Rotavirus VP6 protein nano antibody and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a rotavirus VP6 protein nano antibody and application thereof. The nano antibody can specifically recognize and bind to the rotavirus VP6 protein, has good affinity with the rotavirus VP6 protein, and can be used for preparing products for diagnosing, preventing or treating diseases or symptoms related to the rotavirus VP6 protein or detecting the existence or level of the rotavirus VP6 protein in a sample.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to rotavirus VP6 protein nanobody and application thereof. BACKGROUND

[0002] Rotavirus is a common enterovirus that mainly causes severe diarrhea in infants and young children, belonging to the genus Rotavirus of the family Reoviridae. Rotavirus is transmitted through oral-oral transmission and contaminated food or water, and its pathogenesis is to invade the intestinal epithelial cells, replicate in the cells, cause cell damage and dysfunction, and thus cause diarrhea, dehydration and other systemic symptoms. The incubation period of infection is usually 1-3 days, and rotavirus infection is more common in winter and spring, especially in infants and young children, and repeated infection is also common.

[0003] With the deepening of the study of rotavirus, scientists have a more comprehensive understanding of its structure. The structure of rotavirus is mainly composed of a double-layered capsid, which contains the viral double-stranded RNA genome, the intermediate capsid protein VP6 and other structural proteins. VP6 is the main component of the rotavirus capsid and plays a key role in the replication and assembly of the virus. Its main functions include: binding to the viral RNA genome to form a stable capsid structure, protecting the RNA from degradation. During virus assembly, VP6 participates in the formation of virus particles together with other structural proteins, ensuring the integrity and infectivity of the virus. In addition, VP6, as a strong immunogenic protein, can activate the host's immune system and induce the production of specific antibodies. Due to its conservation, VP6 has high consistency in different types of rotavirus. Therefore, the development of antibodies against rotavirus VP6 protein has become an important research direction in the field of public health, and is expected to provide new ideas for the prevention and detection of viral infection and its related complications. In summary, VP6 plays an important role in the life cycle of rotavirus, and the antibodies produced by it are the key to detecting and resisting viral infection. A deeper understanding of the function of VP6 and the immune response induced by it will help to develop more effective vaccines and treatment strategies to cope with the challenges of rotavirus infection.

[0004] Antibodies are a crucial tool in studying rotaviruses, particularly for their prevention. Developing antibodies against rotavirus VP6 will provide researchers with powerful tools to further study the functions and regulatory mechanisms of rotavirus in various biological processes such as infection, immune evasion, and pathological mechanisms. This will help better understand the infection mechanisms of rotavirus, provide new directions and targets for prevention and treatment strategies, and provide rich materials for basic biological research. Through the study of rotavirus VP6 antibodies, 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 rotavirus 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 rotavirus VP6 antibodies not only promote the progress of rotavirus research, but also provide important support for prevention and control measures in the field of public health. SUMMARY

[0005] The present application aims to develop nanobodies based on the immune system of llamas, obtain nanobodies that can specifically recognize and bind to the intermediate capsid protein VP6 of rotavirus, and mass-produce and prepare them.

[0006] The first aspect of the present application aims to provide nanobodies or antigen-binding fragments thereof that specifically bind to rotavirus VP6 protein.

[0007] The second aspect of the present application aims to provide heavy chain antibodies or antigen-binding fragments thereof that specifically bind to rotavirus VP6 protein.

[0008] The third aspect of the present application aims to provide chimeric antigen receptors.

[0009] The fourth aspect of the present application aims to provide multispecific antibodies or antigen-binding fragments thereof.

[0010] The fifth aspect of the present application aims to provide isolated nucleic acid molecules.

[0011] The sixth aspect of the present application aims to provide a vector.

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

[0013] The eighth aspect of the present application aims to provide a conjugate.

[0014] The ninth aspect of the present application aims to provide a pharmaceutical composition.

[0015] The tenth aspect of the present application aims to provide a diagnostic or therapeutic kit.

[0016] The eleventh aspect of the present application aims to provide 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 eighth aspect, or the pharmaceutical composition of the ninth aspect.

[0017] To achieve the above-mentioned object, the technical solutions adopted by the present application are as follows: The first aspect of the present application provides a nanobody or antigen-binding fragment thereof specifically binding to rotavirus VP6 protein, the nanobody or antigen-binding fragment thereof comprising a heavy chain variable region, the heavy chain variable region comprising: an amino acid sequence of HCDR1, HCDR2 and HCDR3 comprised in the heavy chain variable region as shown in any one of SEQ ID NO: 10, 15, 20 and 25, or an amino acid sequence having substitution, deletion or addition of one or several amino acids compared with the amino acid sequence of HCDR1, HCDR2 or HCDR3 contained in the heavy chain variable region.

[0018] In some embodiments, the heavy chain variable region comprises: a1) HCDR1 having the amino acid sequence shown in SEQ ID NO: 11, HCDR2 having the amino acid sequence shown in SEQ ID NO: 12 and HCDR3 having the amino acid sequence shown in SEQ ID NO: 13; or a2) HCDR1 having the amino acid sequence shown in SEQ ID NO: 16, HCDR2 having the amino acid sequence shown in SEQ ID NO: 17 and HCDR3 having the amino acid sequence shown in SEQ ID NO: 18; or a3) HCDR1 having the amino acid sequence shown in SEQ ID NO: 21, HCDR2 having the amino acid sequence shown in SEQ ID NO: 22 and HCDR3 having the amino acid sequence shown in SEQ ID NO: 23; or a4) HCDR1 having the amino acid sequence shown in SEQ ID NO: 26, HCDR2 having the amino acid sequence shown in SEQ ID NO: 27 and HCDR3 having the amino acid sequence shown in SEQ ID NO: 28.

[0019] In some embodiments, the heavy chain variable region of the nanobody or antigen-binding fragment thereof specifically binding to rotavirus VP6 protein 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, mink, chicken, duck, or goose, or a mutant thereof.

[0021] In some embodiments, the heavy chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 10, 15, 20, and 25, or an amino acid sequence having one or several amino acid substitutions, deletions, or additions compared thereto, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0022] In some embodiments, the rotavirus VP6 protein is a human rotavirus VP6 protein.

[0023] In a second aspect of the present application, there is provided a heavy chain antibody or an antigen-binding fragment thereof that specifically binds to a rotavirus VP6 protein, comprising an immunoglobulin Fc domain and a nanobody or an antigen-binding fragment thereof of the first aspect of the present application.

[0024] In some embodiments, the immunoglobulin Fc domain comprises an Fc domain of an immunoglobulin derived from a murine, primate, bovine, equine, bovine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, mink, chicken, duck, or goose, or a mutant thereof.

[0025] In a third aspect of the present 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 an antigen-binding fragment thereof of the first aspect of the present application or a heavy chain antibody or an antigen-binding fragment thereof of the second aspect of the present application.

[0026] In a fourth aspect of the present application, there is provided a multispecific antibody or an antigen-binding fragment thereof comprising two or more (e.g., three or four) antigen-binding domains, wherein one of the antigen-binding domains comprises a nanobody or an antigen-binding fragment thereof of the first aspect of the present application or a heavy chain antibody or an antigen-binding fragment thereof of the second aspect of the present application.

[0027] In a fifth aspect of the present application, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a nanobody or an antigen-binding fragment thereof of the first aspect of the present application, a heavy chain antibody or an antigen-binding fragment thereof of the second aspect of the present application, a chimeric antigen receptor of the third aspect of the present application, or a multispecific antibody or an antigen-binding fragment thereof of the fourth aspect of the present application.

[0028] It will be appreciated by persons skilled in the art that nucleotides in nucleic acid molecules can be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimised.

[0029] In some embodiments, the nucleotide sequence encoding the Nanobody or antigen binding fragment thereof of the first aspect of the application comprises the nucleotide sequence set forth in any one of SEQ ID NO: 9, 14, 19 or 24, 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.

[0030] In a sixth aspect of the application, there is provided a vector comprising the nucleic acid molecule of the fifth aspect of the application.

[0031] 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 comprises, for example, but is not limited to, a yeast expression vector, a mammalian expression vector, and an insect expression vector. For example, the expression vector can comprise, but is not limited to, a plasmid, a retroviral vector, a lentiviral vector, a bacteriophage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

[0032] In some embodiments, the vector can be selected from a nanoparticle, a liposome, an exosome, a microvesicle, or a gene gun.

[0033] 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.

[0034] In some embodiments, the cell does not involve reproductive material.

[0035] In some embodiments, the cell can be a host cell routinely used in the art, as long as it is capable of stably expressing the nucleic acid molecule carried by the expression vector as the above-mentioned Nanobody or antigen-binding fragment thereof, the heavy chain antibody or antigen-binding fragment thereof, the chimeric antigen receptor or antigen-binding fragment thereof, or the 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 include E. coli, and / or a eukaryotic cell, for example, which can include 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.

[0036] In some embodiments, the cell can be an immune cell. In some embodiments, the immune cell can include, but is not limited to, T cells, NK cells, DC cells, and macrophages. In these embodiments, the immune cell can express the above-mentioned chimeric antigen receptor of the present disclosure (i.e., a modified immune cell).

[0037] The present application also provides 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.

[0038] In an eighth aspect of the present application, a conjugate is provided, which comprises 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.

[0039] In some embodiments, the conjugating moiety can include, but is not limited to, a detectable label, a delivery vehicle, or a therapeutic agent.

[0040] In some embodiments, the detectable label can be any substance that can be detected by 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 (e.g., streptavidin) modified to bind the above labels. In some embodiments, such labels can be suitable for use in immunological detection methods (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, 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 can be linked to the nanobodies or antigen-binding fragments thereof, or heavy chain antibodies or antigen-binding fragments thereof, of the present disclosure via linkers of different lengths to reduce potential steric hindrance.

[0041] 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.

[0042] In some embodiments, the therapeutic agent can include, for example, but is not limited to, a chemotherapeutic agent, an immunosuppressant, a cytotoxic drug.

[0043] 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), which can be, for example, a chemical group such as polyethylene glycol (PEG), a methyl group, an ethyl group, or a sugar group.

[0044] In a ninth aspect of the present disclosure, a pharmaceutical composition is provided, which comprises: the nanobodies or antigen-binding fragments thereof of the first aspect, the heavy chain antibodies or antigen-binding fragments thereof of the second aspect, the chimeric antigen receptors of the third aspect, the multispecific antibodies or antigen-binding fragments thereof of the fourth aspect, the nucleic acid molecules of the fifth aspect, the vectors of the sixth aspect, the cells of the seventh aspect, or the conjugates of the eighth aspect; and a pharmaceutically acceptable carrier.

[0045] In some embodiments, the pharmaceutical composition can further comprise an additional pharmaceutically active agent.

[0046] In some embodiments, the additional pharmaceutically active agent can be a drug having a biological activity, for example, a drug capable of treating a disease or disorder associated with rotavirus VP6 protein.

[0047] In some embodiments, the nanobody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as a mixed component.

[0048] 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.

[0049] 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.

[0050] In a tenth aspect of the present 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 eighth aspect, or the pharmaceutical composition of the ninth aspect.

[0051] In some embodiments, the kit can further comprise an instruction and / or a device for administration.

[0052] In some embodiments, the kit can be used for diagnosing a disease or disorder associated with rotavirus VP6 protein.

[0053] In some embodiments, the kit can be used for preventing or treating a disease or disorder associated with rotavirus VP6 protein.

[0054] In some embodiments, the disease or disorder comprises rotavirus gastroenteritis or rotavirus diarrhea, for example, infant rotavirus gastroenteritis, adult rotavirus diarrhea, etc.

[0055] The eleventh aspect of the present invention provides 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 eighth aspect, or the pharmaceutical composition of the ninth aspect in the preparation of a product, said product being used for any one of b1)-b3): b1) diagnosing a disease or condition related to rotavirus VP6 protein; b2) preventing or treating a disease or condition related to rotavirus VP6 protein; b3) detecting the presence or level of rotavirus VP6 protein in a sample.

[0056] In some embodiments, the disease or condition includes rotavirus gastroenteritis or rotavirus diarrhea.

[0057] In some embodiments, the disease or condition includes infantile rotavirus gastroenteritis, adult rotavirus diarrhea, etc.

[0058] In some embodiments, the sample is selected from at least one of the body fluids, tissues, cells, and excretions of the subject.

[0059] In some embodiments, the body fluid includes at least one of blood and lymph.

[0060] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0061] In some embodiments, the tissue comprises tumor tissue.

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

[0063] In some implementations, the test subject includes mammals such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

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

[0065] This invention provides a nanobody or its antigen-binding fragment that specifically binds to the rotavirus VP6 protein. It can specifically recognize and bind to the rotavirus VP6 protein and has good affinity for it. It can be mass-produced and used to prepare products for the diagnosis, prevention or treatment of diseases or symptoms related to the rotavirus VP6 protein, or for the detection of the presence or level of the rotavirus VP6 protein in samples. Attached Figure Description

[0066] Figure 1 A schematic diagram showing the results of the antigen affinity test of Nanobody 1B5 is shown.

[0067] Figure 2 A schematic diagram showing the results of the antigen affinity test of Nanobody 2E7 is shown.

[0068] Figure 3 A schematic diagram showing the results of the antigen affinity test of Nanobody 2D5 is shown.

[0069] Figure 4 A schematic diagram showing the results of the antigen affinity test of Nanobody 2B9 is shown. DETAILED DESCRIPTION

[0070] In order to make the objectives, technical solutions, and advantages of the present application clearer, the present application will be described in further detail below with reference to embodiments. The specific embodiments described herein are intended for illustration only and are not intended to be limiting in any way. Moreover, in the following description, descriptions of well-known structures and techniques are omitted to avoid obscuring the concept of the present disclosure. Such structures and techniques are described in many publications.

[0071] 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, for the purposes of explanation, throughout this specification, and the

[0072] As used herein, the terms "a" and "an" include plural references unless the context clearly indicates 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.

[0073] As used herein, the term "about" means ±20% of the numerical value of the number that it precedes. In some embodiments, the term "about" means ±10% of the numerical value of the number that it precedes. In some embodiments, the term "about" means ±5% of the numerical value of the number that it precedes.

[0074] Intermediate capsid protein VP6 of Rotavirus: As the main component of the inner capsid, VP6 forms a stable icosahedral symmetrical structure by multimerization, encapsulates the viral core, protects the genomic RNA from the external environment, and provides a basis for the assembly of outer capsid proteins (VP4, VP7). The VP6 protein accounts for about 50% of the total protein of rotavirus, is the most conserved and abundant protein in rotavirus, and provides support for the structural stability of the virus particle. The amino acid sequence of VP6 protein is highly conserved in rotaviruses of different serotypes and different groups (groups A-G).

[0075] K D Value: dissociation constant (dissociation constant, K D ) is a specific type of equilibrium constant, which measures the tendency of a larger object to separate from another smaller component (dissociation), is the inverse of the association constant, and the unit is mol / L (M) or nmol / L (nM). K D The smaller the value, the stronger the binding ability of the two substances.

[0076] Nanobody: A natural antibody lacking light chains existing in the peripheral blood of Camelidae, which only contains one heavy chain variable region (VHH) and two conventional CH2 regions and CH3 regions, but is not easy to stick together or even aggregate into blocks like artificially engineered 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, with a molecular weight of only about 15 kD, so it is also called nanobody (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 produce highly specific and high-affinity nanobodies.

[0077] Unlike traditional techniques that rely on classic model animals such as mice, rabbits, monkeys, and sheep, the technical solution of the present application is to rely on the antibodies produced by the immune system of a llama, which is called a "nanobody". Nanobody is a small antibody fragment isolated from immunoglobulin in animals such as camels, which has the same antigen binding capacity and structural stability as a complete antibody, and is the smallest unit 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 produce highly specific and high-affinity nanobodies.

[0078] The term "complementarity determining region" (CDR) as used herein are the amino acid residues in an antibody variable region that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, e.g., as defined according to the Kabat numbering system, the Chothia numbering system, the IMGT numbering system, the Martin numbering system, the Contact numbering system, the Honegger numbering system, the Gelfand numbering system, or a combination thereof. For a given antibody, one of skill in the art will readily identify the CDRs defined by each numbering system. The correspondence between different numbering systems is well known to those skilled in the art, and some routinely used software can be used to define the HCDR1-3. 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 is an immunoglobulin numbering system proposed by Chothia et al. that identifies CDR region boundaries based on the location of structural loop regions (see, e.g., Chothia & Lesk, (1987), J. Mol. Biol. 196:901-917; Chothia et al., (1989), Nature 342:878-883; Al-Lazikani et al., (1997), JMB 273:927-948). The IMGT numbering system is a numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al. (see, e.g., Lefranc et al., (2003), Dev. Comparat. Immunol. 27:55-77). The Martin numbering system is a new numbering scheme recommended by Martin et al. that focuses on structural alignments of different framework regions of unusual length, which can be corrected from the Chothia numbering system by the ABnum software. The Contact numbering system defines CDR regions based on existing crystal structure data of antibody complexes. The Honegger numbering system (also referred to as the AHo numbering system) is based on structural alignments of 3D structures of immunoglobulin variable regions that cover observed length variations, allowing definition of structurally conserved Cα positions, thus deducing appropriate FR regions and CDR lengths.The Gelfand numbering system is a relatively complex numbering scheme described by Gelfand et al., in which the variable chain sequence is divided into 21 segments called "words", each of which matches an element of secondary structure (either a strand or a loop), a strand is defined by an alphabetically ordered letter (e.g., A, B, C), and a loop is defined by two letters, which correspond to adjacent strands (e.g., AB, BC...), the numbering system does not include gaps or missing points, but allows for precise comparison of secondary structure between aligned sequences (loops and strands).

[0079] According to the technical scheme 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.

[0080] Table 1

[0081] In addition, due to the degeneracy of bases, the bases of the polynucleotide sequence can be substituted without changing the activity or function of the polynucleotide sequence, see Table 2 below.

[0082] Table 2

[0083] The "percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions in a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since gaps are not nucleotides or amino acids, gaps present in the target sequence are not counted. Likewise, since only nucleotides or amino acids from the target sequence are counted, and not nucleotides or amino acids from the reference sequence, gaps present in the reference sequence are not counted.

[0084] The percentage sequence identity can be calculated by determining the number of positions at which the same amino acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. The comparison of sequences and determination of the percentage sequence identity between two sequences can be accomplished using software programs which are readily available online and for download. Suitable software programs are available from various sources for the alignment of protein and nucleotide sequences. One suitable program for determining percentage sequence identity is bl2seq, which is part of the BLAST suite of programs available from the National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparison between two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs, and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0085] The following examples and figures are provided to aid understanding of the present application. It should be understood that these examples and figures are for illustration only and do not constitute any limitation. The actual scope of the present application 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 application.

[0086] Example 1. Preparation of antigen.

[0087] 1.1 Construction of DNA sequence encoding human Rotavirus Intermediate capsid protein VP6 into pet-28a E. coli expression vector to form Rotavirus Intermediate capsid protein VP6 recombinant expression plasmid.

[0088] The amino acid sequence of the complete expressed recombinant Rotavirus Intermediate capsid protein VP6 protein is shown below as SEQ ID NO: 1: MDVLYSLSKTLKDARDKIVEGTLYSNVSDLIQQFNQMIITMNGNEFQTGGIGNLPIRNWNFDFGLLGTTLLNLDANYVETARNTIDYFVDFVDNVCMDEMVRESQRNGIAPQSDSLIKLSGIKFKRINFDNSSEYIENWNLQNRRQRTGFTFHKPNIFPYSASFTLNRSQPAHDNLMGTMWLNAGSEIQVAGFDYSCAINAPANTQQFEHIVQLRRVLTTATITLLPDAERFSFPRVITSADGATTWYFNPVILRPNNVEIEFLLNGQIINTYQARFGTIIARNFDTIRLSFQLMRPPNMTPAVAALFPNAQPFEHHATVGLTLRIESAVCESVLADASETMLANVTSVRQEYAIPVGPVFPPGMNWTDLITNYSPSREDNLQRVFTVASIRSMLVKLEHHHHHH* (SEQ ID NO: 1).

[0089] The corresponding DNA sequence is shown below as SEQ ID NO: 2:

[0090] 1.2 Transfect the Rotavirus Intermediate capsid protein VP6 recombinant expression plasmid into BL21 (DE3) competent cells, and culture to obtain a monoclonal strain expressing Rotavirus Intermediate capsid protein VP6 protein.

[0091] 1.3 Culture the strain at 37°C, and then add 0.4 mM of an inducer (IPTG, isopropyl-β-D-thiogalactoside) to induce expression of the Rotavirus Intermediate capsid protein VP6 protein at 16°C.

[0092] 1.4 Collect all the bacteria, and after processes such as lysis, centrifugation, affinity chromatography, and gel filtration chromatography, obtain the recombinantly expressed Rotavirus Intermediate capsid protein VP6 protein (recombinant VP6 antigen).

[0093] Example 2. Immunization of a llama.

[0094] (1) Immunize the llama a total of 4 times, with 0.2 mg of the recombinant VP6 antigen prepared in Example 1 being injected subcutaneously into the animal each time. The first immunization is on day 1, and the subsequent immunizations are on days 11, 21, and 31.

[0095] (2) On day 30, before the fourth immunization, collect about 200 mL of peripheral venous blood from the llama.

[0096] (3) On day 45, 14 days after the fourth immunization, collect about 200 mL of peripheral venous blood from the llama.

[0097] Compared with traditional immunization techniques using mice, rabbits, and other animals, the technical advantage of the present application is that a large amount of peripheral venous blood from a llama is collected, which is conducive to subsequent screening to obtain highly diverse nanobodies.

[0098] Example 3. Construction of a nanobody library from a llama.

[0099] Using the two batches of llama peripheral venous blood collected in Example 2 as raw materials, a highly diverse nanobody library is constructed. The two batches of llama peripheral venous blood are processed in the same way, and the specific steps are as follows.

[0100] (1) Use density gradient centrifugation and other methods to separate lymphocytes from the llama peripheral venous blood.

[0101] (2) Extract total mRNA of lymphocytes and reverse transcribe into cDNA.

[0102] (3) Use appropriate DNA primers (see Table 3 below) and the cDNA obtained in step (2) above as a template to amplify the VHH fragments of llama immunoglobulin IgG2 and IgG3, i.e. DNA fragments of nanobodies, by polymerase chain reaction (PCR).

[0103] Table 3

[0104] (4) Link the DNA of VHH obtained in step (3) above to a phage surface display screening vector (Phen1) to form a VHH-pIII fusion protein expression vector plasmid library. pIII is a protein present on the flagella of phage.

[0105] (5) Transform the DNA ligation product into TG1 competent bacteria by electroporation method, and after appropriate culture, collect all colonies, which are the nanobody library of llama.

[0106] Compared with the traditional method of separating antibodies from animal serum or lymphocytes of mice, rabbits, etc., the method of the present application can long-term store all nanobody fragments (i.e. library) of the llama, and can continuously support subsequent screening and development of nanobodies.

[0107] Example 4. Phage surface display screening of specific nanobodies.

[0108] Using the nanobody library obtained in Example 3 as a source, antigen-specific nanobodies are obtained by phage surface display screening. The specific steps are as follows.

[0109] (1) Take the frozen nanobody library and inoculate into a culture medium containing host bacteria (TG1 E. coli), and after culture, add helper phage (M13KO7 helper phage, NEB, N0315S) and continue to culture.

[0110] (2) Extract the amplified phage in the bacterial culture supernatant by PEG-NaCl method.

[0111] (3) Incubate the phage with the recombinant VP6 antigen prepared in Example 1, and the antigen is pre-fixed on an immunotube (Maxisorp immunotube, ThermoFisher Scientific).

[0112] (4) Wash. Discard the phage, and then rinse the antigen with PBS buffer for appropriate times (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.

[0113] (5) Elution. The phages were eluted with an acidic glycine solution to dissociate them from the antigen and retain them. At this point, phages expressing specific nanobodies were obtained, and these phages were subjected to the following steps.

[0114] (6) Transformation into a specific nanobody library. The phage is reinfected into E. coli, but no helper phage is added. After the phage infection is complete, the specific nanobody exists in the E. coli in the form of DNA plasmids. Collect all of these E. coli to form an antigen-specific nanobody library. This library is used as raw material to return to step (1) for the next round of phage surface display screening.

[0115] (7) Transform into monoclonal nanobody colonies. Take a small amount of the phage (0.5%) obtained in step (5), dilute it and infect Escherichia 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.

[0116] Example 5. Identification of positive monoclonal nanobodies.

[0117] Bacterial culture dishes with monoclonal colonies were obtained through step (7) of Example 4, and positive monoclonal nanobodies were identified. The specific steps are as follows.

[0118] (1) Pick single colonies and culture them in microplates.

[0119] (2) Adding IPTG (isopropyl-β-D-thiogalactoside) to induce the expression of VHH-pIII (i.e., the fusion protein containing nanobodies).

[0120] (3) Collect the bacterial culture supernatant containing nanobodies and incubate it with the antigen. The antigen is pre-fixed in a 96-well microplate (Maxisorp transparent microplate, ThermoFisher Scientific).

[0121] (4) Use enzyme-linked immunosorbent assay (ELISA) to detect whether monoclonal nanobodies bind to antigens.

[0122] The main experimental steps are as follows.

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

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

[0125] c. Washing: 3 times with PBST, 3 times with PBS, 150 μL / well.

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

[0127] e. Washing: 3 times with PBST, 3 times with PBS, 150 μL / well.

[0128] f. Add diluted anti-myc HRP and incubate at room temperature for 1 hour.

[0129] g. Washing: 3 times with PBST, 3 times with PBS, 150 μL / well.

[0130] h. Add the ELISA substrate and incubate at room temperature in the dark for 30 minutes.

[0131] i. Read OD405nm.

[0132] (5) For the microbial colonies of monoclonal nanobodies that can bind to antigens, after 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.

[0133] Table 4

[0134] Example 6. Recombinant expression and purification of small-batch monoclonal nanobodies.

[0135] (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 could be expressed and purified in small batches using the E. coli expression system, with a batch production capacity of about a few milligrams.

[0136] (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.

[0137] The results of the affinity tests between each nanobody and the rotavirus VP6 protein antigen are as follows: Figures 1-4 As shown, the affinity value k D The results are shown in Table 5 below.

[0138] Table 5

[0139] 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 the VP6 protein of rotavirus, wherein the nanobody or antigen-binding fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprising: It has the amino acid sequences of HCDR1, HCDR2 and HCDR3 included in the heavy chain variable region as shown in any one of SEQ ID NO: 10, 15, 20 and 25, or has an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequences of HCDR1, HCDR2 or HCDR3 contained in the heavy chain variable region.

2. The nanobody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region includes: a1) HCDR1 having the amino acid sequence shown in SEQ ID NO: 11, HCDR2 having the amino acid sequence shown in SEQ ID NO: 12, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 13; or a2) HCDR1 having the amino acid sequence shown in SEQ ID NO: 16, HCDR2 having the amino acid sequence shown in SEQ ID NO: 17, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 18; or a3) HCDR1 having the amino acid sequence shown in SEQ ID NO: 21, HCDR2 having the amino acid sequence shown in SEQ ID NO: 22, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 23; or a4) HCDR1 having the amino acid sequence shown in SEQ ID NO: 26, HCDR2 having the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 28; Preferably, the heavy chain variable region of the nanobody that specifically binds to the rotavirus VP6 protein or its antigen-binding fragment further includes the framework region of the heavy chain variable region; Preferably, 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. Preferably, the heavy chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO: 10, 15, 20 and 25, 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.

3. A heavy chain antibody or antigen-binding fragment thereof that specifically binds to the rotavirus VP6 protein, comprising an immunoglobulin Fc domain and the nanobody or antigen-binding fragment thereof as described in claim 1 or 2.

4. A chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises the nanobody or its antigen-binding fragment as described in claim 1 or 2, or the heavy chain antibody or its antigen-binding fragment as described in claim 3.

5. A multispecific antibody or its antigen-binding fragment thereof, comprising two or more antigen-binding domains, wherein one antigen-binding domain comprises the nanobody or its antigen-binding fragment as described in claim 1 or 2, or the heavy chain antibody or its antigen-binding fragment as described in claim 3.

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

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

8. A cell comprising the nanobody or its antigen-binding fragment as described in claim 1 or 2, the heavy chain antibody or its antigen-binding fragment as described in claim 3, the chimeric antigen receptor as described in claim 4, the multispecific antibody or its antigen-binding fragment as described in claim 5, the nucleic acid molecule as described in claim 6, or the carrier as described in claim 7.

9. A conjugate comprising the nanobody or antigen-binding fragment thereof as claimed in claim 1 or 2, or the heavy chain antibody or antigen-binding fragment thereof as claimed in claim 3; and, a conjugation portion; Preferably, the coupling portion includes a detectable marker, a delivery carrier, or a therapeutic agent; Preferably, the detectable markers include enzymes, radionuclides, fluorescent dyes, luminescent substances, colored substances, and / or biotin; Preferably, the therapeutic agent includes a chemotherapeutic agent, an immunosuppressant, and / or a cytotoxic drug.

10. A pharmaceutical composition comprising: The nanobody or antigen-binding fragment thereof as claimed in claim 1 or 2, the heavy chain antibody or antigen-binding fragment thereof as claimed in claim 3, the chimeric antigen receptor thereof as claimed in claim 4, the multispecific antibody or antigen-binding fragment thereof as claimed in claim 5, the nucleic acid molecule thereof as claimed in claim 6, the carrier thereof as claimed in claim 7, the cell thereof as claimed in claim 8, or the conjugate thereof as claimed in claim 9; and pharmaceutically acceptable carriers; Preferably, the pharmaceutical composition further includes additional pharmaceutically active agents.

11. Diagnostic or therapeutic reagent kits, comprising: The nanobody or antigen-binding fragment thereof as claimed in claim 1 or 2, the heavy chain antibody or antigen-binding fragment thereof as claimed in claim 3, the chimeric antigen receptor thereof as claimed in claim 4, the multispecific antibody or antigen-binding fragment thereof as claimed in claim 5, the nucleic acid molecule thereof as claimed in claim 6, the carrier thereof as claimed in claim 7, the cell thereof as claimed in claim 8, the conjugate thereof as claimed in claim 9, or the pharmaceutical composition thereof as claimed in claim 10. Preferably, the kit further includes instructions and / or a drug delivery device.

12. The use of the nanobody or antigen-binding fragment thereof of claim 1 or 2, the heavy chain antibody or antigen-binding fragment thereof of claim 3, the chimeric antigen receptor thereof of claim 4, the multispecific antibody or antigen-binding fragment thereof of claim 5, the nucleic acid molecule thereof of claim 6, the carrier thereof of claim 7, the cell thereof of claim 8, the conjugate thereof of claim 9, or the pharmaceutical composition thereof of claim 10 in the preparation of a product, wherein the product is used in any one of b1)-b3): b1) Diagnose a disease or condition that is associated with the rotavirus VP6 protein; b2) Prevention or treatment of a disease or condition that is associated with the rotavirus VP6 protein; b3) Detect the presence or level of rotavirus VP6 protein in the sample; Preferably, the disease or condition includes rotavirus gastroenteritis or rotavirus diarrhea; More preferably, the disease or condition includes infantile rotavirus gastroenteritis and adult rotavirus diarrhea.

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