Ercovirus VP1 protein nano antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an echovirus VP1 protein nano antibody as well as a preparation method and application thereof. The nano antibody specifically recognizes and binds the echovirus VP1 protein, has good affinity with the echovirus VP1 protein, and can be used for preparing products for diagnosing, preventing and / or treating echovirus infection or diseases caused by the echovirus infection, or detecting the existence or the level of the echovirus or the VP1 protein of the echovirus in a sample.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an echovirus VP1 protein nanobody and a preparation method and application thereof. BACKGROUND

[0002] Enteric Cytopathogenic human Orphan Virus (ECHO virus) is a common enterovirus belonging to the Picornaviridae family, which is mainly transmitted through the digestive tract. The pathogenesis of ECHO virus is through invading the intestinal epithelial cells of the host, replicating and spreading in the cells, directly causing damage to the infected cells, and may cause local lesions or systemic symptoms. The incubation period of infection is usually 3-7 days, and ECHO virus can be transmitted throughout the year, especially in summer and autumn, and repeated infection is also common. The virus is particularly common in children, and the clinical symptoms may include fever, cough, diarrhea, etc.

[0003] With the deepening of the research on ECHO virus, scientists have a clearer understanding of its structure. The structure of ECHO virus mainly includes a viral genome composed of single-stranded positive-sense RNA, an envelope, and various structural proteins such as capsid protein VP1. VP1, as a key component of the virus structure, plays an important role in the replication and assembly of the virus. Its main functions include: binding to the viral RNA genome to form a virus particle, protecting the RNA from degradation. In the assembly process of the virus, VP1 participates in the formation of the virus particle together with other structural proteins, ensuring the integrity and infectivity of the virus. In addition, VP1, as a strong immunogenic protein, can activate the host's immune system and induce the production of specific antibodies. Due to its conservation, VP1 is relatively consistent in different types of ECHO virus. Therefore, the development of antibodies against ECHO virus VP1 protein has become an important research direction in the field of public health, which is expected to provide new ideas for the prevention and detection of viral infection and its related complications. In summary, the capsid protein VP1 of ECHO virus plays an important role in the life cycle of the virus, and the antibodies produced by it are the key to detecting and resisting viral infection. A deep understanding of the function of VP1 and the immune response induced by it will help to develop more effective vaccines and treatment strategies to cope with the challenges of ECHO virus infection.

[0004] Antibodies are a crucial tool in the study of ECHO viruses, particularly for the prevention of ECHO viruses. Developing antibodies against the ECHO virus VP1 protein will provide researchers with powerful tools to further study the functions and regulatory mechanisms of ECHO viruses in various biological processes such as infection, immune evasion, and pathological mechanisms. This will help better understand the infection mechanisms of ECHO viruses, provide new directions and targets for prevention and treatment strategies, and provide rich materials for basic biological research. Through the study of antibodies against the ECHO virus VP1 protein, scientists can identify and verify the antigenic properties of the virus, assess the neutralizing ability of the antibodies, and explore their potential applications in vaccine development. In addition, antibodies against the ECHO virus VP1 protein can also be used for clinical diagnosis to help doctors quickly identify infected cases and develop more effective treatment plans. In summary, the development and application of antibodies against the ECHO virus VP1 protein not only promote the progress of ECHO virus research, but also provide important support for prevention and control measures in the field of public health. SUMMARY

[0005] The first aspect of the present invention aims to provide an echovirus VP1 protein nanobody or antigen-binding fragment thereof.

[0006] The second aspect of the present invention aims to provide an echovirus VP1 protein heavy chain antibody or antigen-binding fragment thereof.

[0007] The third aspect of the present invention aims to provide a chimeric antigen receptor.

[0008] The fourth aspect of the present invention aims to provide a multispecific antibody or antigen-binding fragment thereof.

[0009] The fifth aspect of the present invention aims to provide an isolated nucleic acid molecule.

[0010] The sixth aspect of the present invention aims to provide a vector.

[0011] The seventh aspect of the present invention aims to provide a cell.

[0012] The eighth aspect of the present invention aims to provide a method for preparing 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, or the multispecific antibody or antigen-binding fragment thereof of the fourth aspect of the present invention.

[0013] The ninth aspect of the present invention aims to provide a conjugate.

[0014] The tenth aspect of the present invention aims to provide a pharmaceutical composition.

[0015] The object of the eleventh aspect of this invention is to provide a diagnostic or therapeutic reagent kit.

[0016] 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.

[0017] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides an echovirus VP1 protein nanobody or an antigen-binding fragment thereof, said echovirus VP1 protein nanobody or antigen-binding fragment comprising: The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3 having the amino acid sequences shown in SEQ ID NO: 12, 17, 22, 26, 30, 35, 40, 45, 50, or 55.

[0018] An echovirus VP1 protein nanobody or its antigen-binding fragment thereof, wherein the echovirus VP1 protein nanobody or its antigen-binding fragment comprises a heavy chain variable region, the heavy chain variable region comprising: a1) CDR-H1 having the amino acid sequence shown in SEQ ID NO: 9, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 10, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 11; or a2) CDR-H1 having the amino acid sequence shown in SEQ ID NO: 14, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 15, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 16; or a3) CDR-H1 having the amino acid sequence shown in SEQ ID NO: 19, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 20, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 21; or a4) CDR-H1 having the amino acid sequence shown in SEQ ID NO: 24, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 15, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 25; or a5) CDR-H1 having an amino acid sequence set forth in SEQ ID NO: 28, CDR-H2 having an amino acid sequence set forth in SEQ ID NO: 10, and CDR-H3 having an amino acid sequence set forth in SEQ ID NO: 29; or a6) CDR-H1 having an amino acid sequence set forth in SEQ ID NO: 32, CDR-H2 having an amino acid sequence set forth in SEQ ID NO: 33, and CDR-H3 having an amino acid sequence set forth in SEQ ID NO: 34; or a7) CDR-H1 having an amino acid sequence set forth in SEQ ID NO: 37, CDR-H2 having an amino acid sequence set forth in SEQ ID NO: 38, and CDR-H3 having an amino acid sequence set forth in SEQ ID NO: 39; or a8) CDR-H1 having an amino acid sequence set forth in SEQ ID NO: 42, CDR-H2 having an amino acid sequence set forth in SEQ ID NO: 43, and CDR-H3 having an amino acid sequence set forth in SEQ ID NO: 44; or a9) CDR-H1 having an amino acid sequence set forth in SEQ ID NO: 47, CDR-H2 having an amino acid sequence set forth in SEQ ID NO: 48, and CDR-H3 having an amino acid sequence set forth in SEQ ID NO: 49; or a10) CDR-H1 having an amino acid sequence set forth in SEQ ID NO: 52, CDR-H2 having an amino acid sequence set forth in SEQ ID NO: 53, and CDR-H3 having an amino acid sequence set forth in SEQ ID NO: 54.

[0019] In some embodiments, the heavy chain variable region of the echovirus VP1 protein nanobody or antigen-binding fragment thereof further comprises a framework region of the heavy chain variable region.

[0020] In some embodiments, the framework region of the heavy chain variable region comprises a framework region of a heavy chain variable region of an immunoglobulin derived from a murine, primate, bovine, equine, bovine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, marten, chicken, duck, or goose, or a mutant thereof.

[0021] In some embodiments, the echovirus VP1 protein Nanobody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, 17, 22, 26, 30, 35, 40, 45, 50, or 55, 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.

[0022] In a second aspect of the application, there is provided an echovirus VP1 protein heavy chain antibody or antigen-binding fragment thereof comprising an immunoglobulin Fc domain and a Nanobody or antigen-binding fragment thereof of the first aspect of the application.

[0023] In some embodiments, the immunoglobulin Fc domain comprises an Fc domain derived from an immunoglobulin of a murine, primate, bovine, equine, bovine, porcine, ovine, caprine, canine, feline, lagomorph, camelid, donkey, cervid, mink, chicken, duck, or goose, or a mutant thereof.

[0024] In a third aspect of the application, there is provided a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, the antigen-binding domain comprising a Nanobody or antigen-binding fragment thereof of the first aspect of the application or a heavy chain antibody or antigen-binding fragment thereof of the second aspect of the application.

[0025] In a fourth aspect of the application, there is provided a multispecific antibody or 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 antigen-binding fragment thereof of the first aspect of the application or a heavy chain antibody or antigen-binding fragment thereof of the second aspect of the application.

[0026] In a fifth aspect of the application, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a Nanobody or antigen-binding fragment thereof of the first aspect of the application, a heavy chain antibody or antigen-binding fragment thereof of the second aspect of the application, a chimeric antigen receptor of the third aspect of the application, or a multispecific antibody or antigen-binding fragment thereof of the fourth aspect of the application.

[0027] It will be understood by those skilled in the art that nucleotides in the nucleic acid molecule can be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0028] In some embodiments, the nucleotide sequence encoding the Nanobody or antigen binding fragment thereof of the first aspect of the application comprises: SEQ ID NO: 13, 18, 23, 27, 31, 36, 41, 46, 51, or 56, 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.

[0029] In a sixth aspect of the application, there is provided a vector comprising the nucleic acid molecule of the fifth aspect of the application.

[0030] In some embodiments, the vector can be an expression vector. In some embodiments, the expression vector can comprise a eukaryotic expression vector and / or a prokaryotic expression vector. In some embodiments, the eukaryotic expression vector can comprise, for example, but not limited to, a yeast expression vector, a mammalian expression vector, and an insect expression vector. For example, the expression vector can comprise, but 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.

[0031] In some embodiments, the vector can be selected from the group consisting of a nanoparticle, a liposome, an exosome, a microvesicle, or a gene gun.

[0032] In a seventh aspect of the application, there is provided a cell comprising the Nanobody or antigen binding fragment thereof of the first aspect of the application, the heavy chain antibody or antigen binding fragment thereof of the second aspect of the application, the chimeric antigen receptor of the third aspect of the application, the multispecific antibody or antigen binding fragment thereof of the fourth aspect of the application, the nucleic acid molecule of the fifth aspect of the application, or the vector of the sixth aspect of the application.

[0033] In some embodiments, the cell does not involve reproductive material.

[0034] In some embodiments, the cell can be a host cell routinely used in the art, as long as the expression vector can stably express the nucleic acid molecule carried thereby as the aforementioned Nanobody or antigen binding fragment thereof, heavy chain antibody or antigen binding fragment thereof, chimeric antigen receptor, or multispecific antibody or antigen binding fragment thereof of the present disclosure. In some embodiments, the host cell can be a prokaryotic cell, for example, which can comprise E. coli, and / or a eukaryotic cell, for example, which can comprise 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.

[0035] In some embodiments, the cell can be an immune cell. In some embodiments, the immune cell can include, but is not limited to, a T cell, an NK cell, a DC cell, and a macrophage. In these embodiments, the immune cell can express the above-mentioned chimeric antigen receptor of the present disclosure (i.e., a modified immune cell).

[0036] In an eighth aspect of the present application, there is provided a method for preparing 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, or the multispecific antibody or antigen-binding fragment thereof of the fourth aspect of the present application, by culturing the cell of the seventh aspect of the present application.

[0037] In a ninth aspect of the present application, there is provided a conjugate comprising the nanobody or antigen-binding fragment thereof of the first aspect, or the heavy chain antibody or antigen-binding fragment thereof of the second aspect of the present application; and, a conjugating moiety.

[0038] In some embodiments, the conjugating moiety can include, but is not limited to, a detectable label or a therapeutic agent.

[0039] In some embodiments, the detectable label can be any substance that can be detected by, for example, fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, chemistry, etc. Such labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-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 dot, or a cyanine dye derivative (e.g., Cy7, Alexa 750)), acridinium esters, magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for use with avidin modified to bind the above labels. In some embodiments, such labels can be suitable for use in immunoassays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). In some embodiments, the detectable label is selected from the group consisting of 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 linked to the nanobody or antigen-binding fragment thereof, or the heavy chain antibody or antigen-binding fragment thereof of the present application via linkers of different lengths to reduce potential steric hindrance.

[0040] In some embodiments, the detectable label can include, but is not limited to, an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), a colored substance, biotin, etc.

[0041] In some embodiments, the therapeutic agent can include, but is not limited to, a drug that prevents and / or treats an echovirus infection or a disease caused thereby.

[0042] In some embodiments, the conjugating moiety is selected from a substance that can improve the biological properties of the antibody (e.g., increase the serum half-life), such as a chemical group, e.g., polyethylene glycol (PEG), methyl, ethyl, or a sugar group.

[0043] In a tenth aspect of the present application, there is provided a pharmaceutical composition comprising: 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, or the conjugate of the ninth aspect; and a pharmaceutically acceptable carrier.

[0044] In some embodiments, the pharmaceutical composition can further comprise an additional pharmaceutically active agent.

[0045] In some embodiments, the additional pharmaceutically active agent can be a drug that is biologically active, such as a drug that prevents and / or treats an echovirus infection or a disease caused thereby.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as a mixture.

[0047] In some embodiments, the pharmaceutical composition can be administered by, for example, parenteral, subcutaneous injection, sublingual, rectal, nasal, intravenous injection, intramuscular injection, oral, ocular, topical, etc.

[0048] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, a suspension, a powder, a tablet, a capsule, a granule, a powder, a pill, a disintegrant, a syrup, a spray, a gel, an emulsion, an injection, an elixir, a lozenge, a suppository, etc.

[0049] In an eleventh aspect of the application, a diagnostic or therapeutic kit is provided, comprising: 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.

[0050] In some embodiments, the kit can further comprise instructions and / or a device for administration.

[0051] In some embodiments, the kit can be used for diagnosing an echovirus or a disease caused thereby, and / or detecting the presence or level of an echovirus or a VP1 protein thereof in a sample.

[0052] In some embodiments, the kit can be used for preventing and / or treating an echovirus infection or a disease caused thereby.

[0053] In a twelfth aspect of the application, 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, for the manufacture of a product for any of c1 )-c4): c1 ) diagnosing an echovirus infection or a disease caused thereby; c2) preventing and / or treating an echovirus infection or a disease caused thereby; c3) detecting the presence or level of an echovirus or a VP1 protein thereof in a sample; c4) detecting the presence or level of an echovirus or a VP1 protein thereof for non-diagnostic purposes.

[0054] In some embodiments, the sample is selected from at least one of a bodily fluid, a tissue, a cell, an excretion of a subject.

[0055] In some embodiments, the bodily fluid comprises at least one of blood, lymph.

[0056] In some embodiments, the blood comprises at least one of serum, plasma, dried blood spot, whole blood.

[0057] In some embodiments, the excretion comprises at least one of urine, feces, tears.

[0058] In some embodiments, the subject to be tested comprises a mammal, such as a human, a non-human primate (e.g., chimpanzee, ape), a rodent (e.g., rat, mouse, guinea pig), a pet (e.g., cat, dog), a livestock (e.g., horse, cow, sheep, pig, rabbit).

[0059] In some embodiments, the subject to be tested comprises a human.

[0060] In the present application, the echovirus is ECHO 1-ECHO 7, ECHO 9, ECHO 11-ECHO 21, ECHO 24-ECHO 27, or ECHO 29-ECHO 33; further ECHO 9.

[0061] In the present application, the amino acid sequence of the echovirus VP1 protein comprises the 1-226 amino acids of SEQ ID NO: 1; further comprises SEQ ID NO: 1.

[0062] In the present application, the disease caused by the echovirus infection comprises at least one of gastrointestinal disease, respiratory disease, hepatitis, myocarditis, acute hemorrhagic conjunctivitis, hand-foot-mouth disease, nervous system disease, rash, fever.

[0063] In the present application, the gastrointestinal disease comprises gastroenteritis.

[0064] In the present application, the respiratory disease is selected from upper respiratory tract infection and / or pneumonia.

[0065] In the present application, the nervous system disease is selected from meningitis and / or encephalitis.

[0066] In the present application, the meningitis is selected from aseptic meningitis, and / or non-suppurative meningitis.

[0067] The beneficial effects of the present application are: The present application provides an echovirus VP1 protein nanobody or antigen-binding fragment thereof, which can specifically recognize and bind to the echovirus VP1 protein and has good affinity therewith, and can be used for preparing products for diagnosing, preventing and / or treating echovirus infection or diseases caused thereby, or detecting the presence or level of echovirus or VP1 protein thereof in a sample. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 The figure shows the results of the affinity detection of nanobody 1C3 and antigen.

[0069] Figure 2 The figure shows the results of the affinity detection of nanobody 2G10 and antigen.

[0070] Figure 3A schematic of the results of the antigen affinity assay for Nanobody 1E5 is shown.

[0071] Figure 4 A schematic of the results of the antigen affinity assay for Nanobody 1A10 is shown.

[0072] Figure 5 A schematic of the results of the antigen affinity assay for Nanobody 1F3 is shown.

[0073] Figure 6 A schematic of the results of the antigen affinity assay for Nanobody 2G5 is shown.

[0074] Figure 7 A schematic of the results of the antigen affinity assay for Nanobody 4D3 is shown.

[0075] Figure 8 A schematic of the results of the antigen affinity assay for Nanobody 2A12 is shown.

[0076] Figure 9 A schematic of the results of the antigen affinity assay for Nanobody 2H6 is shown.

[0077] Figure 10 A schematic of the results of the antigen affinity assay for Nanobody 1F7 is shown. DETAILED DESCRIPTION

[0078] In order to make the objectives, technical solutions, and advantages of the present application clearer, the following further describes the present application with reference to examples. The specific examples described herein are intended to explain the present application and are not intended to constitute any limitation on the present application. In addition, in the following description, descriptions of well-known structures and techniques are omitted to avoid unnecessarily obscuring the concept of the present disclosure. Such structures and techniques are also described in many publications.

[0079] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied to the descriptions and claims herein and, unless otherwise specified, terms used in singular form include the plural and vice versa as appropriate for the purposes of this disclosure.

[0080] The expressions "a" and "an" as used herein include plural references unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so on.

[0081] The term "about" as used herein means ± 20% of the numerical value that it precedes. In some embodiments, the term "about" means ± 10% of the numerical value that it precedes. In some embodiments, the term "about" means ± 5% of the numerical value that it precedes.

[0082] K D values : dissociation constant (K D ) is a specific type of equilibrium constant that measures the tendency of a larger object to separate (dissociate) into smaller components, is the inverse 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.

[0083] Nanobodies : natural missing light chain antibodies existing in the peripheral blood of Camelidae, the antibodies only contain one heavy chain variable region (VHH) and two conventional CH2 regions and CH3 regions, but are not easy to stick to each other, even to aggregate into blocks, unlike artificially modified single-chain antibody fragments; the VHH structure cloned and expressed alone has structural stability comparable to that of the original heavy chain antibody and binding activity with antigens, and is the smallest unit known to bind target antigens; the VHH crystal is 2.5 nm long and 4 nm long, and the molecular weight is only about 15 kD, so it is also called nanobody (Nb). Compared with traditional mice, rabbits and other animals that can only recognize polypeptides flat on the surface of antigens, the immune system in Camelidae can recognize complex spatial structures on the surface of antigens and can produce highly specific and high-affinity nanobodies.

[0084] Unlike traditional technologies that rely on classic model animals such as mice, rabbits, monkeys, and sheep, the technical solution of the present application is an antibody produced by the immune system of a llama, known as a "nanobody". Nanobodies are small antibody fragments isolated from immunoglobulins in animals such as camels, which have the same antigen-binding capacity and structural stability as complete antibodies, and are the smallest units known to bind target antigens, with a relative molecular mass of only about 15 kD. Compared with traditional mice, rabbits and other animals that can only recognize polypeptides flat on the surface of antigens, the immune system in animals such as camels can recognize complex spatial structures on the surface of antigens and can produce highly specific and high-affinity nanobodies.

[0085] According to the technical solution of the present application, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, see Table 1 below.

[0086] Table 1

[0087] In addition, because of the degeneracy of bases, substitutions can be made to the bases of the polynucleotide sequence without changing the activity or function of the polynucleotide sequence, see Table 2 below.

[0088] Table 2

[0089] The following examples and drawings are provided to aid the understanding of the present application, but should not be construed as limiting the present application in any way. The actual scope of the present application is set forth in the appended claims. It should be understood that any modifications and changes can be made to the present application without departing from the spirit thereof.

[0090] Example 1. Preparation of Antigen 1.1 Constructing a DNA sequence encoding a human-derived ECHO 9 (Coxsackie virus A23, CAV-23) virus Capsid protein Vp1 protein (ECHO 9 virus Capsid protein Vp1 protein (partial), ECHO virus type 9 VP1 protein) into a pet-28a E. coli expression vector to form an ECHO virus Capsid protein Vp1 recombinant expression plasmid. The amino acid sequence of the complete expressed recombinant ECHO 9 (Coxsackie virus A23, CAV-23) virus Capsid protein Vp1 protein is as follows (with a His tag): MAKYEARGDPESTDRFDAWEISIRDMVQLRRKCEMFTYLRFDVEVTFVITSYQHQGTINQDMPPMTHQIMYIPPGGPIPKKVDGYEWQTSTNPSIFWTEGNAPPRMSIPFISIGNAYSSFYDGWSHFDSKGAYGFNTLNKMGHIYCRHVNKETPAEVTSYIRIYFKPKHVRAWVPRPPRLCQYKNKANVNFDATAVTETRETINTVPVSNHGSNHRGDLAALNTLEHHHHHH*, SEQ ID NO: 1; The corresponding DNA sequence thereof is as follows: ATGGCAAAATACGAGGCACGTGGCGACCCTGAATCTACCGACCGTTTTGATGCGTGGGAAATTTCTATCCGTGATATGGTTCAGCTGCGCCGTAAATGCGAAATGTTCACCTACCTGCGTTTCGATGTTGAAGTTACCTTCGTGATTACCAGCTACCAGCATCAGGGTACGATCAACCAGGATATGCCTCCGATGACCCACCAGATCATGTATATCCCACCGGGCGGTCCGATTCCGAAGAAAGTGGATGGTTACGAATGGCAGACGTCTACTAACCCGAGCATTTTCTGGACTGAAGGCAACGCACCACCGCGCATGAGCATCCCTTTTATCTCCATCGGCAACGCCTACTCCTCTTTTTACGACGGCTGGTCCCATTTCGATTCTAAAGGTGCGTACGGCTTCAACACCCTGAACAAAATGGGCCACATCTATTGCCGCCACGTGAACAAAGAGACGCCGGCTGAAGTTACCAGCTACATCCGTATCTATTTCAAACCAAAACACGTTCGTGCGTGGGTTCCACGCCCACCGCGTCTGTGTCAGTACAAGAACAAAGCCAACGTAAACTTCGATGCAACCGCTGTTACCGAAACCCGTGAGACCATCAACACCGTGCCAGTGAGCAACCACGGCTCTAACCATCGTGGTGACCTGGCCGCTCTGAACACCctcgagcaccaccaccaccaccactga, SEQ ID NO: 2.

[0091] 1.2 Transfect the ECHO 9 (Coxsackie virus A23, CAV-23) virus Capsid protein Vpl recombinant expression plasmid into BL21 (DE3) competent cells, and culture to obtain a monoclonal strain expressing the ECHO 9 (Coxsackie virus A23, CAV-23) virus Capsid protein Vpl protein; 1.3 The strain was cultured in large quantities at 37℃, and then 0.4 mM of inducer (IPTG, isopropyl-β-D-thiogalactoside) was added at 16℃ to induce the expression of ECHO 9 (Coxsackie virus A23, CAV-23)virus Capsidprotein Vp1 protein.

[0092] 1.4 All bacteria were collected and subjected to processes such as lysis, centrifugation, affinity chromatography, and gel filtration chromatography to obtain the recombinant ECHO 9 (Coxsackie virus A23, CAV-23) virus Capsid protein Vp1 protein (i.e., antigen).

[0093] Example 2. Alpaca Immunization In this embodiment, the antigen prepared in Example 1 is used to immunize alpacas. The specific steps are as follows: (1) The alpacas were immunized a total of 4 times. Each time, 0.2 mg of antigen was injected subcutaneously into the animal. The first immunization was recorded as day 1, and the subsequent immunizations were on day 11, day 21 and day 31, respectively. (2) On day 30, before the fourth immunization injection, about 200 mL of alpaca peripheral blood was collected from the vein; (3) On day 45, that is, 14 days after the fourth immunization, about 200 mL of alpaca peripheral blood was collected.

[0094] 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.

[0095] Example 3. Construction of an alpaca nanobody library 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: (1) Lymphocytes were isolated from peripheral blood of alpaca veins using density gradient centrifugation and other methods; (2) Extract total mRNA from lymphocytes and reverse transcribe it into cDNA; (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. Table 3. Primers used to construct nanobody libraries

[0096] (4) connecting the DNA of VHH to a phage surface display screening vector (Phen1) to form a VHH-pIII fusion protein expression vector plasmid library; wherein, pIII is a protein existing on the flagella of phage; (5) transforming the DNA connection product into TG1 competent E. coli by an electrotransformation method, and collecting all colonies after culture, which is a llama nanobody library.

[0097] Compared with the traditional method of separating antibodies from animal serum or lymphocytes, the application can long-term store all nanobody fragments (i.e. library) of the llama, and can continuously support subsequent screening and development of nanobodies.

[0098] Example 4. Phage surface display screening of specific nanobodies This example takes the nanobody library obtained in Example 3 as the source, and obtains antigen-specific nanobodies through phage surface display screening. The specific steps are as follows: (1) Take an appropriate amount of frozen nanobody library, inoculate into LB medium containing host TG1 E. coli, and after culture, add an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S), and continue to culture under appropriate conditions; (2) Extract the amplified phage in the bacterial culture supernatant by PEG-NaC method; (3) Incubate the phage with the antigen prepared in Example 1, and the antigen is pre-fixed on an immunotube (Maxisorp immunotube, ThermoFisher Scientific); (4) Washing: discard the phage, and then rinse the antigen with PBS buffer for 3-5 times, wash and remove the phage that is not specifically combined with the antigen, and retain the phage that is specifically combined with the antigen; (5) Elution: elute the phage that is specifically combined with the antigen with an acidic glycine solution to dissociate the phage from the antigen and retain it.

[0099] At this point, phage expressing specific nanobodies has been obtained, and these phage can be subjected to the following technical operations: (6) Transform into a specific nanobody library: infect the TG1 competent E. coli with the phage again, but do not add the helper phage, and after the phage infection is complete, the specific nanobodies exist in the E. coli in the form of DNA plasmids. Collecting these E. coli, which becomes an antigen-specific nanobody library, and returning to step (1) with this library as raw material for the next round of phage surface display screening; (7) Transforming into single clone nanobody colony: take a small amount of phage obtained in step (5) (about 0.5%), dilute and then infect TG1 competent E. coli again, but no helper phage is added, after the phage infection is completed, evenly spread these E. coli on a bacterial culture dish, and culture to obtain single clone colonies containing nanobody DNA plasmid. Use these single clone colonies as raw materials to identify positive single clone nanobody.

[0100] Example 5. Identifying positive single clone nanobody and sequencing nanobody This example uses the bacterial culture dish with single clone colonies obtained in step (7) of Example 4 to identify positive single clone nanobody. The specific steps are as follows: (1) Pick single clone colonies to a microplate for culture; (2) Add IPTG (isopropyl-β-D-thiogalactoside) to induce VHH-pIII (i.e. fusion protein containing nanobody) expression; (3) Collect the supernatant of the bacterial culture containing nanobody obtained in (2) and incubate with the antigen prepared in Example 1, which is pre-fixed on a 96-well microplate (Maxisorp transparent microplate, ThermoFisher Scientific). Use enzyme-linked immunosorbent assay (ELISA) to detect whether the single clone nanobody binds to the antigen. The main experimental steps are as follows: a. Coating: dilute the antigen with PBS to 5 μg / mL, 50 μL / well, and incubate for coating overnight at 4°C with shaking; b. Blocking: the next day, discard the antigen, and add 100 μL / well of PBS-2% BSA and incubate at room temperature for 1 hour with shaking; c. Washing: 3 times with PBST and 3 times with PBS, 150 μL / well; d. Add the culture supernatant, 50 μL / well, and incubate at room temperature for 1-2 hours with shaking; e. Washing: 3 times with PBST and 3 times with PBS, 150 μL / well; f. Add diluted anti-myc HRP and incubate at room temperature for 1 hour; g. Washing: 3 times with PBST and 3 times with PBS, 150 μL / well; h. Add ELISA color developing substrate and incubate at room temperature for 30 min in the dark; i. Read OD405nm.

[0101] (4) For the monoclonal nanobodies (1C3, 2G10, 1E5, 1A10, 1F3, 2G5, 4D3, 2A12, 2H6, 1F7) that can bind to the antigen, the TG1 strain expressing the relevant monoclonal nanobody was cultured overnight at 37°C, and then the DNA plasmid was extracted and subjected to DNA sequencing to obtain the nucleotide sequence of the nanobody. After translation, the complete amino acid sequence of the nanobody was obtained, as shown in Table 4-13.

[0102] Table 4: Amino acid sequence and nucleotide sequence of 1C3

[0103] Table 5: Amino acid sequence and nucleotide sequence of 2G10

[0104] Table 6: Amino acid sequence and nucleotide sequence of 1E5

[0105] Table 7: Amino acid sequence and nucleotide sequence of 1A10

[0106] Table 8: Amino acid sequence and nucleotide sequence of 1F3

[0107] Table 9: Amino acid sequence and nucleotide sequence of 2G5

[0108] Table 10: Amino acid sequence and nucleotide sequence of 4D3

[0109] Table 11: Amino acid sequence and nucleotide sequence of 2A12

[0110] Table 12: Amino acid sequence and nucleotide sequence of 2H6

[0111] Table 13: Amino acid sequence and nucleotide sequence of 1F7

[0112] Example 6. Small-batch recombinant expression and purification of monoclonal nanobodies (1) The monoclonal nanobodies capable of specifically recognizing and binding to the antigen were obtained in Example 5. The DNA plasmids encoding the nanobodies (1C3, 2G10, 1E5, 1A10, 1F3, 2G5, 4D3, 2A12, 2H6, 1F7) were transformed into BL21 (DE3) competent cells, and the nanobodies were expressed and purified by means of E. coli recombination, and the batch production capacity was about several milligrams.

[0113] (2) The nanobodies of different concentrations were incubated by means of ELISA method, and the affinities of the nanobodies and the antigen were measured according to the binding ability of the nanobodies and the antigen (the antigen prepared in Example 1).

[0114] The detection results are shown in Table 1. Figures 1-10 The affinity values K D of the monoclonal nanobodies 1C3, 2G10, 1E5, 1A10, 1F3, 2G5, 4D3, 2A12, 2H6, 1F7 were about 27.32 nM, 57.59 nM, 16.79 nM, 31.13 nM, 15.34 nM, 10.00 nM, 32.52 nM, 68.10 nM, 10.30 nM, and 28.53 nM, respectively.

[0115] The technical solutions of the present application are not limited to the above specific examples, and any technical variations made according to the technical solutions of the present application fall within the protection scope of the present application.

Claims

1. An echovirus VP1 protein Nanobody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 12, 17, 22, 26, 30, 35, 40, 45, 50, or 55.

2. The Nanobody or antigen-binding fragment thereof according to claim 1, characterized in that, the echovirus VP1 protein Nanobody or antigen-binding fragment thereof comprises a heavy chain variable region comprising: a1) CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 9, CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 10, and CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 11; or a2) CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 14, CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 15, and CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 16; or a3) CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 19, CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 20, and CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 21; or a4) CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 24, CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 15, and CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 25; or a5) CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 28, CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 10, and CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 29; or a6) CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 32, CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 33, and CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 34; or a7) CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 37, CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 38, and CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 39; or a8) CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 42, CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 43, and CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 44; or a9) CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 47, CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 48, and CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 49; or a10) CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 52, CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 53, and CDR-H3 having an amino acid sequence as set forth in SEQ ID NO:

54. a9) CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 47, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 48, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 49; or a10) CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 52, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 53, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 54; Preferably, the heavy chain variable region of the echovirus VP1 protein nanobody or antigen binding fragment thereof further comprises a framework region of the heavy chain variable region; Preferably, the framework region of the heavy chain variable region comprises a framework region of a heavy chain variable region of an immunoglobulin derived from a murine, primate, bovine, equine, bovine, porcine, ovine, caprine, canine, feline, lagomorph, camelid, donkey, cervid, mink, chicken, duck, or goose, or a mutant thereof; Preferably, the echovirus VP1 protein nanobody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, 17, 22, 26, 30, 35, 40, 45, 50, or 55, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

3. An echovirus VP1 protein heavy chain antibody or antigen binding fragment thereof comprising an immunoglobulin Fc domain and the nanobody or antigen binding fragment thereof of any one of claims 1-2.

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

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

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

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

9. A method of producing 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, 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 1-2, or the heavy chain antibody or antigen-binding fragment thereof according to claim 3; and, a conjugating moiety; Preferably, the conjugating moiety comprises a detectable label or a therapeutic agent; Preferably, the detectable label comprises an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, a colored substance, and / or biotin; Preferably, the therapeutic agent comprises a drug that prevents and / or treats an echovirus infection or a disease caused thereby.

11. A pharmaceutical composition 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, 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 1-2, 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 a device for administration.

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, for the manufacture of a product for any one of c1) - c4): c1) diagnosing an echovirus infection or a disease caused thereby; c2) preventing and / or treating an echovirus infection or a disease caused thereby; c3) detecting the presence or level of an echovirus or of a VP1 protein thereof in a sample; c4) detecting the presence or level of an echovirus or of a VP1 protein thereof for non-diagnostic purposes.

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