Il-18bp nanobodies and methods of making and using the same

Abstract

The application belongs to the field of biotechnology, and particularly relates to IL-18BP nanobody and a preparation method and application thereof. The nanobody specifically recognizes and combines with IL-18BP, and has good affinity with IL-18BP, 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.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to IL-18BP nanobodies, their preparation methods, and applications. Background Technology

[0002] Interleukin-18 binding protein (IL-18BP) is a glycoprotein belonging to the immunoglobulin superfamily. It has a very strong affinity for interleukin-18 (IL-18), binding to IL-18 and inhibiting its potent immune-activating activity. Studies have found that IL-18BP released and secreted by tumor cells competitively binds to IL-18. Because the affinity of IL-18BP for IL-18 is much higher than that of the IL-18 receptor (IL-18R) on lymphocytes and T cells, IL-18 cannot activate innate lymphocytes and T cells or promote the proliferation of immune cells, greatly reducing IL-18's ability to enhance immunity and thus failing to exert its anti-tumor effect in vivo. In other words, IL-18BP produced in the tumor microenvironment may act as a secretory immune checkpoint, limiting the efficacy of IL-18 immunotherapy. In recent years, tumor immunotherapy has achieved frequent successes in the clinical field, with the advent of various tumor immunotherapy drugs. The secretory 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, thus possessing potential application value in tumor treatment. Furthermore, IL-18BP may also serve as a drug target or biomarker, showing broad application prospects in medical research and clinical use. Summary of the Invention

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

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

[0005] A third aspect of the present invention aims to provide a chimeric antigen receptor.

[0006] The fourth aspect of this invention aims to provide a multispecific antibody or an antigen-binding fragment thereof.

[0007] The fifth aspect of this invention aims to provide isolated nucleic acid molecules.

[0008] The sixth aspect of this invention is to provide a carrier.

[0009] 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 a nanobody or antigen-binding fragment thereof according to the first aspect of the present invention, a heavy chain antibody or antigen-binding fragment thereof according to the second aspect, a chimeric antigen receptor according to the third aspect, or a multispecific antibody or antigen-binding fragment thereof according to the fourth aspect.

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

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

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

[0014] The object of the twelfth aspect of the present invention is to provide the use of 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, the carrier of the sixth aspect, the cell of the seventh aspect, the conjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect.

[0015] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0016] In a first aspect, the present invention provides an IL-18BP nanobody or an antigen-binding fragment thereof, said IL-18BP nanobody or antigen-binding fragment comprising:

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

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

[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 includes the framework region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

[0023] In some embodiments, the IL-18BP nanobody or its antigen-binding fragment includes 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 with it.

[0024] A second aspect of the present invention provides an IL-18BP heavy chain antibody or an antigen-binding fragment thereof, comprising an immunoglobulin Fc domain and a nanobody or an antigen-binding fragment thereof of the first aspect of the present invention.

[0025] In some embodiments, the immunoglobulin Fc domain includes the Fc domain of an immunoglobulin derived from a mouse, primate, cow, horse, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, or goose, or a mutant thereof.

[0026] A third aspect of the invention provides a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof of the first aspect of the invention, or a heavy chain antibody or an antigen-binding fragment thereof of the second aspect of the invention.

[0027] A fourth aspect of the present invention provides a multispecific antibody or an antigen-binding fragment thereof comprising two or more (e.g., three or four) antigen-binding domains, wherein one antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof of the first aspect of the present invention or a heavy chain antibody or an antigen-binding fragment thereof of the second aspect of the present invention.

[0028] A fifth aspect of the invention provides isolated nucleic acid molecules comprising nucleotide sequences encoding nanobodies or antigen-binding fragments thereof of the first aspect of the invention, heavy chain antibodies or antigen-binding fragments thereof of the second aspect, chimeric antigen receptors of the third aspect, or multispecific antibodies or antigen-binding fragments thereof of the fourth aspect.

[0029] Those skilled in the art will understand that nucleotides in nucleic acid molecules can be substituted based on 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 its antigen-binding fragment of the first aspect of the invention comprises: 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 with it.

[0031] A sixth aspect of the present invention provides a carrier 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 vector includes, for example, but 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, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0033] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

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

[0035] In some embodiments, the cells do not involve reproductive material.

[0036] In some embodiments, the cell can be a host cell conventionally used in the art, as long as the expression vector stably expresses the carried nucleic acid molecule as the 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 disclosed herein. In some embodiments, the host cell can 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, NSO 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 cells may be immune cells. In some embodiments, the immune cells may include, but are not limited to, T cells, NK cells, dendritic cells (DCs), and macrophages. In these embodiments, the immune cells may express the chimeric antigen receptors described above (i.e., modified immune cells).

[0038] The eighth aspect of the present invention provides a method for preparing 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, or the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, obtained by culturing the cells of the seventh aspect of the present invention.

[0039] A ninth aspect of the invention provides a conjugate comprising a nanobody or an antigen-binding fragment thereof of the first aspect of the invention, or a heavy chain antibody or an antigen-binding fragment thereof of the second aspect of the invention; and a conjugation portion.

[0040] In some implementations, the coupling portion may include, but is not limited to, a detectable marker or a therapeutic agent.

[0041] In some embodiments, the detectable marker can be any substance detectable by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, chemistry, etc. Such markers are well known in the art, and examples 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)), acridine esters, 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 are suitable for immunological assays (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 markers described above can be linked to the nanobodies or their antigen-binding fragments of the present invention, or heavy chain antibodies or their antigen-binding fragments, of different lengths via linkers of varying lengths to reduce potential steric hindrance.

[0042] In some embodiments, the detectable marker may include, but is not limited to, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), colored substances, biotin, etc.

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

[0044] In some embodiments, the coupling portion is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl groups.

[0045] A tenth aspect of the present invention provides a pharmaceutical composition comprising: a nanobody or antigen-binding fragment thereof of the first aspect of the present invention, a heavy chain antibody or antigen-binding fragment thereof of the second aspect of the present invention, a chimeric antigen receptor thereof of the third aspect of the present invention, a multispecific antibody or antigen-binding fragment thereof of the fourth aspect of the present invention, a nucleic acid molecule thereof of the fifth aspect of the present invention, a carrier thereof of the sixth aspect of the present invention, a cell thereof of the seventh aspect of the present invention, or a conjugate thereof of the ninth aspect of the present invention; and a pharmaceutically acceptable carrier.

[0046] In some embodiments, the pharmaceutical composition may also include additional pharmaceutically active agents.

[0047] In some embodiments, the additional pharmaceutically active agent may be a biologically active drug, 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 is provided as a separate component or as a mixed component with the additional pharmaceutically active agent.

[0049] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[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 comprising: a nanobody or antigen-binding fragment thereof of the first aspect of the present invention, a heavy chain antibody or antigen-binding fragment thereof of the second aspect of the present invention, a chimeric antigen receptor thereof of the third aspect of the present invention, a multispecific antibody or antigen-binding fragment thereof of the fourth aspect of the present invention, a nucleic acid molecule thereof of the fifth aspect of the present invention, a carrier thereof of the sixth aspect of the present invention, a cell thereof of the seventh aspect of the present invention, a conjugate thereof of the ninth aspect of the present invention, or a pharmaceutical composition thereof of the tenth aspect of the present invention.

[0052] In some embodiments, the kit may also include instructions and / or a drug delivery device.

[0053] In some implementations, the kit can be used to diagnose IL-18BP-related diseases.

[0054] In some embodiments, the kit can be used to prevent and / or treat IL-18BP-related diseases.

[0055] The twelfth aspect of the present invention provides the use of the nanobody or antigen-binding fragment thereof of the first aspect, 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, the carrier 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, said product being used in any one of c1)-c3):

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

[0057] c2) Prevention and / or treatment of IL-18BP-related diseases;

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

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

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

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

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

[0063] In some implementations, the test subject 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, cattle, sheep, pigs, rabbits).

[0064] In some implementations, the subject of the test includes humans.

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

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

[0067] In this invention, the tumor includes at least one of solid tumors and hematomas.

[0068] In this invention, the solid tumors include liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, gastric cancer, adrenocortical carcinoma, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoma, rhabdomyosarcoma, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt lymphoma, carcinoid tumor, cardiac tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, germinal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germ cell tumor, gonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, and Langerhans cell tissue. Cellular hyperplasia, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal cavity and sinus cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumor, islet cell tumor, papilloma, paraganglioma, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary adenoma, pleural pulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, Cézare 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 single myeloma.

[0069] In this invention, the hematologic malignancy is selected from at least one of the following: B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphoblastic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's 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's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom's macroglobulinemia, and pre-leukemia.

[0070] The beneficial effects of this invention are:

[0071] This invention provides an IL-18BP nanobody or its antigen-binding fragment, which can specifically recognize and bind to IL-18BP and has good affinity for it. It can be used to prepare products for the diagnosis, prevention and / or treatment of IL-18BP-related diseases, or for the detection of the presence or level of IL-18BP in samples. Attached Figure Description

[0072] Figure 1 A schematic diagram showing the results of the affinity test between the nanobody 6H11 and the antigen is presented.

[0073] Figure 2 A schematic diagram showing the results of the affinity test between nanobody 7D1 and the antigen is presented. Detailed Implementation

[0074] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0075] definition

[0076] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0077] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

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

[0079] K D Value: dissociation constant (K) D K is a specific type of equilibrium constant used to measure the tendency of a larger component to separate (dissociate) from another smaller component. It is the reciprocal of the association constant and is measured in mol / L (M) or nmol / L (nM). D The smaller the value, the stronger the binding ability of the two substances.

[0080] Nanobodies: Naturally occurring antibodies lacking the light chain, found in the peripheral blood of camels. These antibodies contain only a single heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Unlike artificially engineered single-chain antibody fragments, they do not easily adhere to each other or aggregate. Individually cloned and expressed VHH structures possess structural stability and antigen-binding activity comparable to the original heavy chain antibody, making them the smallest known unit capable of binding to target antigens. VHH crystals are 2.5 nm in size, 4 nm in length, and have a molecular weight of only about 15 kDa, hence the name nanobody (Nb). Compared to traditional animals like mice and rabbits that can only recognize flat peptides on the surface of antigens, the immune system in camels can recognize the complex spatial structures on the surface of antigens, producing highly specific and high-affinity nanobodies.

[0081] Unlike traditional technologies that rely on classic model animals such as mice, rabbits, monkeys, and sheep, the technical solution of this invention utilizes antibodies produced by the alpaca's immune system, known as "nanobodies." Nanobodies are tiny antibody fragments isolated from immunoglobulins in animals such as camels. They possess the same antigen-binding ability and structural stability as intact antibodies and are the smallest existing units capable of binding target antigens, with a relative molecular mass of only about 15 kDa. Compared to traditional methods where mice and rabbits can only recognize flat polypeptides on the surface of antigens, the immune systems in animals like alpacas can recognize the complex spatial structures on the surface of antigens, enabling the production of 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, as shown in Table 1 below.

[0083] Table 1

[0084]

[0085]

[0086] Furthermore, due to the degeneracy of bases, bases in polynucleotide sequences can be substituted without altering the activity or function of the polynucleotide sequence, as shown in Table 2 below.

[0087] Table 2

[0088]

[0089]

[0090] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection 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. Alpaca Immunization

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

[0093] (1) The recombinant human IL-18BP protein (Yiqiao Shenzhou, 10357-H08H) was divided into 4 portions, each containing approximately 0.2 mg. The alpacas were immunized a total of 4 times. The antigen was injected subcutaneously into the animals. The first immunization was recorded as day 1, and the subsequent immunizations were performed on day 11, day 21, and day 31, respectively.

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

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

[0096] Compared to traditional immunization techniques using animal antibodies from mice, rabbits, etc., the technical advantage of this invention lies in the collection of a large amount of peripheral blood from alpacas, which is beneficial for subsequent screening to obtain highly diverse nanobodies.

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

[0098] Using two batches of alpaca peripheral 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 blood were the same, and the specific steps are as follows:

[0099] (1) Lymphocytes were isolated from peripheral blood of alpaca veins using density gradient centrifugation and other methods;

[0100] (2) Extract total mRNA from lymphocytes and reverse transcribe it into cDNA;

[0101] (3) Using appropriate DNA primers (see Table 3 below), the above cDNA was used as a template to amplify the VHH fragments of alpaca immunoglobulins IgG2 and IgG3 by polymerase chain reaction (PCR), which are the DNA fragments of nanobodies.

[0102] Table 3. Primers used to construct nanobody libraries

[0103]

[0104]

[0105] (4) The DNA of VHH was ligated to the phage surface display selection vector phen1 to form a VHH-pIII fusion protein expression vector plasmid library; where pIII is a protein present on the flagella of the phage surface.

[0106] (5) The DNA ligation product was transformed into TG1 competent Escherichia coli by electroporation. After culturing, all colonies were collected to obtain the alpaca nanobody library.

[0107] Compared to traditional methods of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, this invention can preserve all nanobody fragments (i.e., libraries) of alpacas for a long time, which can continuously support the subsequent screening and development of nanobodies.

[0108] Example 3. Displaying screening-specific nanobodies on the surface of bacteriophages

[0109] This embodiment uses the nanobody library obtained in Example 2 as the source, and antigen-specific nanobodies are obtained through phage surface display screening. The specific steps are as follows:

[0110] (1) Take an appropriate amount of frozen nanobody library, inoculate it into LB medium containing host Escherichia coli TG1, and after culturing, add an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S) and continue to culture under appropriate conditions.

[0111] (2) Phages amplified in bacterial culture supernatant were extracted using the PEG-NaC method;

[0112] (3) Incubate the phage with the antigen (recombinant human IL-18BP protein in Example 1), the antigen being pre-fixed in an immunoassay tube (Maxisorp immunoassay tube, ThermoFisher Scientific);

[0113] (4) Washing: Discard the phages, then wash the antigen 3-5 times with PBS buffer to wash away the phages that are not specifically bound to the antigen, and retain the phages that are specifically bound to the antigen.

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

[0115] At this point, bacteriophages expressing specific nanobodies have been obtained, and these bacteriophages can be used for the following technical operations:

[0116] (6) Transformation into a specific nanobody library: The phage is re-infected into TG1 competent E. coli, but no helper phage is added. After complete phage infection, the specific nanobody exists in the E. coli in the form of DNA plasmids. Collecting all these E. coli results in an antigen-specific nanobody library. This library can be used as raw material to return to step (1) for the next round of phage surface display screening.

[0117] (7) Conversion into monoclonal nanobody colonies: Take a small amount of the phage obtained in step (5), dilute it, and infect TG1 competent E. coli again, but do not add helper phage. After the phage infection is complete, spread these E. coli evenly on bacterial culture dishes and culture to obtain monoclonal colonies containing nanobody DNA plasmids. Use these monoclonal colonies as raw materials to identify positive monoclonal nanobodies.

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

[0119] This embodiment utilizes the bacterial culture dish with monoclonal colonies obtained in step (7) of Example 3 to identify positive monoclonal nanobodies. The specific steps are as follows:

[0120] (1) Pick single colonies and culture them in microplates;

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

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

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

[0124] 2) Blocking: On the second day, discard the antigen and add 100 μL / well of PBS-2% BSA. Incubate at room temperature with shaking for 1 hour.

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

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

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

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

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

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

[0131] 9) Read OD450nm;

[0132] (4) For monoclonal nanobodies (6H11, 7D1) that can specifically recognize and bind to antigens, the TG1 strain expressing the relevant monoclonal nanobodies was cultured overnight at 37°C, and the DNA plasmid was extracted and DNA sequencing was performed to obtain the nucleotide sequence of the nanobodies. After translation, the complete amino acid sequence of the nanobodies was obtained, as shown in Table 4-5.

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

[0134]

[0135]

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

[0137]

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

[0139] (1) In Example 4, a monoclonal nanobody capable of specifically recognizing and binding to the antigen was obtained. The DNA plasmid encoding the above nanobody (6H11, 7D1) was transformed into BL21(DE3) competent cells. The nanobody was recombinantly expressed and purified by Escherichia coli, and the batch production capacity was about a few milligrams.

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

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

[0142] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An IL-18BP nanobody or an antigen-binding fragment thereof, comprising a heavy chain variable region, which comprises: a1) CDR-H1 of the amino acid sequence set forth in SEQ ID NO: 7, CDR-H2 of the amino acid sequence set forth in SEQ ID NO: 8, and CDR-H3 of the amino acid sequence set forth in SEQ ID NO: 9; or a2) CDR-H1 of the amino acid sequence set forth in SEQ ID NO: 12, CDR-H2 of the amino acid sequence set forth in SEQ ID NO: 13, and CDR-H3 of the amino acid sequence set forth in SEQ ID NO:

14.

2. The nanobody or an antigen-binding fragment thereof according to claim 1, wherein 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.

3. The nanobody or an antigen-binding fragment thereof according to claim 2, wherein 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 murine, primate, bovine, equine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervid, mink, chicken, duck, or goose, or a mutant thereof.

4. The nanobody or an antigen-binding fragment thereof according to any one of claims 1-3, wherein the IL-18BP nanobody or an antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth 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.

5. An IL-18BP heavy chain antibody comprising an immunoglobulin Fc domain and the nanobody or an antigen-binding fragment thereof according to any one of claims 1-4.

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

7. A vector comprising the nucleic acid molecule of claim 6.

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

9. A method of producing the nanobody or an antigen-binding fragment thereof according to any one of claims 1-4 or the heavy chain antibody of claim 5 by culturing the cell of claim 8.

10. A conjugate comprising the nanobody or an antigen-binding fragment thereof according to any one of claims 1-4 or the heavy chain antibody of claim 5; and, a conjugating moiety; the conjugating moiety is a detectable label. the detectable label comprises an enzyme, a radionuclide, a luminescent substance, a colored substance, and / or biotin. the detectable label comprises a fluorescent dye. ​ 11. The conjugate of claim 10, wherein, ​ 12. The conjugate of claim 10, wherein, ​ 13. A diagnostic kit comprising: The nanobody or antigen-binding fragment thereof according to any one of claims 1-4, the heavy chain antibody according to claim 5 or the conjugate according to any one of claims 10-12.

14. The diagnostic kit according to claim 13, further comprising instructions.

15. Use of the nanobody or antigen-binding fragment thereof according to any one of claims 1-4, the heavy chain antibody 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 any one of claims 10-12 for the manufacture of a product for any one of c1) - c2): c1) diagnosing an IL-18BP related disease; c2) detecting the presence or level of IL-18BP in a sample. ​

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