Pepsinogen II nano antibody and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a pepsinogen II nano antibody and application thereof. The nano antibody can specifically recognize and bind to pepsinogen II, has good affinity with the pepsinogen II, and can be used for preparing products for diagnosing, preventing or treating stomach lesions and / or pepsinogen II related diseases or symptoms, or detecting the existence or level of pepsinogen II in a sample.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to pepsinogen II nano antibody and application thereof. Background Art

[0002] Pepsinogen II (PGII) is one of the precursors of the two pepsin enzymes secreted by the stomach and involved in digestion. It is mainly synthesized by the gastric enzyme cells and mucous neck cells of the fundic glands and secreted into the gastric cavity. Human PGII is a polypeptide chain composed of 388 amino acids. It is inactive by itself and can be cleaved and processed into mature pepsin by gastric acid or activated pepsin in the gastric cavity. Pepsin can hydrolyze protein in food. It mainly acts on the peptide bonds containing phenylalanine or tyrosine in protein and polypeptide molecules. Under normal circumstances, about 1% of pepsinogen enters the blood circulation through the gastric mucosal capillaries. The pepsinogen that enters the blood circulation is very stable in the blood. When pathological changes occur in the gastric mucosa, the content of pepsinogen in the serum also changes accordingly. PGII is highly correlated with gastric fundus mucosal lesions, and its increase is associated with fundus gland atrophy, gastric epithelial metaplasia or pseudopyloric metaplasia, and atypical proliferation. Therefore, monitoring the concentration of PG II in serum can be used as an important means to monitor gastric lesions: (1) Helicobacter pylori infection screening: the PGII content in the blood of infected patients is significantly increased, and the content decreases after recovery; (2) Superficial gastritis: the serum PGII content of early patients is significantly increased. In addition, in different gastric diseases, clinical diagnosis of gastric lesions is often combined with the results of PGI and PGII content tests.

[0003] At present, scientific research or clinical diagnosis and detection of PGII mainly relies on antibodies. There are many monoclonal antibodies of PGII that are publicly available or sold on the market. The technical solutions for preparing these antibodies are: (1) tissue extraction or recombinant expression of PGII protein antigen; (2) injecting the antigen into animals such as mice, rabbits, and monkeys, and relying on the animal's immune system to produce antibodies; (3) collecting blood from animals and separating and extracting serum, and then further separating the components containing specific antibodies.

[0004] However, the above-mentioned technology based on the immune system of animals such as mice to produce PGII antibodies and separate and extract them has the following disadvantages: (1) The stability of the antibodies is poor, and they need to be stored, transported, tested and verified at low temperature (4°C) to maintain the concentration, specificity and other characteristics of the antibodies, which is not conducive to long-distance transportation and popular testing; (2) The cost of large-scale mass production is high. The above-mentioned monoclonal antibodies are full-length immunoglobulins (IgG), which require repeated immunization of animals and separation and purification of antibodies from animal body fluids, or recombinant expression in mammalian cells and other expensive expression systems before separation, purification and extraction. The operation is complicated and costly, which is not conducive to large-scale mass production. Summary of the invention

[0005] The present invention aims to develop nano antibodies based on the alpaca immune system, obtain nano antibodies that can specifically recognize and bind to PGII, and prepare them in batches.

[0006] The purpose of the first aspect of the present invention is to provide a nanobody or an antigen-binding fragment thereof that specifically binds to pepsinogen II.

[0007] The second aspect of the present invention aims to provide a heavy chain antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen II.

[0008] The third aspect of the present invention aims to provide a chimeric antigen receptor.

[0009] The fourth aspect of the present invention aims to provide a multispecific antibody or an antigen-binding fragment thereof.

[0010] The fifth aspect of the present invention aims to provide an isolated nucleic acid molecule.

[0011] The sixth aspect of the present invention aims to provide a carrier.

[0012] The seventh aspect of the present invention aims to provide a cell.

[0013] The purpose of the eighth aspect of the present invention is to provide 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.

[0014] The ninth aspect of the present invention aims to provide a conjugate.

[0015] The tenth aspect of the present invention aims to provide a pharmaceutical composition.

[0016] The eleventh aspect of the present invention aims to provide a diagnostic or therapeutic kit.

[0017] The object of the twelfth aspect of the present invention is to provide the use of the nanoantibody 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 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.

[0018] In order to achieve the above object, the technical solution adopted by the present invention is:

[0019] In a first aspect of the present invention, a Nanobody or an antigen-binding fragment thereof that specifically binds to pepsinogen II is provided, the Nanobody or the antigen-binding fragment thereof comprising a heavy chain variable region, the heavy chain variable region comprising: an amino acid sequence of CDR-H1, CDR-H2 and CDR-H3 included in the heavy chain variable region as shown in any one of SEQ ID NOs: 29-47, or an amino acid sequence having one or several amino acids substituted, deleted or added compared to the amino acid sequence of CDR-H1, CDR-H2 or CDR-H3 contained in the heavy chain variable region.

[0020] In some embodiments, the heavy chain variable region comprises:

[0021] a1) CDR-H1 having the amino acid sequence of SEQ ID NO:48, CDR-H2 having the amino acid sequence of SEQ ID NO:67, and CDR-H3 having the amino acid sequence of SEQ ID NO:86; or

[0022] a2) CDR-H1 having the amino acid sequence of SEQ ID NO:49, CDR-H2 having the amino acid sequence of SEQ ID NO:68, and CDR-H3 having the amino acid sequence of SEQ ID NO:87; or

[0023] a3) CDR-H1 having the amino acid sequence of SEQ ID NO:50, CDR-H2 having the amino acid sequence of SEQ ID NO:69, and CDR-H3 having the amino acid sequence of SEQ ID NO:88; or

[0024] a4) CDR-H1 having the amino acid sequence of SEQ ID NO:51, CDR-H2 having the amino acid sequence of SEQ ID NO:70, and CDR-H3 having the amino acid sequence of SEQ ID NO:89;

[0025] a5) CDR-H1 having the amino acid sequence of SEQ ID NO:52, CDR-H2 having the amino acid sequence of SEQ ID NO:71, and CDR-H3 having the amino acid sequence of SEQ ID NO:90;

[0026] a6) CDR-H1 having the amino acid sequence of SEQ ID NO:53, CDR-H2 having the amino acid sequence of SEQ ID NO:72, and CDR-H3 having the amino acid sequence of SEQ ID NO:91;

[0027] a7) CDR-H1 having the amino acid sequence of SEQ ID NO:54, CDR-H2 having the amino acid sequence of SEQ ID NO:73, and CDR-H3 having the amino acid sequence of SEQ ID NO:92;

[0028] a8) CDR-H1 having the amino acid sequence of SEQ ID NO:55, CDR-H2 having the amino acid sequence of SEQ ID NO:74, and CDR-H3 having the amino acid sequence of SEQ ID NO:93;

[0029] a9) CDR-H1 having the amino acid sequence of SEQ ID NO:56, CDR-H2 having the amino acid sequence of SEQ ID NO:75, and CDR-H3 having the amino acid sequence of SEQ ID NO:94;

[0030] a10) CDR-H1 having the amino acid sequence of SEQ ID NO: 57, CDR-H2 having the amino acid sequence of SEQ ID NO: 76, and CDR-H3 having the amino acid sequence of SEQ ID NO: 95;

[0031] a11) CDR-H1 having the amino acid sequence of SEQ ID NO: 58, CDR-H2 having the amino acid sequence of SEQ ID NO: 77, and CDR-H3 having the amino acid sequence of SEQ ID NO: 96;

[0032] a12) CDR-H1 having the amino acid sequence of SEQ ID NO:59, CDR-H2 having the amino acid sequence of SEQ ID NO:78, and CDR-H3 having the amino acid sequence of SEQ ID NO:97;

[0033] a13) CDR-H1 having the amino acid sequence of SEQ ID NO: 60, CDR-H2 having the amino acid sequence of SEQ ID NO: 79, and CDR-H3 having the amino acid sequence of SEQ ID NO: 98;

[0034] a14) CDR-H1 having the amino acid sequence of SEQ ID NO:61, CDR-H2 having the amino acid sequence of SEQ ID NO:80, and CDR-H3 having the amino acid sequence of SEQ ID NO:99;

[0035] a15) CDR-H1 having the amino acid sequence of SEQ ID NO: 62, CDR-H2 having the amino acid sequence of SEQ ID NO: 81, and CDR-H3 having the amino acid sequence of SEQ ID NO: 100;

[0036] a16) CDR-H1 having the amino acid sequence of SEQ ID NO: 63, CDR-H2 having the amino acid sequence of SEQ ID NO: 82, and CDR-H3 having the amino acid sequence of SEQ ID NO: 101;

[0037] a17) CDR-H1 having the amino acid sequence of SEQ ID NO: 64, CDR-H2 having the amino acid sequence of SEQ ID NO: 83, and CDR-H3 having the amino acid sequence of SEQ ID NO: 102;

[0038] a18) CDR-H1 having the amino acid sequence of SEQ ID NO: 65, CDR-H2 having the amino acid sequence of SEQ ID NO: 84, and CDR-H3 having the amino acid sequence of SEQ ID NO: 103;

[0039] a19) CDR-H1 having the amino acid sequence shown in SEQ ID NO:66, CDR-H2 having the amino acid sequence shown in SEQ ID NO:85, and CDR-H3 having the amino acid sequence shown in SEQ ID NO:104.

[0040] In some embodiments, the heavy chain variable region of the Nanobody or antigen-binding fragment thereof that specifically binds to pepsinogen II also includes a framework region of the heavy chain variable region.

[0041] In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region of an immunoglobulin derived from mouse, primate, bovine, horse, cattle, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof.

[0042] In some embodiments, the heavy chain variable region includes the amino acid sequence shown in any one of SEQ ID NOs: 29-47, or an amino acid sequence having one or several amino acid substitutions, deletions or additions compared thereto, 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.

[0043] In some embodiments, the pepsinogen II is human pepsinogen II; further, the pepsinogen II has an amino acid sequence as shown in SEQ ID NO: 1.

[0044] The second aspect of the present invention provides a heavy chain antibody or an antigen-binding fragment thereof that specifically binds to pepsinogen II, which comprises an immunoglobulin Fc domain and the Nanobody or an antigen-binding fragment thereof of the first aspect of the present invention.

[0045] In some embodiments, the immunoglobulin Fc domain comprises an Fc domain of an immunoglobulin from mouse, primate, bovine, horse, cattle, porcine, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof.

[0046] In the third aspect of the present invention, a chimeric antigen receptor is provided, which comprises an antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain comprises the nanobody or antigen binding fragment thereof of the first aspect of the present invention or the heavy chain antibody or antigen binding fragment thereof of the second aspect.

[0047] In a fourth aspect of the invention, a multispecific antibody or antigen-binding fragment thereof is provided, which comprises two or more (e.g., three or four) antigen-binding domains, wherein one 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.

[0048] In a fifth aspect of the invention, an isolated nucleic acid molecule is provided, 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, the chimeric antigen receptor of the third aspect, or the multispecific antibody or antigen-binding fragment thereof of the fourth aspect.

[0049] Those skilled in the art will appreciate that nucleotides in a nucleic acid molecule may be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimized.

[0050] In some embodiments, the nucleotide sequence encoding the Nanobody or antigen-binding fragment thereof of the first aspect of the invention comprises: the nucleotide sequence shown in any one of SEQ ID NOs: 10-28, 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.

[0051] The sixth aspect of the present invention provides a vector comprising the nucleic acid molecule of the fifth aspect of the present invention.

[0052] In some embodiments, the vector may be an expression vector. In some embodiments, the expression vector may include a eukaryotic expression vector and / or a prokaryotic expression vector. In some embodiments, the eukaryotic expression vector includes, for example, but is not limited to, a yeast expression vector, a mammalian expression vector, and an insect expression vector. For example, the expression vector may include, but is not limited to, a plasmid, a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

[0053] In some embodiments, the carrier can be selected from nanoparticles, liposomes, exosomes, microbubbles or gene guns.

[0054] In the seventh aspect of the present invention, a cell is provided, which includes the nano antibody 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.

[0055] In some embodiments, the cells are not involved in propagation material.

[0056] In some embodiments, the cell can be a host cell conventionally used in the art, as long as the expression vector can stably express the carried nucleic acid molecule as the above-mentioned nano antibody or antigen-binding fragment thereof, heavy chain antibody or antigen-binding fragment thereof, chimeric antigen receptor or multispecific antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell, the prokaryotic cell can include, for example, Escherichia coli, the eukaryotic cell can 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.

[0057] 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).

[0058] In the eighth aspect of the present invention, a method for preparing the nanoantibody 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 is provided, which is obtained by culturing the cells of the seventh aspect of the present invention.

[0059] In a ninth aspect of the present invention, a conjugate is provided, which comprises the Nanobody or antigen-binding fragment thereof of the first aspect of the present invention, or the heavy chain antibody or antigen-binding fragment thereof of the second aspect; and a coupling portion.

[0060] In some embodiments, the conjugated moiety may include, but is not limited to, a detectable label, a delivery vehicle, or a therapeutic agent.

[0061] In some embodiments, the detectable marker can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, 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.) microbeads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned markers. In some embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme. In some embodiments, the detectable label as described above can be connected to the nanobody or its antigen-binding fragment, or heavy chain antibody or its antigen-binding fragment of the present disclosure by linkers of different lengths to reduce potential steric hindrance.

[0062] In some embodiments, the detectable marker may include, but is not limited to, an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), a colored substance, biotin, and the like.

[0063] In some embodiments, the therapeutic agent may include, for example, but not limited to, chemotherapeutic agents, immunosuppressants, cytotoxic drugs.

[0064] In some embodiments, the coupling moiety is selected from substances that can improve the biological properties of the antibody (eg, increase serum half-life), for example, it can be a chemical group such as polyethylene glycol (PEG), methyl, ethyl or sugar group.

[0065] In the tenth aspect of the present invention, a pharmaceutical composition is provided, comprising: the nanoantibody 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 vector of the sixth aspect, the cell of the seventh aspect or the conjugate of the ninth aspect; and a pharmaceutically acceptable carrier.

[0066] In some embodiments, the pharmaceutical composition may further include additional pharmaceutically active agents.

[0067] In some embodiments, the additional pharmaceutically active agent can be a biologically active drug, such as a drug capable of treating a disease or condition associated with pepsinogen II.

[0068] In some embodiments, the Nanobody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as mixed components.

[0069] In some embodiments, the pharmaceutical composition can be administered, for example, parenterally, subcutaneously, sublingually, rectally, nasally, intravenously, intramuscularly, orally, ophthalmically, topically, or the like.

[0070] 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.

[0071] In the eleventh aspect of the present invention, a diagnostic or therapeutic kit is provided, which comprises: 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 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.

[0072] In some embodiments, the kit may further include instructions and / or an administration device.

[0073] In some embodiments, the kits can be used to diagnose a disease or condition associated with pepsinogen II.

[0074] In some embodiments, the kits can be used to prevent or treat a disease or condition associated with pepsinogen II.

[0075] In the twelfth aspect of the present invention, there is provided 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 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, wherein the product is used for any one of c1) to c3):

[0076] c1) diagnosing a disease or condition, said disease or condition being associated with pepsinogen II;

[0077] c2) preventing or treating a disease or condition associated with pepsinogen II;

[0078] c3) detecting the presence or level of pepsinogen II in the sample.

[0079] In some embodiments, the sample is selected from at least one of body fluids, tissues, cells, and excrement of the subject to be tested.

[0080] In some embodiments, the body fluid comprises at least one of blood and lymph.

[0081] In some embodiments, the blood comprises at least one of serum, plasma, dried blood spots, and whole blood.

[0082] In some embodiments, the tissue comprises tumor tissue.

[0083] In some embodiments, the excreta comprises at least one of urine, feces, and tears.

[0084] In some embodiments, the subject to be tested comprises mammals, such as humans, non-human primates (such as gorillas, apes), rodents (such as rats, mice, guinea pigs), pets (such as cats, dogs), and livestock (such as horses, cows, sheep, pigs, rabbits).

[0085] In some embodiments, the subject comprises a human.

[0086] In the present invention, the disease or condition includes a gastric disease, condition or lesion. In some embodiments, the disease or condition includes one or more of Helicobacter pylori infection, acute gastritis, chronic gastritis, superficial gastritis, erosive gastritis, gastric ulcer, duodenal ulcer, atrophic gastritis or gastric cancer.

[0087] The present invention provides a nanobody or an antigen-binding fragment thereof that specifically binds to pepsinogen II, which can specifically recognize and bind to pepsinogen II and has good affinity thereto, and can be prepared in batches for the preparation of products for diagnosing, preventing or treating diseases or conditions associated with pepsinogen II, or detecting the presence or level of pepsinogen II in a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 A schematic diagram showing the results of affinity testing between nanobody 13A12 and antigen is shown.

[0089] Figure 2 A schematic diagram showing the results of affinity testing between nanobody 12F2 and antigen is shown.

[0090] Figure 3 A schematic diagram showing the results of affinity testing of nanobody 15H5 with antigens is shown.

[0091] Figure 4 A schematic diagram showing the results of affinity testing between nanobody 15C10 and antigen is shown.

[0092] Figure 5 A schematic diagram showing the results of affinity testing between nanobody 9F7 and antigen is shown.

[0093] Figure 6 A schematic diagram showing the results of affinity testing of nanobody 15A5 with antigen is shown.

[0094] Figure 7 A schematic diagram showing the results of affinity testing between nanobody 12H6 and antigen is shown.

[0095] Figure 8 A schematic diagram showing the results of affinity testing of nanobody 10H7 with antigens is shown.

[0096] Fig. 9A schematic diagram showing the results of affinity testing between nanobody 12C2 and antigen is shown.

[0097] Fig.10 A schematic diagram showing the results of affinity testing between nanobody 12G6 and antigen is shown.

[0098] Fig.11 A schematic diagram showing the results of affinity testing between nanobody 9D7 and antigen is shown.

[0099] Fig.12 A schematic diagram showing the results of affinity testing of nanobody 7C11 and antigen is shown.

[0100] Fig.13 A schematic diagram showing the results of affinity testing between nanobody 13C8 and antigen is shown.

[0101] Fig.14 A schematic diagram showing the results of affinity testing of nanobody 6C8 with antigens.

[0102] Fig.15 A schematic diagram showing the results of affinity testing of nanobody 9D10 and antigen is shown.

[0103] Fig.16 A schematic diagram showing the results of affinity testing of Nanobody 9A4 with antigens.

[0104] Fig.17 A schematic diagram showing the results of affinity testing of nanobody 14B7 with antigen is shown.

[0105] Fig.18 A schematic diagram showing the results of affinity testing between nanobody 16G7 and antigen is shown.

[0106] Fig.19 A schematic diagram showing the results of affinity testing between nanobody 16E3 and antigen is shown. DETAILED DESCRIPTION

[0107] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is 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 intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0108] definition

[0109] 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 will also include the plural form, and vice versa.

[0110] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0111] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0112] Pepsinogen II (PGII) It is one of the two precursors of pepsin secreted by the stomach and involved in digestion. It is mainly synthesized by the gastric enzyme cells and mucous neck cells of the fundic glands and secreted into the gastric cavity. Human PGII is a polypeptide chain composed of 388 amino acids. It is not active by itself and can be cleaved and processed into mature pepsin (Pepsin) by gastric acid or activated pepsin in the gastric cavity. Pepsin can hydrolyze protein in food. It mainly acts on the peptide bonds containing phenylalanine or tyrosine in protein and polypeptide molecules. Under normal circumstances, about 1% of pepsinogen enters the blood circulation through the gastric mucosal capillaries. The pepsinogen that enters the blood circulation is very stable in the blood. When pathological changes occur in the gastric mucosa, the content of pepsinogen in the serum also changes accordingly.

[0113] K D value :Dissociation constant (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. It is the reciprocal of the association constant and has units of mol / L (M) or nmol / L (nM). K D The smaller the value, the stronger the binding ability of the two substances.

[0114] Nanobodies: An antibody that naturally lacks light chains and exists in the peripheral blood of camelids. This antibody contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not as easy to stick to each other or even aggregate into clumps as artificially modified single-chain antibody fragments; the VHH structure cloned and expressed separately has the same structural stability and antigen binding activity as the original heavy chain antibody, and is the smallest unit known to bind to the target antigen; the VHH crystal is 2.5nm, 4nm long, and has a molecular weight of only about 15kD, so it is also called a nanobody (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 the complex spatial structure of the antigen surface and can produce highly specific and high-affinity nanobodies.

[0115] Unlike traditional technologies that rely on classic 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, which are called "nanoantibodies". Nanoantibodies are tiny antibody fragments separated from immunoglobulins in animals such as camels. They have the same antigen binding ability and structural stability as complete antibodies. They are the smallest units that can bind to target antigens, with a relative molecular mass of only about 15kD. Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the surface of antigens, the immune systems in animals such as alpacas can recognize the complex spatial structure of the antigen surface and can produce highly specific and high-affinity nanoantibodies.

[0116] As used herein, the term "complementarity determining region (CDR)" is the amino acid residue in the variable region of an antibody that is responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system, the Chothia numbering system, the IMGT numbering system, the Martin numbering system, the Contact numbering system, the Honegger numbering system, the Gelfand numbering system, or a combination thereof. For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. The corresponding relationship between different numbering systems is well known to those skilled in the art, and some conventionally used software can be used to define HCDR1-3. For example, the Kabat numbering system is an immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The Chothia numbering system is an immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of the structural loop region (see, for example, Chothia & Lesk, (1987), J. Mol. Biol. 196: 901-917; Chothia et al., (1989), Nature 342: 878-883; Al-Lazikani et al., (1997), JMB 273: 927-948). The IMGT numbering system is a numbering system based on the international ImMunoGeneTics information (IMGT) initiated by Lefranc et al. (see, for example, Lefranc et al., (2003), Dev. Comparat. Immunol. 27: 55-77). The Martin numbering system is a new numbering scheme recommended by Martin et al. for structural alignment of different framework regions with unconventional lengths, which can be completed by the Chothia numbering system corrected by ABnum software. The Contact numbering system defines CDR regions based on available crystal structure data for antibody complexes. The Honegger numbering system (also known as the AHo numbering system) is based on structural alignments of 3D structures of immunoglobulin variable regions covering the observed length variation, allowing the definition of structurally conserved Cα positions and therefore the deduction of appropriate FR region and CDR lengths.The Gelfand numbering system is a relatively complex numbering scheme described by Gelfand et al., in which variable chain sequences are divided into 21 segments called "words", each of these "words" matches a secondary structure element (chain or loop), chains are defined by alphabetical letters (e.g., A, B, C), and loops are defined by two letters, which correspond to adjacent chains (e.g., AB, BC...), this numbering system does not include gaps or missing points, but allows accurate comparison of secondary structures (loops and chains) between aligned sequences.

[0117] 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:

[0118] Table 1

[0119] Residue Conservative substitution Residue Conservative substitution Ala Ser Leu Ile; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met; Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr Gly Pro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu; Val

[0120] In addition, due to the degeneracy of the bases, the bases of the polynucleotide sequence can be substituted without changing the activity or function of the polynucleotide sequence, as shown in Table 2 below:

[0121] Table 2

[0122]

[0123]

[0124] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide or amino acid, gaps present in the target sequence are not counted. Likewise, since target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted, gaps present in the reference sequence are not counted.

[0125] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The determination of the sequence identity percentage between the comparison of the sequence and the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite that can be obtained from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. Government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0126] 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, but 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.

[0127] Example 1. Preparation of antigens

[0128] The DNA sequence encoding PGII was constructed into the pFASTBac-HTA expression vector, and PGII with a histidine tag (6*His-tag) was recombinantly expressed in SF9 insect cells. After purification by histidine affinity chromatography, gel filtration chromatography, etc., the complete PGII protein was obtained as an antigen.

[0129] The specific steps are as follows:

[0130] 1.1 Construct the DNA sequence encoding PGII (SEQ ID NO: 1) into the pFASTBac-HTA expression vector;

[0131] 1.2 Transform the constructed plasmid into DH10Bac competent cells (Shanghai Weidi, DL1071M);

[0132] 1.3 After blue-white screening, pick the DH10Bac monoclonal clone for culture and extract the Bacmid baculovirus;

[0133] 1.4 Use Cellfectin II transfection reagent (ThermoFisher, 10362100) to transfect Bacmid baculovirus into SF9 insect cells. After culture, collect the cell culture supernatant as P1 virus;

[0134] 1.5 Use P1 virus to infect SF9 insect cells, and collect the cell supernatant after culture to obtain P2 virus;

[0135] 1.6 Use P2 virus to infect SF9 insect cells, and collect the cell supernatant after culture to obtain P3 virus;

[0136] 1.7 Use P3 virus to infect SF9 insect cells and recombinantly express PGII with a histidine tag (6*His-tag) in SF9 insect cells;

[0137] 1.8 Collect SF9 insect cells that recombinantly express PGII, disrupt and lyse them by ultrasonication, centrifuge and collect the supernatant;

[0138] 1.9 Histidine tag affinity chromatography: incubate the supernatant obtained after centrifugation with nickel affinity chromatography filler, and then elute the recombinant PGII with histidine tag bound to the nickel filler;

[0139] 1.10 Gel filtration chromatography: The product obtained after the above affinity chromatography is concentrated and subjected to gel filtration chromatography to obtain purified recombinant PGII.

[0140] The amino acid sequence of PGII is shown in SEQ ID NO: 1:

[0141] MKWMVVVLVCLQLLEAAVVKVPLKKFKSIRETMKEKGLLGEFLRTHKYDPAWKYRFGDLSVTYEPMAYMDAAYFGEISIGTPPQNFLVLFDTGSSSNLWVPSVYCQSQACTSHSRFNPSESSTYSTNGQTFSLQYGSGSLTGFFGYDTLTVQSIQVPNQEFGLSENEPGTNFVYAQFDGIMGLAYPALSVDEATT AMQGMVQEGALTSPVFSVYLSNQQGSSGGAVVFGGVDSSLYTGQIYWAPVTQELYWQIGIEEFLIGGQASGWCSEGCQAIVDTGTSLLTVPQQYMSALLQATGAQEDEYGQFLVNCNSIQNLPSLTFIINGVEFPLPPSSYILSNNGYCTVGVEPTYLSSQNGQPLWILGDVFLRSYYSVYDLGNNRVGFATAA

[0142] The amino acid sequence of the fully expressed recombinant PGII is shown in SEQ ID NO: 2:

[0143] MSYYHHHHHHDYDIPTTENLYFQGAMAMKWMVVVLVCLQLLEAAVVKVPLKKFKSIRETMKEKGLLGEFLRTHKYDPAWKYRFGDLSVTYEPMAYMDAAYFGEI SIGTPPQNFLVLFDTGSSSNLWVPSVYCQSQACTSHSRFNPSESSTYSTNGQTFSLQYGSGSLTGFFGYDTLTVQSIQVPNQEFGLSENEPGTNFVYAQFDGIMG LAYPALSVDEATTAMQGMVQEGALTSPVFSVYLSNQQGSSGGAVVFGGVDSSLYTGQIYWAPVTQELYWQIGIEEFLIGGQASGWCSEGCQAIVDTGTSLLTVP QQYMSALLQATGAQEDEYGQFLVNCNSIQNLPSLTFIINGVEFPLPPSSYILSNNGYCTVGVEPTYLSSQNGQPLWILGDVFLRSYYSVYDLGNNRVGFATAA.

[0144] The DNA sequence encoding the recombinant PGII is shown in SEQ ID NO:3:

[0145]

[0146] Example 2. Alpaca Immunization

[0147] (1) The alpacas were immunized 4 times in total, and 0.2 mg of the recombinant PGII antigen prepared in Example 1 was subcutaneously injected into the animals each time. The first immunization was recorded as day 1, and the subsequent immunizations were recorded on days 11, 21, and 31 respectively;

[0148] (2) On day 30, before the fourth immunization, approximately 200 mL of peripheral venous blood was collected from the alpaca;

[0149] (3) On day 45, i.e. 14 days after the fourth immunization, approximately 200 mL of peripheral venous blood was collected from the alpaca.

[0150] Compared with traditional immunization technology solutions for animal antibodies such as mice and rabbits, the technical advantage of the present invention lies in the collection of a large amount of alpaca venous peripheral blood, which is conducive to subsequent screening to obtain highly diverse nano-antibodies.

[0151] Example 3. Construction of Alpaca Nanobody Library

[0152] The two batches of alpaca venous peripheral blood collected in Example 2 were used as raw materials to construct a highly diverse nanoantibody library. The two batches of alpaca venous peripheral blood were processed in the same way, and the specific steps were as follows:

[0153] (1) Lymphocytes were isolated from alpaca venous peripheral blood using density gradient centrifugation and other methods;

[0154] (2) extracting total mRNA from lymphocytes and reverse transcribed into cDNA;

[0155] (3) using appropriate DNA primers (see Table 3 below) and the above cDNA as a template, amplifying the VHH fragments of alpaca immunoglobulins IgG2 and IgG3, i.e., the DNA fragments of the nanobody, by polymerase chain reaction (PCR);

[0156] Table 3. Primers used to construct the nanobody library

[0157]

[0158] (4) Connect the VHH DNA to the phage surface display screening vector to form a VHH-pIII fusion protein expression vector plasmid library. Among them, pIII is a protein present on the flagella on the surface of phage.

[0159] (5) The DNA ligation product is transformed into TG1 competent bacteria by electroporation, and all colonies are collected after appropriate cultivation, which is the alpaca nanoantibody library.

[0160] Compared with the traditional method of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, the method of the present invention can preserve all the nano-antibody fragments (i.e., the library) of alpacas for a long time, and can continuously support the subsequent screening and development of nano-antibodies.

[0161] Example 4. Screening of specific nanobodies by phage surface display

[0162] This example uses the nanobody library obtained in Example 3 as a source, and obtains antigen-specific nanobodies through phage surface display screening. The specific steps are as follows:

[0163] (1) Taking the frozen nanobody library, inoculating it into a culture medium containing host bacteria (TG1 Escherichia coli), adding helper phage (M13KO7 helper phage, NEB, N0315S) after cultivation, and continuing to culture;

[0164] (2) extracting the amplified phages from the bacterial culture supernatant using the PEG-NaCl method;

[0165] (3) incubating the phage with the recombinant PGII antigen prepared in Example 1, where the antigen was pre-fixed in an immunotube (Maxisorp immunotube, ThermoFisher Scientific);

[0166] (4) Washing. Discard the phages and rinse the antigen with PBS buffer for an appropriate number of times (3-5 times). Wash and remove the phages that non-specifically bind to the antigen and retain the phages that specifically bind to the antigen.

[0167] (5) Elution. Use acidic glycine solution to elute the phage, so that the phage and antigen are dissociated and retained;

[0168] At this point, phages expressing specific nanobodies are obtained, and these phages continue to perform the following steps:

[0169] (6) Transformation into a specific nanoantibody library. The phage is infected with E. coli again, but no helper phage is added. After the phage infection is complete, the specific nanoantibody exists in the E. coli in the form of a DNA plasmid. Collect all these E. coli to form an antigen-specific nanoantibody library. This library is used as the raw material and returns to step (1) for the next round of phage surface display screening;

[0170] (7) Transformation into monoclonal nanoantibody colonies. Take a small amount of the phage obtained in step (5) (e.g., 0.5%), dilute it, and infect E. coli again, but no helper phage is added. After the phage infection is complete, the E. coli are evenly spread on a bacterial culture dish and cultured to obtain monoclonal colonies containing the nanoantibody DNA plasmid.

[0171] Example 5. Identification of positive monoclonal nanobodies

[0172] The bacterial culture dish with monoclonal colonies obtained by step (7) of Example 4 can be used to identify positive monoclonal nanoantibodies. The specific technical scheme is as follows:

[0173] (1) Pick a single clone and culture it in a microplate.

[0174] (2) Add IPTG (isopropyl-β-D-thiogalactoside) to induce the expression of VHH-pIII (i.e., the fusion protein containing the nanobody).

[0175] (3) The bacterial culture supernatant containing the nanobodies was collected and incubated with the antigen, which was pre-immobilized in a 96-well microplate (Maxisorp transparent microplate, ThermoFisher Scientific).

[0176] (4) Using enzyme-linked immunosorbent assay (ELISA), detect whether the monoclonal nanoantibody binds to the antigen.

[0177] The main experimental steps are as follows:

[0178] 1) Coating: dilute the antigen to 5 μg / mL with PBS, 50 μL / well, and incubate overnight at 4°C with shaking;

[0179] 2) Blocking: On the second day, discard the antigen, add 100 μL / well PBS-2% BSA and incubate at room temperature for 1 hour with shaking;

[0180] 3) Washing: 3 times PBST, 3 times PBS, 150 μL / well;

[0181] 4) Add culture supernatant, 50 μL / well, and incubate at room temperature with shaking for 1-2 hours;

[0182] 5) Washing: 3 times PBST, 3 times PBS, 150 μL / well;

[0183] 6) Add diluted anti-myc HRP and incubate at room temperature for 1 hour;

[0184] 7) Washing: 3 times PBST, 3 times PBS, 150 μL / well;

[0185] 8) Add ELISA colorimetric substrate and incubate at room temperature in the dark for 30 min;

[0186] 9) Read OD450nm.

[0187] (5) For the monoclonal nanoantibody microbial colonies that can bind to the antigen, after appropriate culturing again, the DNA plasmid is extracted and DNA sequencing is performed to obtain the nanoantibody nucleic acid sequence. After translation, the complete amino acid sequence of the nanoantibody can be obtained, and the CDR sequence of each nanoantibody is further determined. The specific sequence information is shown in Table 4 below.

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] Example 6. Small batch monoclonal nanobody recombinant expression and purification

[0197] (1) A monoclonal nanobody capable of specifically recognizing and binding to an antigen was obtained in Example 5. The DNA plasmid of the nanobody was transformed into BL21 (DE3) competent cells. The monoclonal nanobody can be expressed and purified in small batches using the E. coli expression system, with a batch production capacity of about several milligrams.

[0198] (2) Using the ELISA method (same as in Example 5), incubate different concentrations of Nanobodies, and measure the affinity of the Nanobodies to the antigen based on the binding ability of the Nanobodies to the antigen (preparation method is the same as in Example 1).

[0199] The results of the affinity test of each nanobody to the pepsinogen II antigen are as follows: Figure 1-19 As shown, the affinity value k D The results are shown in Table 5 below.

[0200] Table 5

[0201] Antibody <![CDATA[K D Value (nM)]]> 13A12 0.9370 12F2 9.624 15H5 15.18 15C10 16.70 9F7 17.20 15A5 18.01 12H6 21.79 10H7 25.60 12C2 26.01 12G6 26.50 9D7 27.27 7C11 28.22 13C8 25.50 6C8 45.59 9D10 48.34 9A4 49.45 14B7 49.80 16G7 54.15 16E3 67.99

[0202] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A Nanobody or antigen-binding fragment thereof that specifically binds to pepsinogen II, wherein the Nanobody or antigen-binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises: An amino acid sequence having CDR-H1, CDR-H2 and CDR-H3 included in the heavy chain variable region as shown in any one of SEQ ID NOs: 29-47, or an amino acid sequence having one or several amino acids substituted, deleted or added compared to the amino acid sequence of CDR-H1, CDR-H2 or CDR-H3 contained in the heavy chain variable region.

2. The nanobody or antigen-binding fragment thereof according to claim 1, characterized in that The heavy chain variable region comprises: a1) CDR-H1 having the amino acid sequence of SEQ ID NO:48, CDR-H2 having the amino acid sequence of SEQ ID NO:67, and CDR-H3 having the amino acid sequence of SEQ ID NO:86; or a2) CDR-H1 having the amino acid sequence of SEQ ID NO:49, CDR-H2 having the amino acid sequence of SEQ ID NO:68, and CDR-H3 having the amino acid sequence of SEQ ID NO:87; or a3) CDR-H1 having the amino acid sequence of SEQ ID NO:50, CDR-H2 having the amino acid sequence of SEQ ID NO:69, and CDR-H3 having the amino acid sequence of SEQ ID NO:88; or a4) CDR-H1 having the amino acid sequence of SEQ ID NO:51, CDR-H2 having the amino acid sequence of SEQ ID NO:70, and CDR-H3 having the amino acid sequence of SEQ ID NO:89; a5) CDR-H1 having the amino acid sequence of SEQ ID NO:52, CDR-H2 having the amino acid sequence of SEQ ID NO:71, and CDR-H3 having the amino acid sequence of SEQ ID NO:90; a6) CDR-H1 having the amino acid sequence of SEQ ID NO:53, CDR-H2 having the amino acid sequence of SEQ ID NO:72, and CDR-H3 having the amino acid sequence of SEQ ID NO:91; a7) CDR-H1 having the amino acid sequence of SEQ ID NO:54, CDR-H2 having the amino acid sequence of SEQ ID NO:73, and CDR-H3 having the amino acid sequence of SEQ ID NO:92; a8) CDR-H1 having the amino acid sequence of SEQ ID NO:55, CDR-H2 having the amino acid sequence of SEQ ID NO:74, and CDR-H3 having the amino acid sequence of SEQ ID NO:93; a9) CDR-H1 having the amino acid sequence of SEQ ID NO:56, CDR-H2 having the amino acid sequence of SEQ ID NO:75, and CDR-H3 having the amino acid sequence of SEQ ID NO:94; a10) CDR-H1 having the amino acid sequence of SEQ ID NO: 57, CDR-H2 having the amino acid sequence of SEQ ID NO: 76, and CDR-H3 having the amino acid sequence of SEQ ID NO: 95; a11) CDR-H1 having the amino acid sequence of SEQ ID NO: 58, CDR-H2 having the amino acid sequence of SEQ ID NO: 77, and CDR-H3 having the amino acid sequence of SEQ ID NO: 96; a12) CDR-H1 having the amino acid sequence of SEQ ID NO:59, CDR-H2 having the amino acid sequence of SEQ ID NO:78, and CDR-H3 having the amino acid sequence of SEQ ID NO:97; a13) CDR-H1 having the amino acid sequence of SEQ ID NO: 60, CDR-H2 having the amino acid sequence of SEQ ID NO: 79, and CDR-H3 having the amino acid sequence of SEQ ID NO: 98; a14) CDR-H1 having the amino acid sequence of SEQ ID NO:61, CDR-H2 having the amino acid sequence of SEQ ID NO:80, and CDR-H3 having the amino acid sequence of SEQ ID NO:99; a15) CDR-H1 having the amino acid sequence of SEQ ID NO: 62, CDR-H2 having the amino acid sequence of SEQ ID NO: 81, and CDR-H3 having the amino acid sequence of SEQ ID NO: 100; a16) CDR-H1 having the amino acid sequence of SEQ ID NO: 63, CDR-H2 having the amino acid sequence of SEQ ID NO: 82, and CDR-H3 having the amino acid sequence of SEQ ID NO: 101; a17) CDR-H1 having the amino acid sequence of SEQ ID NO: 64, CDR-H2 having the amino acid sequence of SEQ ID NO: 83, and CDR-H3 having the amino acid sequence of SEQ ID NO: 102; a18) CDR-H1 having the amino acid sequence of SEQ ID NO: 65, CDR-H2 having the amino acid sequence of SEQ ID NO: 84, and CDR-H3 having the amino acid sequence of SEQ ID NO: 103; a19) CDR-H1 having the amino acid sequence of SEQ ID NO: 66, CDR-H2 having the amino acid sequence of SEQ ID NO: 85, and CDR-H3 having the amino acid sequence of SEQ ID NO: 104; Preferably, the heavy chain variable region of the Nanobody or antigen-binding fragment thereof that specifically binds to pepsinogen II 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 a heavy chain variable region of an immunoglobulin derived from mouse, primate, bovine, horse, cattle, porcine, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof; Preferably, the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 29-47, or an amino acid sequence having one or several amino acid substitutions, deletions or additions compared thereto, 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 antigen-binding fragment thereof that specifically binds to pepsinogen II, comprising an immunoglobulin Fc domain and a nanobody or antigen-binding fragment thereof according to any one of claims 1 to 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 antigen binding fragment thereof according to any one of claims 1 to 2 or the heavy chain antibody or antigen binding fragment thereof according to claim 3.

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

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

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

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

11. A pharmaceutical composition comprising: The nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or antigen-binding fragment thereof 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 comprises an additional pharmaceutically active agent.

12. A diagnostic or therapeutic kit comprising: The nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or antigen-binding fragment thereof 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 comprises instructions and / or an administration device.

13. Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1-2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or antigen-binding fragment thereof 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, wherein the product is used for any one of c1)-c3): c1) diagnosing a disease or condition, said disease or condition being associated with pepsinogen II; c2) preventing or treating a disease or condition associated with pepsinogen II; c3) detecting the presence or level of pepsinogen II in the sample; Preferably, the disease or condition comprises a gastric disease, condition or lesion; Preferably, the disease or condition comprises one or more of Helicobacter pylori infection, acute gastritis, chronic gastritis, superficial gastritis, erosive gastritis, gastric ulcer, duodenal ulcer, atrophic gastritis or gastric cancer.

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