IL-18BP nano antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an IL-18BP nano antibody as well as a preparation method and application thereof. The nano antibody specifically recognizes and binds IL-18BP, has good affinity with the IL-18BP, and can be used for preparing products for diagnosing, preventing and / or treating IL-18BP related diseases or detecting the existence or level of IL-18BP in a sample.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an IL-18BP nanobody and its preparation method and application. Background Art

[0002] Interleukin-18 binding protein (IL-18BP), a glycoprotein, is a member of the immunoglobulin superfamily and has a strong affinity for interleukin-18 (IL-18). It can bind to IL-18 and inhibit its strong immune activation activity. Studies have found that IL-18BP secreted by tumor cells competitively binds to IL-18. Since the affinity of IL-18BP for IL-18 is much higher than that of the interleukin-18 receptor (IL-18R) on lymphocytes and T cells, IL-18 is unable to activate innate lymphocytes and T cells and promote the proliferation of immune cells, greatly reducing the immune-enhancing ability of IL-18 and failing to exert its anti-tumor effect in vivo. In other words, IL-18BP produced in the tumor microenvironment may act as a secreted immune checkpoint, limiting the effect of IL-18 immunotherapy. In recent years, tumor immunotherapy has achieved frequent success in the clinical field, and a variety of tumor immunotherapy drugs have emerged. The secreted immune checkpoint IL-18BP provides a new direction for tumor immunotherapy. IL-18BP is closely related to the occurrence and development of tumors, and its expression level is positively correlated with the progression and prognosis of various tumors. Therefore, it has potential application value in tumor treatment. In addition, IL-18BP may also be used as a drug target or biomarker and has broad application prospects in medical research and clinical applications. Summary of the Invention

[0003] The purpose of the first aspect of the present invention is to provide an IL-18BP nanobody or its antigen-binding fragment.

[0004] The purpose of the second aspect of the present invention is to provide an IL-18BP heavy chain antibody or its antigen-binding fragment.

[0005] The purpose of the third aspect of the present invention is to provide a chimeric antigen receptor.

[0006] The purpose of the fourth aspect of the present invention is to provide a multispecific antibody or its antigen-binding fragment.

[0007] The purpose of the fifth aspect of the present invention is to provide an isolated nucleic acid molecule.

[0008] The purpose of the sixth aspect of the present invention is to provide a vector.

[0009] The purpose of the seventh aspect of the present invention is to provide a cell.

[0010] The object of the eighth aspect of the present invention is to provide a method for preparing the nanobody or its antigen-binding fragment according to the first aspect of the present invention, the heavy-chain antibody or its antigen-binding fragment according to the second aspect, the chimeric antigen receptor according to the third aspect, or the multispecific antibody or its antigen-binding fragment according to the fourth aspect of the present invention.

[0011] The object of the ninth aspect of the present invention is to provide a conjugate.

[0012] The object of the tenth aspect of the present invention is to provide a pharmaceutical composition.

[0013] The object of the eleventh aspect of the present invention is to provide a diagnostic or therapeutic kit.

[0014] The object of the twelfth aspect of the present invention is to provide the use of the nanobody or its antigen-binding fragment according to the first aspect of the present invention, the heavy-chain antibody or its antigen-binding fragment according to the second aspect, the chimeric antigen receptor according to the third aspect, the multispecific antibody or its antigen-binding fragment according to the fourth aspect, the nucleic acid molecule according to the fifth aspect, the vector according to the sixth aspect, the cell according to the seventh aspect, the conjugate according to the ninth aspect, or the pharmaceutical composition according to the tenth aspect of the present invention.

[0015] In order to achieve the above object, the technical solution adopted by the present invention is:

[0016] The first aspect of the present invention provides an IL-18BP nanobody or its antigen-binding fragment, and the IL-18BP nanobody or its antigen-binding fragment includes:

[0017] CDR-H1, CDR-H2, and CDR-H3 included in the heavy-chain variable region having the amino acid sequence shown in SEQ ID NO: 10 or 15.

[0018] An IL-18BP nanobody or its antigen-binding fragment, the IL-18BP nanobody or its antigen-binding fragment includes a heavy-chain variable region, and the heavy-chain variable region includes:

[0019] a1) 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; or

[0020] a2) CDR-H1 having the amino acid sequence shown in SEQ ID NO: 12, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 13, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 14.

[0021] In some embodiments, the heavy-chain variable region of the IL-18BP nanobody or its antigen-binding fragment further includes the framework region of the heavy-chain variable region.

[0022] 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, ovine, porcine, caprine, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof.

[0023] In some embodiments, the IL-18BP nanobody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10 or 15, 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.

[0024] In a second aspect of the invention, there is provided an IL-18BP heavy chain antibody or antigen-binding fragment thereof, which comprises an immunoglobulin Fc domain and the nanobody or antigen-binding fragment thereof of the first aspect of the invention.

[0025] In some embodiments, the immunoglobulin Fc domain comprises the Fc domain of an immunoglobulin derived from a mouse, primate, bovine, equine, ovine, porcine, caprine, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof.

[0026] In a third aspect of the invention, 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 or antigen-binding fragment thereof of the first aspect of the invention or the heavy chain antibody or antigen-binding fragment thereof of the second aspect of the invention.

[0027] In a fourth aspect of the invention, there is provided a multispecific antibody or antigen-binding fragment thereof, which comprises two or more (e.g., three or four) antigen-binding domains, and one of the antigen-binding domains comprises the nanobody or antigen-binding fragment thereof of the first aspect of the invention or the heavy chain antibody or antigen-binding fragment thereof of the second aspect of the invention.

[0028] In a fifth aspect of the invention, there is provided an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the nanobody or antigen-binding fragment thereof of the first aspect of the invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect of the invention, the chimeric antigen receptor of the third aspect of the invention, or the multispecific antibody or antigen-binding fragment thereof of the fourth aspect of the invention.

[0029] Those skilled in the art will 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.

[0030] In some embodiments, the nucleotide sequence encoding the nanobody or antigen-binding fragment thereof of the first aspect of the present invention includes: SEQ ID NO: 11 or 16, 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 therewith.

[0031] The sixth aspect of the present invention provides a vector comprising the nucleic acid molecule of the fifth aspect of the present invention.

[0032] 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.

[0033] In some embodiments, the vector may be selected from nanoparticles, liposomes, exosomes, microbubbles, or gene guns.

[0034] The seventh aspect of the present invention provides a cell comprising the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy-chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, or the vector of the sixth aspect.

[0035] In some embodiments, the cell does not relate to propagating materials.

[0036] 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 antigen-binding fragment thereof, heavy-chain antibody or antigen-binding fragment thereof, chimeric antigen receptor, or multispecific antibody or antigen-binding fragment thereof of the present disclosure. In some embodiments, the host cell may be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, Escherichia coli, and the eukaryotic cell may include, for example, 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.

[0037] In some embodiments, the cell can be an immune cell. In some embodiments, the immune cells can include, but are not limited to, T cells, NK cells, DC cells, and macrophages. In these embodiments, the immune cells can express the chimeric antigen receptor of the present disclosure (i.e., modified immune cells).

[0038] The eighth aspect of the present invention provides a method for preparing the nanobody or its antigen-binding fragment of the first aspect of the present invention, 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 cells of the seventh aspect of the present invention.

[0039] The ninth aspect of the present invention provides a conjugate, which includes the nanobody or its antigen-binding fragment of the first aspect of the present invention, or the heavy-chain antibody or its antigen-binding fragment of the second aspect; and a conjugation moiety.

[0040] In some embodiments, the conjugation moiety can include, but is not limited to, a detectable label or a therapeutic agent.

[0041] In some embodiments, the detectable label can be any substance that can be detected by means such as fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrochemistry, optics, chemistry, etc. Such labels 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 labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microspheres, and biotin for binding avidin (e.g., streptavidin) modified with the above labels. In some embodiments, such labels are suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, the detectable label is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable label 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 invention through linkers of different lengths to reduce potential steric hindrance.

[0042] 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.

[0043] In some embodiments, the therapeutic agent may include, for example, but is not limited to, drugs for preventing and / or treating IL-18BP-related diseases.

[0044] In some embodiments, the conjugate moiety is selected from substances capable of improving the biological properties of the antibody (such as increasing the serum half-life), and may be, for example, a chemical group such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl.

[0045] The tenth aspect of the present invention provides a pharmaceutical composition comprising: the nanobody of the first aspect of the present invention 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, or the conjugate of the ninth aspect; and a pharmaceutically acceptable carrier.

[0046] In some embodiments, the pharmaceutical composition may further include an additional pharmaceutically active agent.

[0047] In some embodiments, the additional pharmaceutically active agent may be a drug having biological activity, such as a drug capable of preventing and / or treating IL-18BP-related diseases.

[0048] 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.

[0049] 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.

[0050] 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, etc.

[0051] The eleventh aspect of the present invention provides a diagnostic or therapeutic kit, which includes: the nanobody of the first aspect of the present invention 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.

[0052] In some embodiments, the kit may further include instructions and / or a dosing device.

[0053] In some embodiments, the kit can be used for diagnosing IL-18BP-related diseases.

[0054] In some embodiments, the kit can be used for preventing and / or treating IL-18BP-related diseases.

[0055] The twelfth aspect of the present invention provides the use of the nanobody of the first aspect of the present invention 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):

[0056] c1) Diagnosing IL-18BP-related diseases;

[0057] c2) Preventing and / or treating IL-18BP-related diseases;

[0058] c3) Detecting the presence or level of IL-18BP in a sample.

[0059] In some embodiments, the sample is selected from at least one of the body fluids, tissues, cells, and excreta of the subject to be tested.

[0060] In some embodiments, the body fluid includes at least one of blood and lymph fluid.

[0061] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0062] In some embodiments, the excreta includes at least one of urine, feces, and tears.

[0063] In some embodiments, the object to be tested includes mammals, such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cows, sheep, pigs, rabbits).

[0064] In some embodiments, the object to be tested includes humans.

[0065] In the present invention, the IL-18BP is human IL-18BP.

[0066] In the present invention, the IL-18BP-related diseases include tumors.

[0067] In the present invention, the tumors include at least one of solid tumors and hematological tumors.

[0068] In the present invention, the solid tumors include at least one of liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, adrenocortical carcinoma, paraganglioma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt lymphoma, carcinoid tumor, cardiac tumor, cholangiocarcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, germ cell tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal and paranasal sinus cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumor, islet cell tumor, papillomatosis, paraganglioma, nasal and paranasal sinus cancer, parathyroid carcinoma, penile cancer, pharyngeal cancer, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer, and multiple myeloma.

[0069] In the present invention, the hematological tumor is selected from at least one of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell - follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and preleukemia.

[0070] The beneficial effects of the present invention are as follows:

[0071] The present invention provides an IL-18BP nanobody or an antigen-binding fragment thereof, which can specifically recognize and bind to IL-18BP and has good affinity with it, and can be used for preparing products for diagnosing, preventing, and / or treating IL-18BP-related diseases, or detecting the presence or level of IL-18BP in a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Shows a schematic diagram of the detection result of the affinity between nanobody 6H11 and the antigen.

[0073] Figure 2 Shows a schematic diagram of the detection result of the affinity between nanobody 7D1 and the antigen. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0074] To make the objectives, technical solutions, and advantages of the present invention clearer, 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 do not constitute any limitation to the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications.

[0075] Definition

[0076] 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 explaining this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0077] Unless the context clearly indicates otherwise, as used herein, the expressions "a" and "an" include plural referents. For example, reference to "a cell" includes a plurality of such cells and equivalents known to those skilled in the art, and so on.

[0078] As used herein, the term "about" means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0079] K D Value: Dissociation constant (K D ) is a specific type of equilibrium constant that measures the tendency of a larger object to separate (dissociate) from another smaller component, and is the reciprocal of the association constant, with units of mol / L (M) or nmol / L (nM). K D The smaller the K value, the stronger the binding ability of the two substances.

[0080] Nanobody: An antibody that is naturally lacking a light chain in the peripheral blood of camelids. This antibody only contains one variable heavy chain region (VHH) and two conventional CH2 and CH3 regions, but it is 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 equivalent to that of the original heavy-chain antibody and binding activity to antigens, and is the smallest known unit that can bind to a target antigen; 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 a nanobody (Nb). Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the surface of antigens, the immune system in camelids can recognize complex spatial structures on the surface of antigens and can produce highly specific and high-affinity nanobodies.

[0081] Different from traditional technologies that rely on classical model animals such as mice, rabbits, monkeys, and sheep, the technical solution of the present invention relies on 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 a complete antibody, is the smallest existing unit that can bind to a target antigen, 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 surface of antigens, the immune system in animals such as alpacas can recognize complex spatial structures on the surface of antigens and can produce highly specific and high-affinity nanobodies.

[0082] According to the technical solution of the present invention, 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.

[0083] Table 1

[0084]

[0085]

[0086] 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.

[0087] Table 2

[0088]

[0089]

[0090] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention and do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0091] Example 1. Immunization injection of alpaca

[0092] In this example, the recombinant human IL-18BP protein (Sino Biological, 10357-H08H) was used as an antigen to immunize alpacas. The specific steps are as follows:

[0093] (1) The recombinant human IL-18BP protein (Sino Biological, 10357-H08H) was evenly divided into 4 portions, each portion being about 0.2 mg; the alpaca was immunized 4 times in total, and the antigen was subcutaneously injected into the animal body. The first immunization was recorded as day 1, and the subsequent immunizations were on day 11, day 21, and day 31 respectively;

[0094] (2) On day 30, about 200 mL of alpaca peripheral venous blood was collected before the fourth immunization injection;

[0095] (3) On day 45, that is, 14 days after the fourth immunization, about 200 mL of alpaca peripheral venous blood was collected.

[0096] Compared with the traditional immunization technical solutions of antibodies of animals such as mice and rabbits, the technical advantage of the present invention is that a large amount of alpaca peripheral venous blood can be collected, which is beneficial to subsequent screening for highly diverse nanobodies.

[0097] Example 2. Construction of a nanobody library of alpaca

[0098] Using the two batches of alpaca peripheral venous blood collected in Example 1 as raw materials, a highly diverse nanobody library was constructed. The processing methods for the two batches of alpaca peripheral venous blood were the same, and the specific steps were as follows:

[0099] (1) Lymphocytes were isolated from alpaca peripheral venous blood using methods such as density gradient centrifugation;

[0100] (2) Total mRNA of lymphocytes was extracted and reverse transcribed into cDNA;

[0101] (3) Using appropriate DNA primers (see Table 3 below), 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);

[0102] Table 3. Primers used for constructing the nanobody library

[0103]

[0104]

[0105] (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 phage surface flagellum;

[0106] (5) The DNA ligation product was transformed into TG1 competent Escherichia coli by electroporation, and after cultivation, all colonies were collected, which was the alpaca nanobody library.

[0107] Compared with the traditional method of isolating antibodies from the sera or lymphocytes of animals such as mice and rabbits, the present invention can long-term preserve all the nanobody fragments (i.e., the library) of alpacas, and can continuously support the subsequent screening and development of nanobodies.

[0108] Example 3. Screening of specific nanobodies by phage surface display

[0109] In this example, using the nanobody library obtained in Example 2 as a source, antigen-specific nanobodies were screened by phage surface display. The specific steps were as follows:

[0110] (1) An appropriate amount of the cryopreserved nanobody library was inoculated into an LB medium containing the host TG1 Escherichia coli. After cultivation, an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S) was added, and cultivation was continued under appropriate conditions;

[0111] (2) The phages amplified in the bacterial culture supernatant were extracted by the PEG-NaC method;

[0112] (3) Incubate the phages with the antigen (the recombinant human IL-18BP protein in Example 1), and the antigen is pre-fixed in an immunotube (Maxisorp immunotube, ThermoFisher Scientific).

[0113] (4) Panning: Discard the phages, and then rinse the antigen with PBS buffer 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.

[0114] (5) Elution: Treat the phages that specifically bind to the antigen with an acidic glycine solution to dissociate the phages from the antigen and retain them.

[0115] At this point, phages expressing specific nanobodies are obtained, and these phages can be subjected to the following technical operations:

[0116] (6) Transformation into a specific nanobody library: Infect the phages into TG1 competent Escherichia coli again for culture, but no helper phages are added. After the phages are completely infected, the specific nanobodies exist in the form of DNA plasmids in Escherichia coli. Collect all these Escherichia coli, which becomes an antigen-specific nanobody library. Using this library as a raw material, return to step (1) for the next round of phage display screening.

[0117] (7) Transformation into monoclonal nanobody colonies: Take a small amount of the phages obtained in step (5), dilute them and then infect TG1 competent Escherichia coli again for culture, but no helper phages are added. After the phages are completely infected, 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, identify positive monoclonal nanobodies.

[0118] Example 4. Identification of positive monoclonal nanobodies and nanobody sequencing

[0119] In this example, the bacterial culture dish with monoclonal colonies obtained in step (7) of Example 3 is used to identify positive monoclonal nanobodies. The specific steps are as follows:

[0120] (1) Pick monoclonal colonies into a microplate for culture.

[0121] (2) Add IPTG (isopropyl-β-D-thiogalactoside) to induce the expression of VHH-pIII (i.e., the fusion protein containing the nanobody).

[0122] (3) Collect the bacterial culture supernatant containing nanobodies and incubate it with the antigen (the recombinant human IL-18BP protein in Example 1). The antigen is pre-fixed on a 96-well microplate (Maxisorp clear microplate, ThermoFisher Scientific). Use enzyme-linked immunosorbent assay (ELISA) to detect whether the monoclonal nanobody binds to the antigen. The main experimental steps are as follows:

[0123] 1) Coating: Dilute the antigen with PBS to 5 μg / mL, add 50 μL per well, and incubate with shaking at 4 °C overnight for coating;

[0124] 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;

[0125] 3) Washing: Wash 3 times with PBST and 3 times with PBS, 150 μL per well;

[0126] 4) Add the culture supernatant, 50 μL per well, and incubate with shaking at room temperature for 1 - 2 hours;

[0127] 5) Washing: Wash 3 times with PBST and 3 times with PBS, 150 μL per well;

[0128] 6) Add diluted anti-myc HRP and incubate at room temperature for 1 hour;

[0129] 7) Washing: Wash 3 times with PBST and 3 times with PBS, 150 μL per well;

[0130] 8) Add the ELISA chromogenic substrate and incubate in the dark at room temperature for 30 min;

[0131] 9) Read OD450nm;

[0132] (4) For the monoclonal nanobodies (6H11, 7D1) that can specifically recognize and bind to the antigen, after culturing the TG1 strain expressing the relevant monoclonal nanobody overnight at 37 °C, extract the DNA plasmid and perform DNA sequencing to obtain the nucleotide sequence of the nanobody, and then obtain the complete amino acid sequence of the nanobody after translation, as shown in Table 4 - 5 specifically.

[0133] Table 4: Amino acid sequence and nucleotide sequence of 6H11

[0134]

[0135]

[0136] Table 5: Amino acid sequence and nucleotide sequence of 7D1

[0137]

[0138] Example 5. Recombinant Expression and Purification of Small Batch Monoclonal Nanobodies

[0139] (1) Monoclonal nanobodies capable of specifically recognizing and binding to antigens were obtained in Example 4. The DNA plasmids encoding the above nanobodies (6H11, 7D1) were transformed into BL21(DE3) competent cells, and the nanobodies were recombinantly expressed and purified using Escherichia coli, with a batch production capacity of approximately several milligrams.

[0140] (2) Using the ELISA method, different concentrations of nanobodies were incubated, and the affinity between the nanobodies and the antigen (the recombinant human IL-18BP protein in Example 1) was measured based on the binding ability of the nanobodies to the antigen.

[0141] The detection results are as Figure 1-2 shown, and the affinity values K D of the monoclonal nanobodies 6H11 and 7D1 are 58.88 nM and 4.016 nM, respectively.

[0142] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. An IL-18BP nanobody or an antigen-binding fragment thereof, wherein the IL-18BP nanobody or an antigen-binding fragment thereof comprises: CDR-H1, CDR-H2, and CDR-H3 included in the heavy-chain variable region having the amino acid sequence shown in SEQ ID NO: 10 or 15.

2. The nanobody or an antigen-binding fragment thereof according to claim 1, wherein the IL-18BP nanobody or an antigen-binding fragment thereof comprises a heavy-chain variable region, and the heavy-chain variable region comprises: a1) 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; or a2) CDR-H1 having the amino acid sequence shown in SEQ ID NO: 12, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 13, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 14; Preferably, the heavy-chain variable region of the IL-18BP nanobody or an antigen-binding fragment thereof further comprises a framework region of the heavy-chain variable region; Preferably, the framework region of the heavy-chain variable region comprises a 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 IL-18BP nanobody or an antigen-binding fragment thereof comprises a heavy-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10 or 15, 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. An IL-18BP heavy-chain antibody or an antigen-binding fragment thereof, which comprises 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, which comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and 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, which comprises two or more antigen-binding domains, and one of the antigen-binding domains 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, which comprises 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, which comprises the nucleic acid molecule described in claim 6.

8. A cell, which comprises the nanobody or its antigen-binding fragment described in any one of claims 1-2, the heavy-chain antibody or its antigen-binding fragment described in claim 3, the chimeric antigen receptor described in claim 4, the multispecific antibody or its antigen-binding fragment described in claim 5, the nucleic acid molecule described in claim 6, or the vector described in claim 7.

9. A method for preparing the nanobody or its antigen-binding fragment described in any one of claims 1-2, the heavy-chain antibody or its antigen-binding fragment described in claim 3, the chimeric antigen receptor described in claim 4, or the multispecific antibody or its antigen-binding fragment described in claim 5, which is obtained by culturing the cell described in claim 8.

10. A conjugate, which comprises the nanobody or its antigen-binding fragment described in any one of claims 1-2, or the heavy-chain antibody or its antigen-binding fragment described in claim 3; and a conjugation moiety; Preferably, the conjugation 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 comprises a drug for preventing and / or treating IL-18BP-related diseases.

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

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

13. Use of the nanobody or its antigen-binding fragment described in any one of claims 1-2, the heavy-chain antibody or its antigen-binding fragment described in claim 3, the chimeric antigen receptor described in claim 4, the multispecific antibody or its antigen-binding fragment described in claim 5, the nucleic acid molecule described in claim 6, the vector described in claim 7, the cell described in claim 8, the conjugate described in claim 10, or the pharmaceutical composition described in claim 11 in the preparation of a product for any one of c1)-c3): c1) Diagnosing IL-18BP-related diseases; c2) Preventing and / or treating IL-18BP-related diseases; c3) Detecting the presence or level of IL-18BP in a sample.

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