camel-derived nanobody.

[0067] In a specific embodiment of the present application, the antibody is an alpaca-derived nanobody.

[0068] Nucleic acid molecules, vectors, host cells

[0069] The present application provides an isolated nucleic acid comprising a nucleic acid sequence encoding any of the aforementioned antibodies.

[0070] In a specific embodiment of the present application, the nucleic acid molecule is an RNA or DNA molecule.

[0071] In a specific embodiment of the present application, the nucleic acid is single-stranded or double-stranded.

[0072] In a specific embodiment of the present application, the nucleic acid is linear or circular. The nucleic acid can be synthesized by cells or chemically synthesized.

[0073] In a specific embodiment of the present application, the nucleic acid is a chemically modified nucleic acid molecule.

[0074] The present application also provides a vector comprising the above-mentioned nucleic acid.

[0075] In a specific embodiment of the present application, the vector is a plasmid.

[0076] The present application also provides a host cell, which comprises the above-mentioned nucleic acid or vector.

[0077] Pharmaceutical compositions, conjugates and methods of treatment

[0078] The present application provides a pharmaceutical composition, which comprises any one of the above-mentioned anti-NKp46 nanobodies, or the above-mentioned nucleic acid-expressed antibodies, or the above-mentioned vector-prepared antibodies, or the above-mentioned host cell-produced antibodies, and a pharmaceutically acceptable carrier.

[0079] The present application also provides a pharmaceutical composition for treating or preventing NKp46-mediated related diseases. In some embodiments, the pharmaceutical composition comprises the aforementioned nano antibody. In some embodiments, the pharmaceutical composition comprises a complex of the nano antibody or its antigen-binding fragment. In some embodiments, the pharmaceutical composition comprises a nucleic acid molecule encoding the aforementioned nano antibody or its antigen-binding fragment. In some embodiments, the pharmaceutical composition comprises the virus, bacteriophage or cell. In some specific embodiments, the virus, bacteriophage or cell comprises a nucleic acid encoding the nano antibody or its antigen-binding fragment, and after entering the animal body and reaching the target organ, the nano antibody is expressed.

[0080] The pharmaceutical composition can be used to treat or prevent NKp46-mediated related diseases.

[0081] The present application also provides a conjugate, which comprises any one of the above-mentioned anti-NKp46 nanoantibodies, or the above-mentioned nucleic acid-expressed antibodies, or the above-mentioned vector-prepared antibodies, or the above-mentioned host cell-produced antibodies.

[0082] Furthermore, the conjugate further comprises a drug, a cytokine, a radionuclide or an enzyme connected to the antibody or the antigen-binding fragment thereof.

[0083] The pharmaceutical composition of the present application can be administered alone or in combination with one or more therapeutic agents (or second therapeutic agents). In the combined administration described herein, the pharmaceutical composition herein can be administered before, after or simultaneously with one or more additional agents.

[0084] The present application also provides use of the above monoclonal antibody in preparing a drug for treating or preventing NKp46-mediated related diseases.

[0085] Detection Kits

[0086] The present application also provides a kit for detecting NKp46. In some specific embodiments, the kit is an in vitro detection kit. In some embodiments, the kit is an immunoassay kit, such as an ELISA kit or an immunohistochemistry kit, which contains the aforementioned nano antibody or antigen-binding fragment of NKp46. In some embodiments, the kit is an in vivo non-invasive diagnostic kit, which contains the aforementioned conjugate. In some embodiments, the in vivo non-invasive diagnostic kit can be used for an imaging method selected from the following: radioimmunoimaging or targeted ultrasound angiography. When used for radioimmunoimaging, the complex is a conjugate of the aforementioned nano antibody or antigen-binding fragment of NKp46 and a radionuclide. When used for targeted ultrasound angiography, the complex is a conjugate of the aforementioned nano antibody or antigen-binding fragment of NKp46 and an ultrasound contrast agent.

[0087] In a preferred embodiment, the NKp46 contained in the detection kit is a nanobody. In the immunoassay kit, due to the small size of the nanobody, it can be firmly bound to the solid phase carrier at a high density to capture trace amounts of antigens, thereby improving the sensitivity of the detection. In the in vivo non-invasive diagnostic kit, on the one hand, the nanobody has a short half-life in the body, is easy to metabolize and quickly clear, and thus can reduce the time that the contrast agent or imaging agent complexed with it remains in the body to improve safety; on the other hand, the nanobody has good tissue permeability and a high signal-to-noise ratio due to its small molecule, good penetrability, and high affinity.

[0088] In the present application, human and cynomolgus monkey NKp46 proteins are used as immunogens to immunize alpacas, with two human antigens, one monkey antigen, and then one human antigen in order, each immunization protein amount is 0.5 mg, the immunization interval is two weeks, and the titer of four immunizations meets the requirements. Total RNA is extracted from alpaca peripheral blood and reverse transcribed to obtain cDNA, and the variable region is amplified using specific primers to construct an antibody library. Human or monkey NKp46-His protein is used for screening to obtain monoclonal antibodies that simultaneously bind to human and monkey NKp46 antigens. Monoclonal phages are sequenced to obtain antibody variable region sequences, and alpaca monoclonal antibodies are transformed into recombinant antibodies with Fc regions of hIgG1 (L234A / L235A) subtypes through in vitro recombinant expression. ELISA and FACS prove the binding of NKp46-1 antibodies to human and monkey antigens and to cells expressing human NKp46; Luciferase reporter cell line activation experiments prove that antibody NKp46-1 can induce NKp46 activation of cells.

[0089] Example 1

[0090] Antibodies bind to human or cynomolgus monkey NKp46

[0091] In this example, the variable region protein sequence obtained by phage library screening was combined with the hIgG1 (L234A / L235A) Fc sequence to obtain the full-length NKp46-1 antibody sequence. A DNA sequence was synthesized according to the antibody protein sequence, an expression vector was constructed, and the antibody was expressed in CHO cells. After protein A affinity purification, a recombinant antibody with an Fc region of hIgG1 (L234A / L235A) subtype was finally prepared.

[0092] The ELISA method is used to verify the binding of candidate antibodies to human or cynomolgus monkey NKp46 molecules. The steps are as follows:

[0093] (1) Protein coating: Coat the plate with antigen protein at a concentration of 2 μg / mL, 100 μL / well, incubate at 37°C for 1 h, wash the plate 6 times, and pat dry.

[0094] (2) Blocking: Use 3% BSA-PBS blocking solution, 200 μL / well, incubate at 25°C for 1 h, wash the plate 6 times, and pat dry.

[0095] (3) Primary antibody incubation: The first well of the sample to be tested is diluted 5-fold with blocking solution at 100 nM, and eight gradients are performed. The last well is added with blocking solution as a blank, 100 μL / well, incubated at 25°C for 1 h, washed 6 times, and patted dry.

[0096] (4) Secondary antibody incubation: The secondary antibody (Invitrogen, A18817) was diluted with blocking buffer, 100 μL / well, reacted at 25°C for 1 h, washed 6 times, and patted dry.

[0097] (5) Color development: Add 100 μL / well TMB (Invitrogen, 002023) and develop color at room temperature in the dark for 3-5 min.

[0098] (6) Termination: Add 50 μL / well ELISA stop solution (BBI, E661066-0500).

[0099] (7) Reading: Read OD450 using a microplate reader.

[0100] (8) Data processing: The data were fitted using the log(agonist) vs. response--Variable slope (four parameters) equation of GraphPad Prism 8.

[0101] The binding effect of the antibody on human or cynomolgus monkey NKp46 molecules was verified by ELISA method. The results are as follows Figure 1 and Figure 2 As shown, antibody NKp46-1 binds to recombinant human NKp46-His protein (ARCO, NC1-H52H4) and recombinant cynomolgus monkey NKp46-His protein (ARCO, NC1-C52H4).

[0102] Example 2

[0103] Antibody binding to cells expressing human NKp46

[0104] In this example, the variable region protein sequence obtained by phage library screening was combined with the hIgG1(LALA) Fc sequence to obtain the full-length NKp46-1 antibody sequence. A DNA sequence was synthesized according to the antibody protein sequence, an expression vector was constructed, and the antibody was expressed in CHO cells. After protein A affinity purification, a recombinant antibody with an Fc region of hIgG1(LALA) subtype was finally prepared.

[0105] The detection protocol for detecting the binding of candidate antibodies to human NKp46 cells by flow cytometry on Jurkat cells expressing human NKp46 is as follows:

[0106] (1) Collect Jurkat cells and adjust the cell density to 3×10 6 cells / mL, 50 μl / well was added to a 96-well plate.

[0107] (2) The antibody was diluted 3-fold with 0.5% BSA-PBS diluent, with a final concentration of 100 nM in the first well. The dilution was diluted 8 times and the dilution in the last well was used as a blank. The dilution was added to a 96-well plate at 50 μL / well and incubated at 4°C for 1 h.

[0108] (3) Centrifuge at 400 g and 4°C for 5 min, discard the supernatant, and wash twice with 150 μL of 0.5% BSA-PBS diluent.

[0109] (4) Add 100 μL of diluted fluorescent secondary antibody (Invitrogen, A18818).

[0110] (5) Incubate at 4°C for 30 min.

[0111] (6) Centrifuge at 400 g and 4°C for 5 min, discard the supernatant, and wash twice with 150 μL of 0.5% BSA-PBS diluent.

[0112] (7) Resuspend in 100 μL 0.5% BSA-PBS diluent and load onto an analyzer (Attune NxT, Invitrogen).

[0113] (8) Data processing: The data were fitted using the log(agonist) vs. response--Variable slope (four parameters) equation of GraphPad Prism 8.

[0114] The antibody binding activity to Jurkat cells expressing human NKp46 was verified by flow cytometry. Figure 3 As shown, the antibody NKp46-1 has an excellent binding effect on cells expressing human NKp46.

[0115] Example 3

[0116] NKp46-1 antibody-mediated Jurkat-NKp46 activation assay

[0117] The method of Example 2 was used to prepare a recombinant antibody having an Fc region of hIgG1LALA subtype.

[0118] Based on the activation validation of the Jurkat-NKp46-luciferase reporter cell line, the detection antibody pair mediated cell activation detection scheme has the following steps:

[0119] (1) NKp46-1 was diluted to 100 nM with DPBS, 100 μl / well was added to a 96-well plate, and incubated at 4°C overnight.

[0120] (2) Wash the 96-well plate three times with DPBS and dry it.

[0121] (3) Jurkat reporter cells were collected, resuspended in culture medium to 1E6 / ml, 200 μl / well, and incubated at 37°C for 24 h.

[0122] (4) Take 20 μl of supernatant and add 50 μl of detection substrate (InvivoGen, rep-qlc4lg1) to a white opaque 96-well plate, and use an ELISA reader to detect the fluorescence intensity.

[0123] (5) Data processing: The data were fitted using a conventional bar graph in GraphPad Prism 8.

[0124] The activation effect of the antibody on NKp46 was verified based on Jurkat-NKp46-luciferase activation, and the results were shown in Figure 4 In this study, the results showed that the antibody NKp46-1 activated cells via NKp46.

Claims

1. A nanobody, characterized in that The nanobody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3), wherein: CDR-H1 has an amino acid sequence of SEQ ID NO 2: GRTFSNYA, CDR-H2 has an amino acid sequence of SEQ ID NO 3: ISRSGDT, CDR-H3 has an amino acid sequence of SEQ ID NO 4: AAGPSLYATNIFTAGRWYDY.

2. The Nanobody according to claim 1, characterized in that The Nanobody comprises an antibody heavy chain variable region VHH, wherein: VHH has the sequence SEQ ID NO: 1: The amino acid sequence of EVQLVESGGGLVQAGASLTLSCLVPGRTFSNYAMGWFRQAPGKDRD VVAAISRSGDTFYIDSVKGRFTISRDDAKNTVYLQMNSLKPEDKALYYCA AGPS LYATNIFTAGRWYDYWGQGTQVTVSS, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO:

1.

3. The Nanobody according to claim 1, characterized in that The Nanobody comprises a heavy chain, wherein: The heavy chain amino acid sequence has the sequence SEQ ID NO: 5: EVQLVESGGGLVQAGASLTLSCLVPGRTFSNYAMGWFRQAPGKDRDVVAAISRSGDTFYIDSVKGRFTISRDDAKNTVYLQMNSLKPEDKALYYCAAGPSLYATNIFTAGRWYDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC The amino acid sequence of LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, or the same as SEQ ID NO:5 has an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.

4. A nucleic acid molecule comprising a sequence encoding the Nanobody according to any one of claims 1 to 3. A vector comprising the nucleic acid according to claim 4 . A host cell comprising the nucleic acid of claim 4 or the vector of claim 5.

7. A pharmaceutical composition, wherein: The pharmaceutical composition comprises: The Nanobody of any one of claims 1 to 3, or the antibody expressed by the nucleic acid of claim 4, or the antibody prepared by the vector of claim 5, or the antibody produced by the host cell of claim 6, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, wherein The pharmaceutical composition is used to treat or prevent NKp46-mediated related diseases. Preferably, the NKp46-mediated related diseases are selected from one or more of breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, esophageal cancer, pancreatic cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, ovarian cancer, kidney cancer, thyroid cancer, lymphoma, leukemia, endometrial cancer, melanoma, osteosarcoma, follicular lymphoma, relapsed / refractory multiple myeloma, relapsed / refractory light chain amyloidosis, rheumatoid arthritis, and systemic lupus erythematosus.

9. A product or kit for detecting NKp46, wherein: Comprising the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 3.

10. A conjugate, wherein: The conjugate comprises: The Nanobody according to any one of claims 1 to 3, or the antibody expressed by the nucleic acid according to claim 4, or the antibody prepared by the vector according to claim 5, or the antibody produced by the host cell according to claim 6, preferably, the conjugate further comprises a drug, cytokine, radionuclide or enzyme linked to the antibody.

11. A method for treating or preventing NKp46-mediated related diseases, comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 7 to 9 to a user in need of treatment or prevention, preferably, the NKp46-mediated related diseases are selected from one or more of breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, esophageal cancer, pancreatic cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, ovarian cancer, kidney cancer, thyroid cancer, lymphoma, leukemia, endometrial cancer, melanoma, osteosarcoma, follicular lymphoma, relapsed / refractory multiple myeloma, relapsed / refractory light chain amyloidosis, rheumatoid arthritis, and systemic lupus erythematosus.

12. Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a product or kit for detecting NKp46.

13. Use of a Nanobody according to any one of claims 1 to 3 in the preparation of a medicament for treating or preventing NKp46-mediated related diseases, preferably, the NKp46-mediated related diseases are selected from one or more of breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, esophageal cancer, pancreatic cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, ovarian cancer, kidney cancer, thyroid cancer, lymphoma, leukemia, endometrial cancer, melanoma, osteosarcoma, follicular lymphoma, relapsed / refractory multiple myeloma, relapsed / refractory light chain amyloidosis, rheumatoid arthritis, and systemic lupus erythematosus.

Images