Pepsinogen ii nanobodies and uses thereof

Abstract

The present application belongs to the field of biotechnology, and particularly relates to nanobody of pepsinogen II and application thereof. The nanobody can specifically recognize and combine with pepsinogen II, and has good affinity therewith, and can be used for preparing products for diagnosing, preventing or treating gastric diseases and / or diseases or disorders related to pepsinogen II, or detecting the presence or level of pepsinogen II in a sample.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to pepsinogen II nanobodies and their applications. Background Technology

[0002] Pepsinogen II (PGII) is one of the precursors of two pepsin proteins secreted by the stomach and involved in digestion. It is mainly synthesized and secreted into the gastric lumen by gastric enzyme cells and mucus neck cells in the gastric fundus glands. Human PGII is a polypeptide chain composed of 388 amino acids. It is inactive on its own but can be processed into mature pepsin by gastric acid or activated pepsin in the gastric lumen. Pepsin hydrolyzes proteins in food, primarily acting on peptide bonds containing phenylalanine or tyrosine in proteins and polypeptide molecules. Normally, about 1% of pepsinogen enters the bloodstream through the gastric mucosal capillaries. Once in the bloodstream, pepsinogen is very stable. When pathological changes occur in the gastric mucosa, the serum pepsinogen level also changes accordingly. PGII is highly correlated with gastric fundus mucosal lesions. Its elevation is associated with atrophy of gastric fundic glands, gastric metaplasia or pseudopyloric gland metaplasia, and atypical proliferation. Therefore, monitoring the concentration of serum PGII can be used as an important means of monitoring 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 is significantly increased in early patients. In addition, in different gastric diseases, the clinical diagnosis of gastric lesions is often made by combining the results of PGI and PGII content detection.

[0003] Currently, scientific research or clinical diagnostic testing of PGII mainly relies on antibodies. There are already a variety of monoclonal antibodies for PGII available on the market. The technical solutions for preparing these antibodies are: (1) extracting or recombinantly expressing PGII protein antigen from tissues; (2) injecting the antigen into animals such as mice, rabbits, and monkeys, relying on the animal's immune system to produce antibodies; (3) collecting the animal's blood and separating and extracting serum, and then further separating the components containing specific antibodies.

[0004] However, the above-mentioned techniques for producing and extracting PGII antibodies based on the immune systems of animals such as mice have the following disadvantages: (1) The antibodies are relatively unstable and require storage, transportation, testing, and verification at low temperatures (4°C) to maintain their concentration, specificity, and other characteristics, which is not conducive to long-distance transportation and widespread detection; (2) Large-scale mass production is costly. 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 relatively expensive expression systems such as mammalian cells followed by separation, purification, and extraction. The operation is complex and costly, which is not conducive to large-scale mass production. Summary of the Invention

[0005] The present invention aims to develop nanobodies based on the alpaca immune system, obtain nanobodies that can specifically recognize and bind to PGII, and mass-produce them.

[0006] The first aspect of the present invention aims to provide nanobodies or antigen-binding fragments thereof that specifically bind to pepsinogen II.

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

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

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

[0010] The fifth aspect of this invention aims to provide isolated nucleic acid molecules.

[0011] The sixth aspect of this invention is to provide a carrier.

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

[0013] 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.

[0014] The object of the ninth aspect of the present invention is to provide a coupling.

[0015] The object of the tenth aspect of this invention is to provide a pharmaceutical composition.

[0016] The object of the eleventh aspect of this invention is to provide a diagnostic or therapeutic reagent kit.

[0017] 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.

[0018] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0019] In a first aspect, the present invention provides a nanobody or antigen-binding fragment thereof that specifically binds to pepsinogen II, said nanobody or antigen-binding fragment comprising a heavy chain variable region, said heavy chain variable region comprising: 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 NO: 29-47, or an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of CDR-H1, CDR-H2 or CDR-H3 contained in said heavy chain variable region.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0038] a18) CDR-H1 having the amino acid sequence shown in SEQ ID NO:65, CDR-H2 having the amino acid sequence shown in SEQ ID NO:84 and CDR-H3 having the amino acid sequence shown in 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 that specifically binds to pepsinogen II or its antigen-binding fragment further includes the 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 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.

[0042] In some embodiments, the heavy chain variable region includes an amino acid sequence shown in any one of SEQ ID NO:29-47, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, 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.

[0043] In some embodiments, the pepsinogen II is human pepsinogen II; more specifically, it is pepsinogen II with the amino acid sequence shown in SEQ ID NO:1.

[0044] A second aspect of the present invention provides 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 of the first aspect of the present invention.

[0045] 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.

[0046] A third aspect of the present 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 present invention or a heavy chain antibody or an antigen-binding fragment thereof of the second aspect of the present invention.

[0047] 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.

[0048] A fifth aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a nanobody or antigen-binding fragment thereof of the first aspect of the invention, a heavy chain antibody or antigen-binding fragment thereof of the second aspect, a chimeric antigen receptor of the third aspect, or a multispecific antibody or antigen-binding fragment thereof of the fourth aspect.

[0049] 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.

[0050] In some embodiments, the nucleotide sequence encoding the nanobody or antigen-binding fragment thereof of the first aspect of the present invention comprises: the nucleotide sequence shown in any one of SEQ ID NO: 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 with it.

[0051] A sixth aspect of the present invention provides a carrier 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 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.

[0053] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0054] 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 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, or a carrier thereof of the sixth aspect of the present invention.

[0055] In some embodiments, the cells do not involve reproductive material.

[0056] 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.

[0057] 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 in this disclosure (i.e., modified immune cells).

[0058] 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.

[0059] 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.

[0060] In some implementations, the coupling portion may include, but is not limited to, a detectable marker, a delivery carrier, or a therapeutic agent.

[0061] 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, or heavy chain antibodies or their antigen-binding fragments, of this disclosure using linkers of different lengths to reduce potential steric hindrance.

[0062] 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.

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

[0064] 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.

[0065] In a tenth aspect of the present invention, a pharmaceutical composition is provided 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.

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

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

[0068] In some embodiments, the nanobody or its antigen-binding fragment is provided as a separate component or as a mixed component with the additional pharmaceutically active agent.

[0069] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[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] 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, a chimeric antigen receptor thereof of the third aspect, a multispecific antibody or antigen-binding fragment thereof of the fourth aspect, a nucleic acid molecule thereof of the fifth aspect, a carrier thereof of the sixth aspect, a cell thereof of the seventh aspect, a conjugate thereof of the ninth aspect, or a pharmaceutical composition thereof of the tenth aspect.

[0072] In some embodiments, the kit may also include instructions and / or a drug delivery device.

[0073] In some embodiments, the kit can be used to diagnose diseases or conditions related to pepsinogen II.

[0074] In some embodiments, the kit can be used to prevent or treat diseases or conditions related to pepsinogen II.

[0075] 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):

[0076] c1) Diagnose a disease or condition that is related to pepsinogen II;

[0077] c2) Prevention or treatment of a disease or condition that is related to pepsinogen II;

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

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

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

[0081] In some embodiments, the blood includes 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 excrement includes at least one of urine, feces, and tears.

[0084] 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).

[0085] In some implementations, the subject of the test includes humans.

[0086] In this invention, the disease or symptom includes gastric diseases, conditions, or lesions. In some embodiments, the disease or symptom 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] This invention provides nanobodies or antigen-binding fragments thereof that specifically bind to pepsinogen II. These nanobodies can specifically recognize and bind to pepsinogen II and have good affinity for it. They can be mass-produced and used to prepare products for the diagnosis, prevention or treatment of diseases or conditions related to pepsinogen II, or for the detection of the presence or level of pepsinogen II in samples. Attached Figure Description

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

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

[0090] Figure 3 A schematic diagram showing the results of the affinity test between nanobody 15H5 and the antigen is presented.

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

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

[0093] Figure 6 A schematic diagram showing the results of the affinity test between nanobody 15A5 and the antigen is presented.

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

[0095] Figure 8 A schematic diagram showing the results of the affinity test between nanobody 10H7 and the antigen is presented.

[0096] Figure 9A schematic diagram showing the results of the affinity test between nanobody 12C2 and the antigen is presented.

[0097] Figure 10 A schematic diagram showing the results of the affinity test between nanobody 12G6 and the antigen is presented.

[0098] Figure 11 A schematic diagram showing the results of the affinity test between nanobody 9D7 and the antigen is presented.

[0099] Figure 12 A schematic diagram showing the results of the affinity test between the nanobody 7C11 and the antigen is presented.

[0100] Figure 13 A schematic diagram showing the results of the affinity test between nanobody 13C8 and the antigen is presented.

[0101] Figure 14 A schematic diagram showing the results of the affinity test between the nanobody 6C8 and the antigen is presented.

[0102] Figure 15 A schematic diagram showing the results of the affinity test between nanobody 9D10 and the antigen is presented.

[0103] Figure 16 A schematic diagram showing the results of the affinity test between nanobody 9A4 and the antigen is presented.

[0104] Figure 17 A schematic diagram showing the results of the affinity test between nanobody 14B7 and the antigen is presented.

[0105] Figure 18 A schematic diagram showing the results of the affinity test between nanobody 16G7 and the antigen is presented.

[0106] Figure 19 A schematic diagram showing the results of the affinity test between nanobody 16E3 and the antigen is presented. Detailed Implementation

[0107] 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.

[0108] definition

[0109] 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.

[0110] 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.

[0111] 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.

[0112] Pepsinogen II (PGII) Human PGII is one of the precursors of two pepsin proteins secreted by the stomach and involved in digestion. It is mainly synthesized and secreted into the gastric lumen by pepsinocytes and mucus neck cells of the gastric fundus glands. Human PGII is a polypeptide chain composed of 388 amino acids. It is inactive on its own but can be processed into mature pepsin by gastric acid or activated pepsin in the gastric lumen. Pepsin hydrolyzes proteins in food, primarily acting on peptide bonds containing phenylalanine or tyrosine in proteins and polypeptide molecules. Normally, about 1% of pepsinogen enters the bloodstream through the gastric mucosal capillaries. Once in the bloodstream, pepsinogen is very stable. When pathological changes occur in the gastric mucosa, the serum pepsinogen level also changes accordingly.

[0113] 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.

[0114] NanobodyNaturally occurring antibodies lacking the light chain, found in the peripheral blood of camels, 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 readily adhere to each other or aggregate. Individually cloned and expressed VHH structures exhibit structural stability and antigen-binding activity comparable to the original heavy chain antibody, representing 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 antigen surface, producing 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 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.

[0116] As used herein, the term “complementarity-determining region (CDR)” refers to the amino acid residues in the variable region of an antibody 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 combinations thereof. For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. The correspondences between different numbering systems are well known to those skilled in the art, and some commonly used software can be used to define HCDRs 1-3. For example, the Kabat numbering system is an immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., 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, proposed by Chothia et al., is a classic rule for identifying CDR boundaries based on the location of structural loop regions (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 based on the international ImMunoGeneTics information (IMGT) system 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., focusing on the structural alignment of different frame regions of unconventional lengths; it can be completed using the Chothia numbering system corrected by ABnum software. The Contact numbering system defines the CDR region based on existing antibody complex crystal structure data. The Honegger numbering system (also known as the AHo numbering system) is based on structural alignment of 3D structures covering observed length variations of immunoglobulin variable regions, allowing the definition of structurally conserved Cα positions, thus deriving appropriate FR regions and CDR lengths.The Gelfand numbering system is a relatively complex numbering scheme described by Gelfand et al., in which a variable chain sequence is divided into 21 segments called “words”, each of which matches a secondary structure element (chain or loop). Chains are defined by alphabetical order of letters (e.g., A, B, C), and loops are defined by two letters corresponding to adjacent chains (e.g., AB, BC…). This numbering system does not include gaps or missing points, but allows for precise comparison of the 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 conservedly substituted without changing the activity or function of the protein, as shown in Table 1 below:

[0118] Table 1

[0119] 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] 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:

[0121] Table 2

[0122]

[0123]

[0124] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0125] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (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 percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0126] 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.

[0127] Example 1. Preparation of antigen

[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 and gel filtration chromatography, the complete PGII protein was obtained as an antigen.

[0129] The specific steps are as follows:

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

[0131] 1.2 The constructed plasmid was transformed into DH10Bac competent cells (Shanghai Weidi, DL1071M);

[0132] 1.3 After blue-white screening, DH10Bac single clones were selected and cultured, and then Bacmid baculovirus was extracted;

[0133] 1.4 Using Cellfectin II transfection reagent (ThermoFisher, 10362100), Bacmid baculovirus was transfected into SF9 insect cells. After culturing, the cell culture supernatant was collected as the P1 generation virus.

[0134] 1.5 P1 virus was used to infect SF9 insect cells, and the cell supernatant was collected after culture to obtain P2 generation virus;

[0135] 1.6 P2 virus was used to infect SF9 insect cells, and the cell supernatant was collected after culture to obtain P3 generation virus;

[0136] 1.7 Using P3 virus to infect SF9 insect cells, PGII with a histidine tag (6*His-tag) was recombinantly expressed in SF9 insect cells;

[0137] 1.8 Collect SF9 insect cells expressing recombinant PGII, sonicate to disrupt and lyse them, centrifuge, and collect the supernatant;

[0138] 1.9 Histidine-tagged affinity chromatography: The supernatant obtained after centrifugation was incubated with nickel affinity chromatography packing material, and then recombinant PGII with histidine tags bound to the nickel packing material was eluted;

[0139] 1.10 Gel filtration chromatography: The product obtained after the above affinity chromatography was concentrated and then purified recombinant PGII was obtained by gel filtration chromatography.

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

[0141] MKWMVVVLVCLQLLEAAVVKVPLKKFKSIRETMKEKGLLGEFLRTHKYDPAWKYRFGDLSVTYEPMAYMDAAYFGEISIGTPPQNFLVLFDTGSSNLWVPSVYCQSQACTSHSRFNPSESSTYSTNGQTFSLQYGSGSLTGFFGYDTLTVQSIQVPNQEFGLSENEPGTNFVYAQFDGIMGLAYPALSVDEATT AMQGMVQEGALTSPVFSVYLSNQQGSSGGAVVFGGVDSSLYTGQIYWAPVTQELYWQIGIEEFLIGGQASGWCSEGCQAIVDTGTSLLTVPQQYMSALLQATGAQEDEYGQFLVNCNSIQNLPSLTFIINGVEFPLPPSSYILSNNGYCTVGVEPTYLSSQNGQPLWILGDVFLRSYYSVYDLGNNRVGFATAA

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

[0143] MSYYHHHHHHDYDIPTTENLYFQGAMAMKWMVVVLVCLQLLEAAVVKVPLKKFKSIRETMKEKGLLGEFLRTHKYDPAWKYRFGDLSVTYEPMAYMDAAYFGEI SIGTPPQNFLVLFDTGSSSNLWVPSVYCQSQACTSHSRFNPSESSTYSTNGQTFSLQYGSGSLTGFFGYDTLTVQSIQVPNQEFGLSENEPGTNFVYAQFDGIMG LAYPALSVDEATTAMQGMVQEGALTSPVFSVYLSNQQGSSGGAVVFGGVDSSLYTGQIYWAPVTQELYWQIGIEEFLIGGQASGWCSEGCQAIVDTGTSLLTVP QQYMSALLQATGAQEDEYGQFLVNCNSIQNLPSLTFIINGVEFPLPPSSYILSNNGYCTVGVEPTYLSSQNGQPLWILGDVFLRSYYSVYDLGNNRVGFATAA.

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

[0145]

[0146] Example 2. Alpaca Immunization Injection

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

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

[0149] (3) On day 45, that is, 14 days after the fourth immunization, about 200 mL of alpaca peripheral blood was collected.

[0150] 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.

[0151] Example 3. Construction of an alpaca nanobody library

[0152] Using two batches of alpaca peripheral blood collected in Example 2 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:

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

[0154] (2) Extract total mRNA from lymphocytes and reverse transcribe it into cDNA;

[0155] (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.

[0156] Table 3. Primers used to construct nanobody libraries

[0157]

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

[0159] (5) The DNA ligation product was transformed into TG1 competent bacteria by electroporation. After appropriate culture, all colonies were collected to form the alpaca nanobody library.

[0160] Compared to traditional methods of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, the method of 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.

[0161] Example 4. Displaying screening-specific nanobodies on the surface of bacteriophages

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

[0163] (1) Take the frozen nanobody library and inoculate it into a culture medium containing host bacteria (TG1 Escherichia coli). After culturing, add helper phage (M13KO7 helper phage, NEB, N0315S) and continue culturing.

[0164] (2) Phages amplified in bacterial culture supernatant were extracted using the PEG-NaCl method;

[0165] (3) The phage was incubated with the recombinant PGII antigen prepared in Example 1. The antigen was pre-fixed in an immunoassay tube (Maxisorp immunoassay tube, ThermoFisher Scientific).

[0166] (4) Washing. Discard the phages, then rinse the antigen with PBS buffer an appropriate number of times (3-5 times) to remove the phages that are not specifically bound to the antigen, and retain the phages that are specifically bound to the antigen;

[0167] (5) Elution. The phages were eluted with an acidic glycine solution to dissociate them from the antigens and retain them.

[0168] At this point, phages expressing specific nanobodies have been obtained. These phages then proceed to the following steps:

[0169] (6) Transformation into a specific nanobody library. The phage is reinfected into *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* bacteria forms an antigen-specific nanobody library. This library serves as the raw material for returning to step (1) for the next round of phage surface display screening.

[0170] (7) Transform into monoclonal nanobody colonies. Take a small amount of the phage obtained in step (5) (e.g., 0.5%), dilute it, and infect E. coli again, but do not add helper phage. After the phage infection is complete, spread these E. coli evenly on a bacterial culture dish and culture to obtain monoclonal colonies containing nanobody DNA plasmids.

[0171] Example 5. Identification of positive monoclonal nanobodies

[0172] The bacterial culture dish with monoclonal colonies obtained through step (7) of Example 4 can be used for the identification of positive monoclonal nanobodies. The specific technical solution is as follows:

[0173] (1) Pick single colonies and culture them in microplates.

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

[0175] (3) Collect the bacterial culture supernatant containing nanobodies and incubate it with the antigen, which is pre-fixed in a 96-well microplate (Maxisorp transparent microplate, ThermoFisher Scientific).

[0176] (4) Use enzyme-linked immunosorbent assay (ELISA) to detect whether monoclonal nanobodies bind to antigens.

[0177] The main experimental steps are as follows:

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

[0179] 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.

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

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

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

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

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

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

[0186] 9) Read OD450nm.

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

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] Example 6. Recombinant Expression and Purification of Small-Batch Monoclonal Nanobodies

[0197] (1) In Example 5, a monoclonal nanobody that can specifically recognize and bind to the antigen was obtained. The DNA plasmid of the nanobody was transformed into BL21(DE3) competent cells. The monoclonal nanobody was expressed and purified in small batches using the E. coli expression system, with a batch production capacity of about a few milligrams.

[0198] (2) Using the ELISA method (same as in Example 5), nanobodies of different concentrations were incubated, and the affinity between the nanobodies and the antigen (prepared by the same method as in Example 1) was measured.

[0199] The results of the affinity tests between each nanobody and 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] 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 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. A nanobody or antigen-binding fragment thereof that specifically binds to pepsinogen II, wherein the nanobody or antigen-binding fragment thereof includes a heavy chain variable region, the heavy chain variable region comprising: CDR-H1 with the amino acid sequence shown in SEQ ID NO: 48, CDR-H2 with the amino acid sequence shown in SEQ ID NO: 67, and CDR-H3 with the amino acid sequence shown in SEQ ID NO:

86.

2. The nanobody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region of the nanobody that specifically binds to pepsinogen II or its antigen-binding fragment also includes the framework region of the heavy chain variable region.

3. The nanobody or its antigen-binding fragment according to claim 2, characterized in that, The frame region of the heavy chain variable region includes the frame 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.

4. The nanobody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:

29.

5. 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 as described in any one of claims 1-4.

6. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a nanobody or an antigen-binding fragment thereof as described in any one of claims 1-4, or a heavy chain antibody or an antigen-binding fragment thereof as described in claim 5.

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

8. A cell comprising the nanobody or antigen-binding fragment thereof as described in any one of claims 1-4, the heavy chain antibody or antigen-binding fragment thereof as described in claim 5, the nucleic acid molecule as described in claim 6, or the carrier as described in claim 7.

9. The method for preparing the nanobody or its antigen-binding fragment according to any one of claims 1-4, and the heavy chain antibody or its antigen-binding fragment according to claim 5, is obtained by culturing the cells according to claim 8.

10. A pharmaceutical composition comprising: The nanobody or its antigen-binding fragment according to any one of claims 1-4, the heavy chain antibody or its antigen-binding fragment according to claim 5, the nucleic acid molecule according to claim 6, the carrier according to claim 7, or the cell according to claim 8; and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10, characterized in that, The pharmaceutical composition also includes additional pharmaceutically active agents.

12. The use of the nanobody or antigen-binding fragment thereof according to any one of claims 1-4, the heavy chain antibody or antigen-binding fragment thereof according to claim 5, the nucleic acid molecule according to claim 6, the carrier according to claim 7, or the cell according to claim 8 in the preparation of a product, wherein the product is used to detect the presence or level of pepsinogen II in a sample.

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