Influenza B N protein specific antibody as well as preparation method and application thereof

Abstract

The invention provides a nano antibody specifically binding to influenza B N protein or an antigen binding fragment thereof, which can be used for clinical diagnosis to help doctors to quickly recognize infected cases so as to formulate a more effective treatment scheme. In a word, development and application of the influenza B virus antibody not only promote progress of influenza virus research, but also provide important support for prevention and control measures in the public health field.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to influenza B N protein-specific antibodies and preparation methods and applications thereof. Background Art

[0002] Influenza B virus is a type of influenza virus that mainly infects humans and belongs to the family Orthomyxoviridae. It can cause influenza. The incubation period of influenza B virus is usually 1 to 4 days. Recurrent fever is the most common symptom, and high fever can even reach 39℃~40℃, accompanied by chills, chills, headache, body aches, fatigue, loss of appetite and other symptoms; respiratory symptoms are generally mild, usually manifested as dry throat, sore throat, dry cough and other symptoms. In addition to typical symptoms, some children with influenza B may also have diarrhea. Influenza B virus usually has a more obvious impact on humans, especially in children and the elderly, and its potential complications need to be given enough attention.

[0003] As the study of influenza B virus continues to deepen, people have a clearer understanding of its structure. The structure of influenza B virus mainly consists of 8 single-stranded negative-sense RNAs that form the viral genome, a double-layer lipid envelope, a nucleoprotein (NP) that wraps RNA, and other proteins. As an important component of the virus structure, NP plays an important role in the replication and transcription of the virus. The main functions include: binding to the negative-sense single-stranded RNA of the virus to form a ribonucleoprotein complex (RNP) to protect RNA from degradation. During the transcription and replication of the virus, NP can help the viral RNA polymerase recognize and bind to the RNA template, thereby promoting the transcription and replication of viral genes. In addition, during the viral assembly process, NP and other structural proteins participate in the formation of viral particles to ensure the integrity and infectivity of the virus. Finally, as a strong immunogenic protein, NP can activate the host's immune system and induce the production of specific antibodies. Due to its high conservatism, NP is relatively consistent in different subtypes of influenza viruses. Therefore, the development of antibodies against the NP protein of influenza B virus has become an important research direction in the field of public health, and is expected to provide new ideas for the prevention and detection of influenza and its related complications. In conclusion, the nucleoprotein of influenza B virus plays an important role in the life cycle of the virus, and the antibodies produced by it are key to detecting and resisting viral infection. A deeper understanding of the function of nucleoprotein and the immune response it induces will help develop more effective influenza vaccines and treatment strategies to meet the challenges of influenza epidemics.

[0004] In the process of studying influenza B virus, antibodies are a very important research tool, especially for the prevention of influenza, and have great value and significance. Summary of the invention

[0005] To solve at least one of the above problems, the present disclosure provides an antibody specific for influenza B N protein. Using the specific antibody provided by the present disclosure, it is capable of specifically recognizing and binding to influenza B N protein and having a good affinity for it.

[0006] According to a first aspect of the present disclosure, there is provided a nanobody specifically binding to influenza B N protein or an antigen-binding fragment thereof.

[0007] In some embodiments, the nanobody specifically binding to influenza B N protein or an antigen-binding fragment thereof includes CDR-H1, CDR-H2, and CDR-H3 included in the heavy chain variable region having the amino acid sequences shown in SEQ ID NOs: 23 to 30.

[0008] In some embodiments, each of the CDRs is defined by any numbering system conventionally used by those skilled in the art. Exemplary numbering systems include, but are not limited to, Kabat, AbM, Chothia, Contact, IMGT, or a combination thereof.

[0009] In some embodiments, for the nanobody specifically binding to influenza B N protein or an antigen-binding fragment thereof, the nanobody specifically binding to influenza B N protein or an antigen-binding fragment thereof includes:

[0010] a1) a heavy chain variable region including the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 2, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 3;

[0011] a2) a heavy chain variable region including the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 4, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 5, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 6;

[0012] a3) a heavy chain variable region including the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 7, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 8, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 9;

[0013] a4) a heavy chain variable region including the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 10, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 11, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 12;

[0014] a5) The heavy chain variable region comprising the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO:13, CDR-H2 having the amino acid sequence shown in SEQ ID NO:14, and CDR-H3 having the amino acid sequence shown in SEQ ID NO:15;

[0015] a6) The heavy chain variable region comprising the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO:16, CDR-H2 having the amino acid sequence shown in SEQ ID NO:17, and CDR-H3 having the amino acid sequence shown in SEQ ID NO:18;

[0016] a7) The heavy chain variable region comprising the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO:19, CDR-H2 having the amino acid sequence shown in SEQ ID NO:20, and CDR-H3 having the amino acid sequence shown in SEQ ID NO:21; or

[0017] a8) The heavy chain variable region comprising the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO:4, CDR-H2 having the amino acid sequence shown in SEQ ID NO:5, and CDR-H3 having the amino acid sequence shown in SEQ ID NO:22.

[0018] In some embodiments, the heavy chain variable region of the nanobody specifically binding to influenza B N protein or its antigen-binding fragment further comprises the framework region of the heavy chain variable region.

[0019] In some embodiments, the framework region of the heavy chain variable region comprises the framework region of the heavy chain variable region of an immunoglobulin derived from a mouse, primate, bovine, equine, porcine, ovine, caprine, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck or goose or a mutant thereof.

[0020] In some embodiments, the nanobody specifically binding to influenza B N protein or its antigen-binding fragment comprises:

[0021] b1) A heavy chain variable region, which comprises the amino acid sequence shown in SEQ ID NO:23, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity therewith;

[0022] b2) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:24, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto;

[0023] b3) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto;

[0024] b4) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:26, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto;

[0025] b5) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:27, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto;

[0026] b6) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:28, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto;

[0027] b7) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:29, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; or

[0028] b8) A heavy chain variable region, which comprises the amino acid sequence shown in SEQ ID NO:30, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0029] In some embodiments, the influenza B N protein is a human influenza B N protein; further, it is an influenza B N protein with an amino acid sequence as shown in SEQ ID NO:39.

[0030] According to the second aspect of the present disclosure, there is provided a heavy chain antibody specifically binding to an influenza B N protein or an antigen-binding fragment thereof, which comprises an immunoglobulin Fc domain and the nanobody of the first aspect of the present disclosure or an antigen-binding fragment thereof.

[0031] In some embodiments, the immunoglobulin Fc domain comprises an Fc domain of an immunoglobulin derived from a mouse, a primate, a bovine, a horse, a bovine, a pig, a sheep, a goat, a dog, a cat, a rabbit, a camel, a donkey, a deer, a mink, a chicken, a duck or a goose, or a mutant thereof.

[0032] According to the third aspect of the present disclosure, there is provided a chimeric antigen receptor, which comprises an antigen-binding domain, a transmembrane domain and an intracellular signaling domain, and the antigen-binding domain comprises the nanobody of the first aspect of the present disclosure or an antigen-binding fragment thereof, or the heavy chain antibody of the second aspect of the present disclosure or an antigen-binding fragment thereof.

[0033] According to the fourth aspect of the present disclosure, there is provided a multispecific antibody or an antigen-binding fragment thereof, which comprises two or more (such as three or four) antigen-binding domains, and one of the antigen-binding domains comprises the nanobody of the first aspect of the present disclosure or an antigen-binding fragment thereof, or the heavy chain antibody of the second aspect of the present disclosure or an antigen-binding fragment thereof.

[0034] According to the fifth aspect of the present disclosure, there is provided an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the nanobody of the first aspect of the present disclosure or an antigen-binding fragment thereof, the heavy chain antibody of the second aspect of the present disclosure or an antigen-binding fragment thereof, the chimeric antigen receptor of the third aspect, or the multispecific antibody of the fourth aspect or an antigen-binding fragment thereof.

[0035] Those skilled in the art should understand that the nucleotides in the nucleic acid molecule can be replaced according to codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0036] In some embodiments, the nucleotide sequence encoding the nanobody or its antigen-binding fragment of the first aspect of the present disclosure includes: SEQ ID NO: 31-38, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0037] According to the sixth aspect of the present disclosure, there is provided a vector comprising the nucleic acid molecule of the fifth aspect of the present disclosure.

[0038] In some embodiments, the vector may be an expression vector. In some embodiments, the expression vector may include eukaryotic cell expression vectors and / or prokaryotic cell expression vectors. In some embodiments, the eukaryotic expression vectors include, for example, but are not limited to yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, phage vectors, adenoviral vectors, adeno-associated vectors, or herpes simplex vectors.

[0039] In some embodiments, the vector may be selected from nanoparticles, liposomes, exosomes, microbubbles, or gene guns.

[0040] According to the seventh aspect of the present disclosure, there is provided a cell comprising the nanobody or its antigen-binding fragment of the first aspect of the present disclosure, the heavy-chain antibody or its antigen-binding fragment of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or its antigen-binding fragment of the fourth aspect, the nucleic acid molecule of the fifth aspect, or the vector of the sixth aspect.

[0041] In some embodiments, the cell does not relate to propagation materials.

[0042] In some embodiments, the cell may be a host cell conventionally used in the art, as long as it can stably express the nucleic acid molecule carried by the expression vector as the above-mentioned nanobody or its antigen-binding fragment, heavy-chain antibody or its antigen-binding fragment, chimeric antigen receptor, or multispecific antibody or its antigen-binding fragment of the present disclosure. In some embodiments, the host cell may be a prokaryotic cell and / or a eukaryotic cell. Examples of the prokaryotic cell may include Escherichia coli, and examples of the eukaryotic cell may include CHO cells, HEK293 cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0043] In some embodiments, the cell can be an immune cell. In some embodiments, the immune cell can include, but is not limited to, T cells, NK cells, DC cells, and macrophages. In these embodiments, the immune cell can express the above-mentioned chimeric antigen receptor of the present disclosure (i.e., a modified immune cell).

[0044] According to the eighth aspect of the present disclosure, there is provided a method for preparing the nanobody or its antigen-binding fragment of the first aspect of the present disclosure, the heavy-chain antibody or its antigen-binding fragment of the second aspect, the chimeric antigen receptor of the third aspect, or the multispecific antibody or its antigen-binding fragment of the fourth aspect, which is obtained by culturing the cell of the seventh aspect of the present disclosure.

[0045] According to the ninth aspect of the present disclosure, there is provided a conjugate, which includes the nanobody or its antigen-binding fragment of the first aspect of the present disclosure, or the heavy-chain antibody or its antigen-binding fragment of the second aspect; and a conjugate part.

[0046] In some embodiments, the conjugate part can include, but is not limited to, a detectable marker or a therapeutic agent.

[0047] In some embodiments, the detectable marker can be any substance that can be detected by means such as fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrochemistry, optics, chemistry, etc. Such markers are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers can be applied to immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable marker as described above can be linked to the nanobody or its antigen-binding fragment, or the heavy-chain antibody or its antigen-binding fragment of the present disclosure through linkers of different lengths to reduce potential steric hindrance.

[0048] In some embodiments, the detectable label may include, but is not limited to, enzymes (such as horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (such as chemiluminescent substances), colored substances, biotin, and the like.

[0049] In some embodiments, the therapeutic agent may include, for example, but is not limited to, chemotherapeutic agents, immunosuppressive agents, cytotoxic drugs.

[0050] In some embodiments, the coupling moiety is selected from substances that can improve the biological properties of the antibody (such as increasing the serum half-life), and may be, for example, chemical groups such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl.

[0051] According to the tenth aspect of the present disclosure, there is provided a pharmaceutical composition comprising: the nanobody or its antigen-binding fragment of the first aspect of the present disclosure, the heavy-chain antibody or its antigen-binding fragment of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or its antigen-binding fragment of the fourth aspect, the nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the cell of the seventh aspect, or the conjugate of the ninth aspect; and a pharmaceutically acceptable carrier.

[0052] In some embodiments, the pharmaceutical composition may further include an additional pharmaceutically active agent.

[0053] In some embodiments, the additional pharmaceutically active agent may be a biologically active drug, such as a drug capable of treating influenza B.

[0054] In some embodiments, the antibody or its antigen-binding fragment and the additional pharmaceutically active agent are provided as separate components or as a mixed component.

[0055] In some embodiments, the pharmaceutical composition may be administered, for example, by parenteral, subcutaneous injection, sublingual, rectal, nasal, intravenous injection, intramuscular injection, oral, ocular, topical, and other means.

[0056] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, suspension, powder, tablet, capsule, granule, powder, pill, disintegrant, syrup, spray, gel, emulsion, injection, elixir, lozenge, suppository, and the like.

[0057] According to the eleventh aspect of the present disclosure, there is provided a diagnostic or therapeutic kit, which comprises: the nanobody of the first aspect of the present disclosure or its antigen-binding fragment, the heavy-chain antibody of the second aspect or its antigen-binding fragment, the chimeric antigen receptor of the third aspect, the multispecific antibody of the fourth aspect or its antigen-binding fragment, the nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the cell of the seventh aspect, the conjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect.

[0058] In some embodiments, the kit may further comprise instructions and / or a dosing device.

[0059] In some embodiments, the kit can be used for diagnosing influenza B.

[0060] In some embodiments, the kit can be used for preventing or treating influenza B.

[0061] According to the twelfth aspect of the present disclosure, there is provided the use of the nanobody of the first aspect of the present disclosure or its antigen-binding fragment, the heavy-chain antibody of the second aspect or its antigen-binding fragment, the chimeric antigen receptor of the third aspect, the multispecific antibody of the fourth aspect or its antigen-binding fragment, the nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the cell of the seventh aspect, the conjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect in the preparation of a product for any one of c1)-c3):

[0062] c1) Diagnosing influenza B;

[0063] c2) Preventing or treating influenza B;

[0064] c3) Detecting the presence or level of influenza B N protein in a sample.

[0065] In some embodiments, the sample is selected from at least one of the body fluids, tissues, cells, and excreta of a subject to be tested.

[0066] In some embodiments, the body fluid comprises at least one of blood and lymph fluid.

[0067] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0068] In some embodiments, the excreta comprises at least one of urine, feces, and tears.

[0069] In some embodiments, the subject to be tested includes mammals, such as humans, non-human primates (such as orangutans, apes), rodents (such as rats, mice, guinea pigs), pets (such as cats, dogs), and livestock (such as horses, cows, sheep, pigs, rabbits).

[0070] In some embodiments, the object to be measured includes humans.

[0071] Beneficial effects:

[0072] The present disclosure provides a nanobody specifically binding to the N protein of influenza B virus or an antigen-binding fragment thereof, which will provide a powerful tool for researchers and contribute to the in-depth study of the functions and regulatory mechanisms of influenza virus in multiple biological processes such as infection, immune escape, and pathological mechanisms. This will help better understand the infection mechanism of influenza virus, provide new directions and targets for the prevention and treatment strategies of influenza B virus, and also provide rich materials for basic biological research. Through the research on influenza B virus antibodies, scientists can identify and verify the antigenic characteristics of the virus, evaluate the neutralizing ability of the antibodies, and explore their application potential in vaccine development. In addition, antibodies against influenza virus can also be used for clinical diagnosis to help doctors quickly identify infection cases, thereby formulating more effective treatment plans. In summary, the development and application of influenza B virus antibodies not only promote the progress of influenza virus research but also provide important support for the prevention and control measures in the public health field. Brief Description of the Drawings

[0073] Figure 1 Shows the affinity determination results of the nanobody 2C2 against the nucleoprotein of influenza B virus.

[0074] Figure 2 Shows the affinity determination results of the nanobody 4A1 against the nucleoprotein of influenza B virus.

[0075] Figure 3 Shows the affinity determination results of the nanobody 2E4 against the nucleoprotein of influenza B virus.

[0076] Figure 4 Shows the affinity determination results of the nanobody 2H3 against the nucleoprotein of influenza B virus.

[0077] Figure 5 Shows the affinity determination results of the nanobody 2A7 against the nucleoprotein of influenza B virus.

[0078] Figure 6 Shows the affinity determination results of the nanobody 3G5 against the nucleoprotein of influenza B virus.

[0079] Figure 7 Shows the affinity determination results of the nanobody 2A6 against the nucleoprotein of influenza B virus.

[0080] Figure 8 Shows the affinity determination results of the nanobody 6H11 against the nucleoprotein of influenza B virus. Detailed Embodiments

[0081] The present invention aims to develop nanobodies based on the alpaca immune system, design and implement an effective and feasible technical scheme for screening and preparing nanobodies, and obtain nanobodies that can specifically recognize the nucleoprotein of influenza B virus.

[0082] Definitions

[0083] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0084] Unless the context clearly indicates otherwise, the expressions "a" and "an" as used herein include plural referents.

[0085] The expression "about" as used herein is as understood by those of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If those of ordinary skill in the art do not understand the use of this term according to the context in which it is used, "about" will mean a specific value plus or minus 10%.

[0086] The terms "variable region" or "variable domain" as used herein refer to the domains of the heavy or light chains of an antibody that participate in the binding of the antigen-binding molecule to an antigen. The variable domains of the heavy and light chains of a natural antibody (VH and VL, respectively) generally have a similar structure, each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The term "variable" in the present invention means that certain segments of the variable domain are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three segments within the variable domains of the light and heavy chains called hypervariable regions (HVRs). The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions, mostly adopting a β-sheet configuration, connected by three HVRs, which form loop connections and, in some cases, part of the β-sheet structure. The HVRs in each chain are held tightly together by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. The constant domain does not directly participate in the binding of the antibody to the antigen and has other effector functions, such as participating in antibody-dependent cell cytotoxicity.

[0087] The term "nanobody" as used herein"Also known as single-domain antibody (sdAb), heavy-chain single-domain antibody (VHH), or camelid antibody, it is an antibody naturally lacking a light chain in the peripheral blood of camelids. This antibody contains only one variable heavy-chain region (VH) and two conventional CH2 and CH3 regions. The variable heavy-chain region includes four conserved framework regions (FR) and three hypervariable regions (HVR), or three Complementarity Determining Regions (CDR). However, nanobodies are not as prone to sticking to each other or even aggregating into clumps as artificially engineered single-chain antibody fragments. The individually cloned and expressed VHH structure has structural stability comparable to that of the original heavy-chain antibody and binding activity to antigens, and is the smallest known unit capable of binding to target antigens. The VHH crystal is 2.5 nm in size, 4 nm in length, and has a molecular weight of only about 15 kD, so it is also called Nanobody (Nb). Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the antigen surface, the immune system in camelids can recognize complex spatial structures on the antigen surface and produce highly specific and high-affinity nanobodies.

[0088] Different from traditional technologies that rely on classic model animals such as mice, rabbits, monkeys, and sheep, the technical solution of the present disclosure relies on the antibodies produced by the immune system of alpacas and is called "nanobody". Nanobodies are tiny antibody fragments isolated from immunoglobulins in animals such as camels. It has the same antigen-binding ability and structural stability as intact antibodies, is the smallest existing unit capable of binding to target antigens, and has a relative molecular mass of only about 15 kD. Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the antigen surface, the immune system in animals such as alpacas can recognize complex spatial structures on the antigen surface and produce highly specific and high-affinity nanobodies.

[0089] According to the technical solution of the present disclosure, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein. See Table 1 below:

[0090] Table 1

[0091]

[0092] In addition, due to the degeneracy of bases, the bases of the polynucleotide sequence can be substituted without changing the activity or function of the polynucleotide sequence. See Table 2 below:

[0093] Table 2

[0094]

[0095]

[0096] In the present disclosure, the term "affinity" or "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (such as an antibody) and its binding ligand (such as an antigen). Binding affinity can generally be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Therefore, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance (SPR). The smaller the equilibrium dissociation constant, the tighter the binding between the antibody or its antigen-binding fragment of the present disclosure and the influenza B N protein to each other. In certain embodiments, the dissociation constant (KD) of the molecule binding to the antigen is ≤10 nM or ≤100 nM.

[0097] In the present disclosure, the term "specific binding" means having binding selectivity for an antigen and being distinguishable from unwanted or non-specific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (such as surface plasmon resonance (SPR) techniques and traditional binding assays). In one embodiment, for example, as measured by SPR, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen.

[0098] The term "% sequence identity" or "sequence identity" as used herein in the context of the present invention is used to describe the degree of similarity between two nucleotide sequences or two amino acid sequences and has the same meaning as "percent identity". The percent homology of two sequences can be calculated as follows: after aligning the two sequences, divide the number of positions with identical residues by the total length of the aligned sequences and multiply by 100%. Methods and tools for aligning two amino acid sequences or nucleotide sequences are well known in the art, such as the BLAST suite provided on the NCBI website (Altschul, S.F. et al. (1990) J. Mol. Biol. 215: 403-410). As used herein, "at least 80% sequence identity" means having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the sequence.

[0099] To make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to constitute any limitation to the present invention. The actual protection scope of the present invention is set forth in the claims. In the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications. The equipment, instruments, reagents and / or kits used in the following embodiments that are not mentioned as to their sources are all obtained through commercial purchases in the market or obtained by conventional methods known to those skilled in the art.

[0100] Example

[0101] Example 1. Preparation of antigen

[0102] (1) Construct a DNA sequence encoding the human influenza B virus NP protein into the pet-28a Escherichia coli expression vector to form an influenza B virus NP recombinant expression plasmid.

[0103] (2) Transfect the influenza B virus NP recombinant expression plasmid into BL21(DE3) competent cells and culture to obtain a monoclonal strain expressing the influenza B virus NP protein;

[0104] (3) Culture a large amount of this strain at 37°C, and then add 0.4 mM inducer (IPTG, isopropyl-β-D-thiogalactoside) at 16°C to induce the expression of the influenza B virus NP protein;

[0105] (4) Collect all the bacteria, and after processes such as lysis, centrifugation, affinity chromatography, and gel filtration chromatography, obtain the recombinantly expressed human influenza B virus NP protein.

[0106] The amino acid sequence of the fully expressed recombinant influenza B virus NP protein is as shown in SEQ ID NO: 39:

[0107]

[0108] Its corresponding DNA sequence is as shown in SEQ ID NO: 40:

[0109]

[0110]

[0111] Example 2. Alpaca immunization injection

[0112] The specific technical solution is:

[0113] (1) The alpaca was immunized 4 times with the human influenza B virus NP protein (antigen) obtained in Example 1. Each time, 0.5 mg of the antigen was subcutaneously injected into the animal. The first immunization was on the 1st day, and the subsequent immunizations were on the 11th day, 21st day, and 31st day respectively;

[0114] (2) On the 30th day, before the fourth immunization injection, approximately 200 mL of peripheral venous blood of the alpaca was collected;

[0115] (3) On the 45th day, that is, 14 days after the fourth immunization, approximately 200 mL of peripheral venous blood of the alpaca was collected.

[0116] Compared with the traditional immunization technical solutions of antibodies in animals such as mice and rabbits, the advantage of the present disclosure is that a large amount of peripheral venous blood of alpacas can be collected, which is beneficial to subsequent screening for highly diverse nanobodies.

[0117] Example 3. Construction of an alpaca nanobody library

[0118] Using the 2 batches of peripheral venous blood of alpacas collected in Example 2 as raw materials, a highly diverse nanobody library was constructed. The processing methods of the 2 batches of peripheral venous blood of alpacas are the same. The specific technical solution is as follows:

[0119] (1) Lymphocytes were isolated from the peripheral venous blood of alpacas by methods such as density gradient centrifugation;

[0120] (2) Total mRNA of lymphocytes was extracted and reverse transcribed into cDNA;

[0121] (3) Using DNA primers (see Table 3), with the above cDNA as a template, the VHH fragments of alpaca immunoglobulin IgG2 and IgG3, that is, the DNA fragments of nanobodies, were amplified by polymerase chain reaction (PCR);

[0122] Table 3. DNA primers

[0123]

[0124]

[0125] (4) The DNA of VHH was ligated to the phage surface display screening vector (phen1) to form a plasmid library of VHH-pIII fusion protein expression vectors. Among them, pIII is a protein present on the flagellum on the surface of the phage.

[0126] (5) The DNA ligation product was transformed into TG1 competent bacteria by electroporation method. After appropriate culture, all the colonies were collected, which is the alpaca nanobody library.

[0127] Compared with the traditional method of isolating antibodies from the sera or lymphocytes of animals such as mice and rabbits, the solution of the present invention can store all the nanobody fragments (i.e., the library) of alpacas for a long time, and can continuously support the subsequent screening and development of nanobodies.

[0128] Example 4. Screening of specific nanobodies by phage surface display

[0129] Using the nanobody library obtained in Example 3 as the source, antigen-specific nanobodies were screened by phage surface display. The specific steps are as follows:

[0130] (1) Take an appropriate amount of the cryopreserved nanobody library, inoculate it into an LB medium containing the host Escherichia coli TG1, and after appropriate culture, add an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S), and continue to culture under appropriate conditions;

[0131] (2) Extract the phages amplified in the bacterial culture supernatant by the PEG-NaC method;

[0132] (3) Incubate the phages with the antigen (the recombinant human influenza B virus NP protein prepared in Example 1), and the antigen is pre-fixed to an immunosorbent tube (Maxisorp immunosorbent tube, ThermoFisher Scientific).

[0133] (4) Washing. Discard the phages, and then rinse the antigen with PBS buffer for an appropriate number of times (3-5 times) to wash away the phages that non-specifically bind to the antigen and retain the phages that specifically bind to the antigen.

[0134] (5) Elution. Incubate at room temperature for 30 minutes with 0.1M hydrochloric acid solution (adjusted to pH 2.2 with glycine and containing 0.1% BSA). After obtaining the phages dissociated from the antigen, immediately add an equal volume of 2M Tris buffer (pH 8.0) to dissociate and retain the phages from the antigen.

[0135] So far, phages expressing specific nanobodies have been obtained, and these phages can be subjected to the following technical operations:

[0136] (6) Convert to a specific nanobody library. Infect the phages into Escherichia coli TG1 again for culture, but do not add helper phage. After the phages are completely infected, the specific nanobodies exist in Escherichia coli in the form of DNA plasmids. Collect all these Escherichia coli, which becomes an antigen-specific nanobody library. Using this library as the raw material, return to step (1) for the next round of phage surface display screening;

[0137] (7) Transformation into monoclonal nanobody colonies. Take a small amount of the phage obtained in step (5) (such as 0.5%), dilute it and then infect the cultured TG1 Escherichia coli again, but do not add helper phage. After the phage infection is complete, spread these Escherichia coli evenly on a bacterial culture dish and culture to obtain monoclonal colonies containing nanobody DNA plasmids. Using these monoclonal colonies as raw materials, positive monoclonal nanobodies can be identified.

[0138] Example 5. Identification of positive monoclonal nanobodies

[0139] The bacterial culture dish with monoclonal colonies obtained through step (7) of Example 4 can be used for the identification of positive monoclonal nanobodies. The specific technical solution is as follows:

[0140] (1) Pick monoclonal colonies and culture them in a microplate.

[0141] (2) Add IPTG to induce the expression of VHH-pIII (i.e., the fusion protein containing the nanobody).

[0142] (3) Collect the bacterial culture supernatant containing the nanobody and incubate it with the antigen, which is pre-fixed on a 96-well microplate (Maxisorp transparent microplate, ThermoFisher Scientific).

[0143] (4) Use enzyme-linked immunosorbent assay (ELISA) to detect whether the monoclonal nanobody binds to the antigen.

[0144] The main experimental steps are as follows:

[0145] 1) Coating: Dilute the antigen with PBS to 5 μg / mL, add 50 μL per well, and incubate with shaking at 4°C overnight.

[0146] 2) Blocking: The next day, discard the antigen, add 100 μL per well of PBS-2% BSA, and incubate with shaking at room temperature for 1 hour.

[0147] 3) Washing: Wash 3 times with PBST and 3 times with PBS, 150 μL per well.

[0148] 4) Add the culture supernatant, 50 μL per well, and incubate with shaking at room temperature for 1 - 2 hours.

[0149] 5) Washing: Wash 3 times with PBST and 3 times with PBS, 150 μL per well.

[0150] 6) Add diluted anti-myc HRP and incubate at room temperature for 1 hour.

[0151] 7) Washing: Wash 3 times with PBST and 3 times with PBS, 150 μL per well.

[0152] 8) Add the ELISA chromogenic substrate and incubate in the dark at room temperature for 30 min;

[0153] 9) Read OD450 nM.

[0154] (5) For the monoclonal nanobody microbial colonies that can bind to the antigen, after re-culturing, extract the DNA plasmid and perform DNA sequencing to obtain the nanobody nucleic acid sequence, as shown in Table 6. After translation, the complete amino acid sequence of the nanobody can be obtained, as shown in Tables 4 and 5.

[0155] Table 4. Amino acid sequences of nanobody CDRs

[0156]

[0157]

[0158] Table 5. Amino acid sequences of nanobody heavy chain variable regions

[0159]

[0160] Table 6. Nucleotide sequences of nanobody heavy chain variable regions

[0161]

[0162]

[0163]

[0164] Example 6. Small-scale recombinant expression and purification of monoclonal nanobodies

[0165] (1) Through Example 5, monoclonal nanobodies that can specifically recognize and bind to the antigen were obtained. The DNA plasmid of the nanobody was transformed into BL21(DE3) competent cells, and the monoclonal nanobodies can be expressed and purified in small batches with the help of the Escherichia coli expression system, and the batch production capacity is about several milligrams.

[0166] (2) Using the ELISA method described in Example 5, incubate nanobodies at different concentrations, and measure the affinity between the nanobody and the antigen according to the binding ability of the nanobody to the antigen. The results are as Figures 1-8 shown.

[0167] The technical solutions of the present invention are not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solutions of the present invention falls within the protection scope of the present invention.

Claims

1. A nanobody specifically binding to influenza B N protein or an antigen-binding fragment thereof.

2. The nanobody or antigen-binding fragment thereof according to claim 1, characterized in that, The nanobody specifically binding to influenza B N protein or an antigen-binding fragment thereof includes: CDR-H1, CDR-H2, and CDR-H3 included in the heavy-chain variable region having the amino acid sequences shown in SEQ ID NOs: 23 to 30; each of the CDRs is defined by the numbering system of Kabat, AbM, Chothia, Contact, IMGT, or a combination thereof; Preferably, the nanobody specifically binding to influenza B N protein or an antigen-binding fragment thereof includes a heavy-chain variable region, and the heavy-chain variable region includes: a1) A heavy-chain variable region including the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 2, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 3; a2) A heavy-chain variable region including the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 4, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 5, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 6; a3) A heavy-chain variable region including the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 7, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 8, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 9; a4) A heavy-chain variable region including the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 10, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 11, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 12; a5) A heavy-chain variable region including the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 13, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 14, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 15; a6) A heavy-chain variable region including the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 16, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 17, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 18; a7) A heavy-chain variable region including the following 3 CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 19, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 20, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 21; or a8) It comprises the heavy chain variable regions of the following 3 CDRs: CDR-H1 with the amino acid sequence shown in SEQ ID NO:4, CDR-H2 with the amino acid sequence shown in SEQ ID NO:5, and CDR-H3 with the amino acid sequence shown in SEQ ID NO:22; Preferably, the heavy chain variable region of the nanobody specifically binding to influenza B N protein or its antigen-binding fragment further comprises the framework region of the heavy chain variable region; Preferably, the framework region of the heavy chain variable region comprises the framework region of the heavy chain variable region of an immunoglobulin derived from a mouse, a primate, a bovine, a horse, a bovine, a pig, a sheep, a goat, a dog, a cat, a rabbit, a camel, a donkey, a deer, a mink, a chicken, a duck or a goose or a mutant thereof; Preferably, the nanobody specifically binding to influenza B N protein or its antigen-binding fragment comprises: b1) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:23, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; b2) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:24, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; b3) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; b4) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:26, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; b5) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:27, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; b6) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:28, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; b7) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; or b8) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

3. A heavy chain antibody or an antigen-binding fragment thereof that specifically binds to influenza B N protein, comprising an immunoglobulin Fc domain and the nanobody or an antigen-binding fragment thereof according to any one of claims 1-2.

4. A chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen-binding domain comprises the nanobody or an antigen-binding fragment thereof according to any one of claims 1-2 or the heavy chain antibody or an antigen-binding fragment thereof according to claim 3.

5. A multispecific antibody or an antigen-binding fragment thereof, comprising two or more antigen-binding domains, wherein one antigen-binding domain comprises the nanobody or an antigen-binding fragment thereof according to any one of claims 1-2 or the heavy chain antibody or an antigen-binding fragment thereof according to claim 3.

6. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the nanobody or an antigen-binding fragment thereof according to any one of claims 1-2, the heavy chain antibody or an antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, or the multispecific antibody or an antigen-binding fragment thereof according to claim 5.

7. A vector comprising the nucleic acid molecule according to claim 6.

8. A cell comprising the nanobody or an antigen-binding fragment thereof according to any one of claims 1-2, the heavy chain antibody or an antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or an antigen-binding fragment thereof according to claim 5, the nucleic acid molecule according to claim 6, or the vector according to claim 7.

9. A method for preparing the nanobody or an antigen-binding fragment thereof according to any one of claims 1-2, the heavy chain antibody or an antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, or the multispecific antibody or an antigen-binding fragment thereof according to claim 5, obtained by culturing the cell according to claim 8.

10. A conjugate comprising the nanobody or an antigen-binding fragment thereof according to any one of claims 1-2, or the heavy chain antibody or an antigen-binding fragment thereof according to claim 3; and a conjugate moiety; Preferably, the conjugate moiety comprises a detectable label or a therapeutic agent; Preferably, the detectable label comprises an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, a colored substance, and / or biotin; Preferably, the therapeutic agent includes a chemotherapeutic agent, an immunosuppressant, and / or a cytotoxic drug.

11. A pharmaceutical composition, comprising: The nanobody or its antigen-binding fragment according to any one of claims 1-2, the heavy-chain antibody or its antigen-binding fragment according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or its antigen-binding fragment according to claim 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the cell according to claim 8, or the conjugate according to claim 10; and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further includes an additional pharmaceutically active agent.

12. Diagnostic or therapeutic kit, comprising: The nanobody or its antigen-binding fragment according to any one of claims 1-2, the heavy-chain antibody or its antigen-binding fragment according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or its antigen-binding fragment according to claim 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the cell according to claim 8, the conjugate according to claim 10, or the pharmaceutical composition according to claim 11; Preferably, the kit further includes an instruction manual and / or a dosing device.

13. Use of the nanobody or its antigen-binding fragment according to any one of claims 1-2, the heavy-chain antibody or its antigen-binding fragment according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or its antigen-binding fragment according to claim 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the cell according to claim 8, the conjugate according to claim 10, or the pharmaceutical composition according to claim 11 in the preparation of a product for any one of c1)-c3): c1) Diagnosing influenza B; c2) Preventing or treating influenza B; c3) Detecting the presence or level of influenza B N protein in a sample.

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