AQP4 antibodies and uses thereof

Abstract

The invention relates to the technical field of antibodies, in particular to an AQP4 antibody and application thereof. The invention constructs and obtains an antibody or antigen binding molecule of AQP4, provides a coding nucleic acid, an expression vector and a host cell of the antibody or antigen binding molecule, and provides a marker or a conjugate related to the antibody or antigen binding molecule. The antibody or the antigen binding molecule can be combined with nerve cells in a targeted manner, so that the cells are protected from being damaged by autoantibodies, and the effect of treating neuromyelitis optica pedigree diseases is further achieved.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and particularly to AQP4 antibodies and their uses. Background Art

[0002] Neuromyelitis optica (NMO), also known as Devic disease, is an inflammatory demyelinating disease of the central nervous system mainly involving the optic nerve and spinal cord. It mainly attacks the optic nerve and spinal cord, causing primary central nervous system inflammatory demyelinating lesions. This disease can cause a sharp decline in the patient's vision and even complete blindness, and can also cause serious damage to the spinal cord function, greatly reducing the patient's quality of life. At present, there is still a lack of effective treatment methods globally, and it has always been a difficult problem to be solved in the medical field.

[0003] Aquaporin (AQP), also known as water pore protein, forms pores on the cell membrane to control the entry and exit of water into and out of cells. Among them, AQP4 is expressed in the central nervous system, especially in the retina, optic nerve, hypothalamus, periventricular area, aqueduct of the midbrain and spinal cord, and has the dual functions of maintaining water balance and osmoreceptor. When the blood-brain barrier is permeable and has a high affinity for the orthogonal array particles of AQP4, the anti-AQP4 autoantibodies present in the body enter the central nervous system after crossing the blood-brain barrier, and attack the astrocytes in the central nervous system through specific cytotoxic effects, causing nutritional deficiency and apoptosis of oligodendrocytes, and then causing demyelination in the central nervous system. Finally, extensive axonal damage leads to lesions in the optic nerve and / or spinal cord.

[0004] In the patient population of neuromyelitis optica spectrum disorders (NMOSDs), AQP4 autoantibodies can be detected in the blood of the vast majority of patients, and IgG-AQP4 has become one of the important markers for diagnosing this disease. In theory, blocking the binding of AQP4 autoantibodies to their antigen AQP4 protein is an ideal method for treating NMOSDs. However, there are currently no effective blocking drugs available.

[0005] Although antibody technology has made great progress in recent years and shown great potential in the treatment of various diseases, with advantages such as strong specificity, high sensitivity, and large-scale preparation, the current situation in the research and application field of antibodies against AQP4 is not optimistic. At present, there is still a lack of an ideal antibody drug that can effectively block the binding of AQP4 autoantibodies to AQP4 protein. The existing antibody research results cannot meet the urgent clinical needs for treating NMO. Therefore, developing a highly efficient and specific AQP4 antibody has become a key problem to be solved in the medical field. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide an AQP4 antibody and its uses.

[0007] The heavy chain CDR1-3 of the AQP4 antibody or antigen-binding moiety provided by the present invention sequentially has the amino acid sequences shown in SEQ ID NO: 1-3, or has a sequence with substitution, deletion and / or addition of one or more amino acids based on the amino acid sequences shown in SEQ ID NO: 1-3; or has a sequence with an identity of more than 80% (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) with any one of the amino acid sequences shown in SEQ ID NO: 1-3.

[0008] In some embodiments, the amino acid sequences of the heavy chain CDR1-3 of the AQP4 antibody or antigen-binding molecule are sequentially GFTFSNYG, ISSGGDT and TRGNSREPY.

[0009] The light chain CDR1-3 of the AQP4 antibody or antigen-binding moiety provided by the present invention sequentially has the amino acid sequences shown in SEQ ID NO: 4-6, or has a sequence with substitution, deletion and / or addition of one or more amino acids based on the amino acid sequences shown in SEQ ID NO: 4-6; or has a sequence with an identity of more than 80% (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) with any one of the amino acid sequences shown in SEQ ID NO: 4-6.

[0010] In some embodiments, the amino acid sequences of the light chain CDR1-3 of the AQP4 antibody or antigen-binding molecule are sequentially QDINGY, RAN and LQSDDFPLT.

[0011] The AQP4 antibody provided by the present invention can target and inhibit the allergic reaction induced by the binding of IgG-AQP4 autoantibody and AQP4 antigen in vivo, thereby protecting astrocytes from immune damage and inhibiting central nervous system inflammatory demyelination, and further achieving the effect of treating NMO. The generation of the main activity of the antibody provided by the present invention lies in the sequence of the CDR region, and it can be prepared into a complete antibody, single-chain antibody, single-domain antibody, Fab antibody according to the sequence of the CDR region, and the present invention does not make any limitation thereto. It can also be prepared into a humanized antibody, chimeric antibody or multivalent antibody according to the CDR region sequence described above, and the present invention also does not make any limitation thereto. In the implementation process of the present invention, in order to clearly demonstrate the feasibility and effectiveness of the technical solution, the scFV antibody is taken as an example for activity verification.

[0012] In the present invention, the FR1-4 of the heavy chain of the AQP4 antibody sequentially has the amino acid sequences shown in SEQ ID NOs: 7-10, or has a sequence with 1 or more amino acids substituted, deleted, and / or added on the basis of the amino acid sequences shown in SEQ ID NOs: 7-10; or has a sequence with an identity of more than 80% to any of the amino acid sequences shown in SEQ ID NOs: 7-10.

[0013] In some embodiments, the amino acid sequences of the FR1-4 of the heavy chain of the AQP4 antibody or antigen-binding molecule are EVKMVESGGGLVKPGESLKVSCAAS, MSWVRQTPEKRLEWVAS, YYLESVKGRFTISRENDRNILYLQMSSLRSEDTAMYYC, and WGQGTLVTVSA in sequence.

[0014] In the present invention, the FR1-4 of the light chain of the AQP4 antibody sequentially has the amino acid sequences shown in SEQ ID NOs: 11-14, or has a sequence with 1 or more amino acids substituted, deleted, and / or added on the basis of the amino acid sequences shown in SEQ ID NOs: 11-14; or has a sequence with an identity of more than 80% to any of the amino acid sequences shown in SEQ ID NOs: 11-14.

[0015] In some embodiments, the amino acid sequences of the FR1-4 of the light chain of the AQP4 antibody or antigen-binding molecule are DIKMTQSPSSMYASLGERVTITCKAS, LSWFQQKPGKSPRTLIY, RLIDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYSC, and FGAGTKLELR in sequence.

[0016] In some specific embodiments, the heavy chain variable region of the antibody of the present invention has the amino acid sequence shown in SEQ ID NO: 15.

[0017] In some specific embodiments, the light chain variable region of the antibody of the present invention has the amino acid sequence shown in SEQ ID NO: 16.

[0018] In the present invention, the heavy chain variable region and the light chain variable region of the antibody are connected by a linker to form an scFV antibody fragment. The heavy chain variable region is located at the C-terminus or the N-terminus, and the present invention does not limit this. In a specific embodiment, the heavy chain variable region of the antibody is located at the C-terminus. Specifically, in the present invention, the sequence of the linker is GGGGSGGGGSGGGGS.

[0019] In a specific embodiment of the present invention, the N-terminus of the antibody further comprises a signal peptide. Specifically, the sequence of the signal peptide is MKHLWFFLLLVAAPRWVLS.

[0020] In a specific embodiment of the present invention, the C-terminus of the antibody further comprises a tag. The sequence of the tag is GSHHHHHH.

[0021] In a specific embodiment, the amino acid sequence of the antibody of the present invention is as shown in SEQ ID NO:17.

[0022] Furthermore, the present invention also provides a nucleic acid encoding the AQP4 antibody or antigen-binding molecule as described above.

[0023] The nucleic acid of the present invention is a nucleic acid encoding the CDR region in the antibody as described above, or a nucleic acid encoding the variable region, or a nucleic acid encoding the full-length antibody, and the present invention does not make a limitation thereto.

[0024] As a feasible example, the nucleic acid encoding the heavy chain variable region has a nucleic acid sequence as shown in SEQ ID NO:18;

[0025] As a feasible example, the nucleic acid encoding the light chain variable region has a nucleic acid sequence as shown in SEQ ID NO:19.

[0026] As a feasible example, the nucleic acid encoding the AQP4 antibody or antigen-binding molecule as described above has a nucleic acid sequence as shown in SEQ ID NO:20.

[0027] Furthermore, the present invention also provides a plasmid vector containing the nucleic acid as described above.

[0028] Furthermore, the present invention also provides a host cell, in whose genome the nucleic acid as described above is integrated, or which is transformed or transfected with the plasmid vector as described above.

[0029] In the present invention, the host cell can be a eukaryotic cell or a prokaryotic cell. The eukaryotic cells are selected from mammalian cells, insect cells, yeast cells, plant cells, etc., and the prokaryotic cells are selected from Escherichia coli, Bacillus subtilis, etc. In a specific embodiment, mammalian cells are taken as an example for antibody expression. Specifically, the mammalian cells are Chinese hamster ovary cells (CHO cells), human embryonic kidney cells (HEK293 cells) or mouse myeloma cells (SP2 / 0 cells), etc.

[0030] Furthermore, the present invention also provides a method for preparing the antibody or antigen-binding molecule as described above, which comprises: culturing the host cell to obtain a culture containing the antibody or antigen-binding molecule.

[0031] In the preparation method of the present invention, a purification step of the antibody or antigen-binding molecule is further included. The purification includes collecting the cell culture supernatant, passing it through an anion chromatography column, and collecting the antibody or antigen-binding molecule. The molecular weight of the antibody or antigen-binding molecule is 26.0 kDa.

[0032] More specifically, the preparation method of the antibody or antigen-binding molecule in the embodiment includes:

[0033] (1) Construction, fermentation and recovery of AQP4 ScFV antibody mammalian cell line: Subclone the artificially synthesized nucleic acid fragment encoding the antibody or antigen-binding molecule as described above into a mammalian cell expression vector, transfect the mammalian cell line for expression, screen for a highly expressing cell line, and express the AQP4 ScFV antibody recombinant protein;

[0034] (2) Preparation of AQP4 ScFV antibody sample: Take the cell culture supernatant of the highly expressing cell line, slowly add an equal volume of saturated ammonium sulfate, mix well and place at 4°C for more than 24 hours, centrifuge at 50000g for 30 min at 4°C to collect the precipitate, dissolve the precipitate in 100 ml of equilibration buffer, and dialyze against 5000 ml of equilibration buffer for 48 hours, changing the buffer 5 times in the middle; Store the sample at -20°C for later use.

[0035] (3) Purification of AQP4 ScFV antibody protein sample: Purify with an anion chromatography column equilibrated with equilibration buffer. Pass the sample prepared in (2) above through the chromatography column, elute with equilibration buffers containing 0.1 mol / L, 0.2 mol / L, 0.5 mol / L, and 1 mol / L NaCl respectively, collect the elution peaks, identify by SDS electrophoresis, take the part containing the protein with a molecular weight of 26.0 kD, and concentrate; Equilibrate a Separose 12 chromatography column with equilibration buffer; Load the concentrated 26.0 kD protein part at a ratio of 2% - 5% of the column volume, with a flow rate of 1.5 ml / min; Collect in fractions; After identification by SDS electrophoresis, collect the part with a high purity of the protein with a molecular weight of 26.0 kDa, which is the AQP4 ScFV antibody histone.

[0036] (4) Identification of the purified AQP4 ScFV antibody protein sample: Determine the protein purity by SDS-PAGE electrophoresis or high performance liquid method, with a purity above 95%, a molecular weight of 26.0 kD, and determine the protein content using an ultraviolet spectrophotometer, approximately 0.2 mg / ml.

[0037] Furthermore, the present invention also provides a labeled antibody, which includes a labeling agent and the AQP4 antibody or antigen-binding molecule as described above, and the labeling agent is a chemical label or a biological label.

[0038] In the present invention, the chemical label is a fluorescent indicator, a chemiluminescent indicator, a radioactive label, and / or a colloidal indicator;

[0039] In the present invention, the biomarker is biotin, avidin or enzyme-labeled.

[0040] As a feasibility case, the enzyme-labeled is selected from one or more of horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-anti-peroxidase conjugate, alkaline phosphatase-anti-alkaline phosphatase conjugate, β-galactosidase-anti-β-galactosidase conjugate;

[0041] As a feasibility case, the fluorescent indicator is selected from one or more of fluorescein isothiocyanate, tetramethyl rhodamine isothiocyanate, rhodamine B isothiocyanate, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor647, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, phycoerythrin, propidium iodide, 4,6-diamidino-2-phenylindole, acridine orange, Texas red, luciferase, green fluorescent protein or red fluorescent protein;

[0042] As a feasibility case, the chemiluminescent indicator is selected from one or more of luminol or its derivatives, isoluminol or its derivatives, acridinium ester, acridinium sulfonamide, acridinium carboxylate ester, lucigenin, o-phenylenediamine, 4,5-diamino-phthalohydrazide or aminobutylethyl phthalhydrazide;

[0043] As a feasibility case, the isotope is selected from one or more of iodine-125, iodine-131, iodine-123, phosphorus-32, sulfur-35, tritium, carbon-14, gallium-67, gallium-68, technetium-99m, indium-111, thallium-201, fluorine-18, copper-64, copper-67, zirconium-89, yttrium-90, bismuth-213, radium-223 or actinium-225;

[0044] As a feasibility case, the colloid indicator is selected from one or more of colloidal gold, colloidal carbon, colloidal selenium, magnetic colloid or quantum dot colloid.

[0045] Furthermore, the present invention also provides a conjugate, which comprises a matrix material and the AQP4 antibody or antigen-binding molecule as described above.

[0046] As a feasibility case, the matrix material is an enzyme-linked immunosorbent assay (ELISA) plate, culture plate, glass slide, magnetic bead or latex microsphere.

[0047] In the present invention, the antibody and the matrix material can be linked by an amide bond or by a streptavidin-biotin system, and the present invention does not make a limitation thereto.

[0048] Furthermore, the present invention also provides the use of the AQP4 antibody or antigen-binding molecule, the nucleic acid, the plasmid vector, the host cell, the labeled antibody or the conjugate as described above in at least one of the following:

[0049] I), preparing a detection reagent for AQP4 protein;

[0050] II), preparing an imaging reagent for nerve cells;

[0051] III), preparing a blocking reagent for blocking the binding of AQP4 protein to the complete AQP4 antibody;

[0052] IV), preparing a drug for preventing and treating neuromyelitis spectrum disorders.

[0053] In the present invention, the neuromyelitis spectrum disorders include optic neuritis, acute myelitis, dorsal medullary syndrome, acute brainstem syndrome, acute diencephalic syndrome and / or cerebral syndrome.

[0054] Furthermore, the present invention also provides a detection reagent, which comprises the AQP4 antibody or antigen-binding molecule, the labeled antibody and / or the conjugate as described above.

[0055] The detection reagent described in the present invention comprises a detection reagent for AQP4 protein and / or an imaging reagent for nerve cells.

[0056] In the present invention, the detection reagent for AQP4 protein comprises the AQP4 antibody or antigen-binding molecule as described above, and also comprises a secondary antibody and / or a tertiary antibody that specifically binds to the AQP4 antibody or antigen-binding molecule.

[0057] In a specific embodiment, the secondary antibody in the detection reagent for AQP4 protein is a rabbit antibody that recognizes the C-terminus of the AQP4 antibody or antigen-binding molecule. The tertiary antibody is HRP-labeled goat anti-rabbit.

[0058] In this embodiment, the detection reagent for AQP4 protein further comprises a TMB reagent and a buffer required for the reaction. The buffer is preferably a PBS buffer, a Tris-HCl buffer, a carbonate buffer or a borate buffer.

[0059] In the present invention, the imaging reagent for nerve cells comprises the labeled antibody as described above, or contains the AQP4 antibody or antigen-binding molecule as described above.

[0060] In the present invention, the imaging reagent for nerve cells comprises the AQP4 antibody or antigen-binding molecule as described above, and also comprises a secondary antibody and / or a tertiary antibody that specifically binds to the AQP4 antibody or antigen-binding molecule.

[0061] In a specific embodiment, the secondary antibody in the imaging reagent for nerve cells is a rabbit antibody that recognizes the C-terminus of the AQP4 antibody or antigen-binding molecule. The tertiary antibody is FITC-labeled goat anti-rabbit.

[0062] In this embodiment, the imaging reagent for nerve cells further includes a buffer required for the reaction. The buffer is preferably PBS buffer, Tris-HCl buffer, carbonate buffer or borate buffer.

[0063] Furthermore, the present invention also provides a method for detecting AQP4 protein or optic nerve cells expressing AQP4 protein, which includes detecting a sample with the reagent as described above.

[0064] The present invention does not limit the method for detecting AQP4 protein expression. Any method involving the AQP4 antibody as described above is within the scope of protection. Preferably, the detection method is an immunological method, such as ELISA, IFA, SDS-PAGE or Western blot.

[0065] Furthermore, the present invention also provides a drug, which includes the AQP4 antibody or antigen-binding molecule as described above, the nucleic acid as described above, the plasmid vector as described above, the host cell as described above, or the culture obtained by the preparation method as described above.

[0066] In the drug of the present invention, it may only include the AQP4 antibody or antigen-binding molecule as described above, or may include the nucleic acid or related materials encoding the antibody or antigen-binding molecule as described above. The present invention does not limit this.

[0067] Furthermore, the present invention also provides a drug combination, which includes the AQP4 antibody or antigen-binding molecule as described above, the nucleic acid as described above, the plasmid vector as described above, the host cell as described above, or the culture obtained by the preparation method as described above, and also includes other therapeutic agents.

[0068] The other therapeutic agents include drugs for treating neuromyelitis optica spectrum disorders. As a feasible case, the drugs for treating neuromyelitis optica spectrum disorders include, but are not limited to, methylprednisolone, prednisone, azathioprine, mycophenolate mofetil, rituximab, ocrelizumab, inebilizumab, tocilizumab, satralizumab, eculizumab, ravulizumab, pregabalin or carbamazepine.

[0069] In the pharmaceutical composition, multiple drugs may be mixed or exist independently of each other. The present invention does not limit this. Multiple drugs may be administered simultaneously or sequentially. The present invention also does not limit this.

[0070] Furthermore, the present invention also provides a method for treating neuromyelitis optica spectrum disorders, which includes administering the aforementioned drug or drug combination.

[0071] The administration methods of the aforementioned drug or drug composition include but are not limited to: topical administration (such as application on the skin or mucosal surface), systemic administration (such as oral administration, inhaled administration, buccal mucosal absorption), canal administration (such as nasal, vaginal, rectal administration), and invasive administration routes (including subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection, or achieved through infusion devices and implantable drug storage systems).

[0072] The present invention constructs and obtains an antibody or antigen-binding molecule of AQP4, and provides its encoding nucleic acid, expression vector, host cell, and also provides markers or conjugates related to the antibody or antigen-binding molecule. This antibody or antigen-binding molecule can target and bind to nerve cells, protect the cells from damage by autoantibodies, and thus play a role in treating neuromyelitis optica spectrum disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Showing the purification result of AQP4 ScFV antibody: M molecular weight 1 2 purified sample;

[0074] Figure 2 Showing the reaction of AQP4 ScFV antibody with nerve cells, immunofluorescence method, A: ScFV antibody B: cell control;

[0075] Figure 3 Showing the inhibitory effect of AQP4 ScFV antibody, immunofluorescence method, A: AQP4 monoclonal antibody B: blocking of AQP4 monoclonal antibody by ScFV antibody;

[0076] Figure 4 Showing the inhibitory effect of AQP4 ScFV antibody, Western blot method, 1: AQP4 monoclonal antibody 2: blocking of AQP4 monoclonal antibody by ScFV antibody). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0077] The present invention provides an AQP4 antibody and its uses. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0078] Unless otherwise defined in this invention, scientific and technical terms related to this invention shall have the meanings understood by those of ordinary skill in the art.

[0079] In this application, the term "and / or" describes the relationship between associated objects and indicates three possible relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, or B exists alone. Here, A and B can be singular or plural.

[0080] In this application, the terms "comprise", "include" and "have" can be used interchangeably, aiming to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that using "comprise", "include" and "have" to describe in this article also provides the solution of "consisting of...".

[0081] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or its similar expressions refer to any combination of these items, including any combination of single item or plural items.

[0082] In this application, "antibody" refers to a class of globulins produced by the body's immune system after being stimulated by an antigen and capable of specifically binding to the corresponding antigen. "Antigen-binding molecule" refers to a molecule that specifically binds to an antigen. For example, the antigen-binding molecule includes but is not limited to an antibody or an antibody mimetic. "Antibody mimetic" refers to a biomolecule that mimics the structure and function of a natural antibody through a non-natural synthetic method. For example, antibody mimetics include but are not limited to affibody, affitin, affilin, designed ankyrin repeat protein (DARPin), aptamer or Kunitz-type domain peptide.

[0083] Globulins with antibody activity or a chemical structure similar to that of an antibody are collectively called immunoglobulins (Ig). The basic structure of Ig is a monomer composed of four peptide chains, that is, it is composed of two identical heavy chains (H chains) and two identical light chains (L chains). There are five classes of heavy chains of Ig, which are represented by the Greek letters γ, α, μ, δ, and ε respectively. The Igs composed of them are called IgG, IgA, IgM, IgD, and IgE respectively. The light chains are divided into two types according to their different antigenicity, namely the κ type and the λ type. The blood of the vast majority of patients with neuromyelitis optica spectrum disorders (NMOSDs) can detect AQP4 autoantibodies, that is, IgG-AQP4.

[0084] In this application, "ScFV antibody" is short for Single-Chain Fragment Variable antibody, which is a small antibody fragment modified by genetic engineering. It is formed by connecting the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of the antibody through a flexible linker peptide, forming a single polypeptide chain structure. Among them, both the variable region of the light chain and the variable region of the heavy chain are composed of 4 relatively conserved framework regions (FR) and 3 highly variable complementary determining regions (CDR), and the linker peptide is generally composed of 15-25 amino acids. Different from the complete antibody, ScFv does not contain the constant region of the antibody (Fc segment).

[0085] In this application, the "antibody" is prepared by immunizing mammals with specific antigen peptides. Commonly used mammals include mice, rats, rabbits, goats, sheep, etc. The antibody in this case is obtained by screening after immunizing mice with specific antigen peptides. The screening of the ScFv antibody of AQP4 described in the present invention can be carried out through phage display screening, recombinant production in prokaryotic or eukaryotic expression systems, and is widely used as a core functional module in the biomedical field, such as constructing bispecific antibodies by tandem or heterologous combination, conjugating with toxins or radioisotopes to form antibody-drug conjugates (ADCs), or integrating into chimeric antigen receptors (CARs) as a target recognition domain.

[0086] In this application, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0087] In this application, the CDR regions of the AQP4 antibody are divided based on the Kabat, Chothia or IMGT systems. There may be differences in the amino acid sequences of the CDR regions divided by different systems.

[0088] In this application, "identity" can be calculated as follows: To determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced into one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). Subsequently, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecule is identical at this position. Considering the number of gaps introduced for optimal alignment of the two sequences and the length of each gap, the percentage of identity between the two sequences varies with the positions shared by the sequences.

[0089] In the present application, "nucleic acid" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide consists of a base, in particular a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Generally, a nucleic acid molecule is described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. In the present application, the sequence of bases is generally represented as 5' end → 3' end.

[0090] In the present application, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and hybrid polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleobases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules that are suitable as vectors for direct expression of the antibodies of the present invention in vitro and / or in vivo, e.g., in a host or a patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo (see, for example, Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2 101 823B1).

[0091] In the present application, "plasmid vector" refers to a nucleic acid molecule that has the ability to amplify other nucleic acids linked thereto. This term encompasses both vectors that exist as self-replicating nucleic acid structures and vector types that have been integrated into the genome of the host cell into which the vector has been introduced. Some vectors have the function of directing the expression of nucleic acids operably linked thereto, and for such vectors, they are collectively referred to as "expression vectors" herein.

[0092] In the present application, "host" or "host cell" specifically refers to a cell into which exogenous nucleic acid has been introduced, and also encompasses the progeny cells derived from the proliferation of such a cell. The scope of host cells includes "transformants" and "cells that have completed transformation", which include both the primary cells that initially achieved transformation and all progeny cells derived from such primary cells, regardless of the number of passages. The progeny cells may differ from the parental cells in terms of the composition and content of nucleic acids and other substances, and mutations may occur. Within the scope of the description herein, it also includes mutant progeny cells that have the same function or biological activity as the cells obtained through screening or selection in the cells that initially completed transformation.

[0093] In the present application, a "drug" is allowed to exist in a biologically active form of the active ingredient contained therein and does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is administered.

[0094] In the present application, "prevention and treatment" covers the related behaviors of prevention and (or) treatment. "Treatment" specifically refers to surgical means or pharmaceutical treatment, the core purpose of which is to prevent the occurrence of unwanted physiological changes or pathological conditions in the subject to be treated, or to slow down (reduce) their development. The beneficial or desired clinical effects that can be achieved include, but are not limited to, the following aspects: the alleviation of related symptoms, the reduction of the severity of the disease, the maintenance of the disease state in a stable condition (i.e., no worsening trend), the delay or slowdown of the progression of the disease, the improvement or alleviation of the disease state, and the achievement of remission (including partial remission and complete remission), regardless of whether these effects can be detected by detection means or are difficult to detect. The subjects in need of treatment include not only individuals who already suffer from specific diseases or disorders, but also those who are prone to developing diseases or disorders, and those who have a need to prevent specific diseases or disorders. When expressions such as slow down, alleviate, weaken, relieve, and remission are mentioned herein, their meanings also include the elimination, complete disappearance, or non-occurrence of the related situations. "Prevention" refers to taking a series of measures for the subject to be treated to avoid the occurrence of unwanted physiological changes or pathological conditions. For subjects who are prone to developing diseases or disorders, by means such as adjusting lifestyle, inoculating specific vaccines, using preventive drugs, etc., the possibility of their onset is reduced. For subjects who intend to prevent diseases or disorders, it is to intervene in advance when there are no signs of the disease and maintain the body in a healthy state to prevent the occurrence of the disease. This is different from "treatment" for the situation where physiological changes or pathological conditions have already occurred. Prevention focuses more on proactive intervention before the occurrence of the disease to achieve the beneficial clinical result of not having the disease.

[0095] In this application, "imaging" refers to the process of converting certain substances or energy into observable or measurable signals to display their forms, properties, structures, or distributions. For example, in the present invention, after treating a sample with an antibody carrying a chemical or biological label, common medical imaging techniques such as X-ray, MRI, CT scan, and ultrasound are used to distinguish cells expressing AQP4 from other cells, or tissues expressing AQP4 from other tissues.

[0096] All the test materials used in the present invention are common commercially available products and can be purchased on the market. Among them:

[0097] The accession number of the AQP4 protein is NP_001295570.1;

[0098] AQP4 complete antibody

[0099] The amino acid sequences of the various regions of the antibody involved in the present invention are shown in Table 1:

[0100] Table 1

[0101]

[0102] Amino acid sequence of the light chain + heavy chain of 30 - B8 - F12 - 1 - A11 - E2

[0103]

[0104] Amino acid sequence of the light chain of 30 - B8 - F12 - 1 - A11 - E2

[0105]

[0106] Linker between the light chain and the heavy chain: GGGGSGGGGSGGGGS

[0107] Signal peptide at the N - terminus: MKHLWFFLLLVAAPRWVLS

[0108] Amino acid sequence of the heavy chain of 30 - B8 - F12 - 1 - A11 - E2

[0109]

[0110] Nucleic acid sequence encoding the light chain + heavy chain of 30 - B8 - F12 - 1 - A11 - E2

[0111] GAATTCGCCGCCACCATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCTAGATGGGTGCTGTCCGACATCAAGATGACCCAGTCTCCATCCTCTATGTACGCCAGCCTGGGCGAGAGAGTGACCATTACCTGCAAGGCCAGCCAGGACATCAACGGCTACCTGTCCTGGTTCCAGCAGAAGCCTGGCAAGTCTCCCCGGACACTGATCTACAGAGCCAACAGACTGATCGACGGCGTGCCCTCCAGATTCTCCGGATCTGGATCTGGCCAGGACTACTCCCTGACCATCTCCAGCCTGGAATACGAGGATATGGGCATCTACAGCTGCCTGCAGTCCGACGACTTCCCACTGACATTTGGCGCTGGCACCAAGCTGGAATTGAGAGGCGGCGGAGGATCTGGCGGAGGTGGAAGCGGAGGCGGTGGATCTGAAGTGAAGATGGTGGAATCTGGCGGCGGACTGGTTAAGCCAGGCGAGTCCCTGAAAGTGTCCTGTGCCGCTTCTGGCTTCACCTTCTCCAACTACGGCATGTCCTGGGTCCGACAGACCCCTGAGAAGAGACTGGAATGGGTCGCCTCCATCTCTTCCGGCGGAGACACCTACTACCTGGAATCCGTGAAGGGCCGCTTTACCATCAGCCGCGAGAACGACCGGAACATCCTGTACCTGCAGATGTCCTCTCTGCGGAGCGAGGACACCGCCATGTACTACTGTACCCGGGGCAACTCCAGAGAGCCTTATTGGGGACAGGGCACCCTGGTCACAGTGTCTGCTGGCTCTCACCACCACCAT CACCATTGAGCGGCCGC(SEQ IDNO.20)

[0112] 30 - B8 - F12 - 1 - A11 - E2, Nucleic acid sequence encoding the light chain

[0113] GAATTCGCCGCCACCATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCTAGATGGGTGCTGTCCGACATCAAGATGACCCAGTCTCCATCCTCTATGTACGCCAGCCTGGGCGAGAGAGTGACCATTACCTGCAAGGCCAGCCAGGACATCAACGGCTACCTGTCCTGGTTCCAGCAGAAGCCTGGCAAGTCTCCCCGGACACTGATCTACAGAGCCAACAGACTGATCGACGGCGTGCCCTCCAGATTCTCCGGATCTGGATCTGGCCAGGACTACTCCCTGACCATCTCCAGCCTGGAATACGAGGATATGGGCATCTACAGCTGCCTGCAGTCCGACGACTTCCCACTGACATTTGGCGCTGGCACCAAGCTGGAATTGAGA(SEQ ID NO.19)

[0114] Nucleic acid sequence encoding the linker

[0115] GGCGGCGGAGGATCTGGCGGAGGTGGAAGCGGAGGCGGTGGATCT

[0116] 30 - B8 - F12 - 1 - A11 - E2, nucleic acid sequence encoding the heavy chain

[0117] GAAGTGAAGATGGTGGAATCTGGCGGCGGACTGGTTAAGCCAGGCGAGTCCCTGAAAGTGTCCTGTGCCGCTTCTGGCTTCACCTTCTCCAACTACGGCATGTCCTGGGTCCGACAGACCCCTGAGAAGAGACTGGAATGGGTCGCCTCCATCTCTTCCGGCGGAGACACCTACTACCTGGAATCCGTGAAGGGCCGCTTTACCATCAGCCGCGAGAACGACCGGAACATCCTGTACCTGCAGATGTCCTCTCTGCGGAGCGAGGACACCGCCATGTACTACTGTACCCGGGGCAACTCCAGAGAGCCTTATTGGGGACAGGGCACCCTGGTCACAGTGTCTGCTGGCTCTCACCACCACCATCACCATTGAGCGGCCGC(SEQ ID NO.18)

[0118] Note: The underlined part is the CDR region, is the signal peptide, is the tag.

[0119] The present invention will be further described below in conjunction with embodiments:

[0120] Example 1 Monoclonal Antibody Screening:

[0121] GNWENHWTINWGGTEKPLP was prepared by the method of polypeptide synthesis, and then conjugated with KLH as an antigen. The synthesized polypeptide was fully mixed with complete adjuvant in equal volume, and 50 μg of protein per mouse was injected subcutaneously at 4 - 6 points on the back of female BALB / c mice, immunizing a total of 5 mice. Two weeks after the primary immunization, a booster immunization was carried out with 25 μg per mouse. After another two weeks, a booster immunization was carried out again with 25 μg per mouse. One week later, the titer of the antiserum was detected. Mice with a titer greater than 32000 were selected. One week later, 10 μL of blood was collected from the tail of the mice after the fourth immunization and added to 990 μL of PBS buffer, mixed well and centrifuged at 5000 r / min for 10 min, and the supernatant was reserved for use. The serum titer was determined by indirect ELISA, and mice with high titer and good sensitivity were selected for the next cell fusion.

[0122] Mice with better serum immune results were selected for cell fusion, fused 1 - 2 times, and each fusion was subjected to 2 - 3 rounds of ELISA screening. After obtaining monoclonal antibodies, cell lines were established, and 3 cell lines were established (denoted as 1 - A11 - E2, 12 - E11 - F7, 26 - A6 - B1 respectively). The obtained positive monoclonal cell lines were injected into the abdominal cavity of mice to produce ascites, and 2 - 5 mg of antibody was purified from each cell line, and the antibody titer was detected by ELISA. The synthetic polypeptide at 2.5 μg / L was used to coat a 96 - well plate, 100 μL per well, at 37°C for 2 h or overnight at 4°C. Then it was rinsed 5 times with PBS, and blocked overnight at 4°C with 3% BSA - PBS, 300 μL per well, washed 3 - 5 times with PBST, 300 μL per well. Each monoclonal antibody was added to the enzyme - labeled plate, 100 μL per well, reacted at room temperature for 1 h, and washed 3 times with PBST. A secondary antibody conjugated with horseradish peroxidase (HRP) - labeled affinity - purified goat anti - mouse IgG was added, 100 μL per well, at 37°C for 30 min, and washed 3 times with PBST. Substrate chromogenic solution 3,3',5,5' - Tetramethylbenzidine (TMB) was added, 100 μL per well, and developed color at room temperature for 5 - 15 min, and the reaction was terminated by adding dilute sulfuric acid, 50 μL per well. The OD450 value was measured with an enzyme - labeled instrument. The results are shown in Table 2:

[0123] Table 2 Detection Results of the Titers of Three Antibodies

[0124]

[0125]

[0126] According to the OD450 value, the antibody secreted by the 1-A11-E2 cell line was selected from the three cell lines for subsequent experiments.

[0127] Example 2 Preparation and Purity Determination of AQP4 ScFV Antibody

[0128] The antibody secreted by the 1-A11-E2 cell line was sequenced. After clarifying the gene sequence, the antibody was artificially synthesized:

[0129] (1) The gene fragment of the AQP4 ScFV antibody was directly obtained by artificial synthesis, and the AQP4 ScFV antibody gene was constructed. It was subcloned into a mammalian cell expression vector and transfected into mammalian cells by the liposome method for expression. High-expression cell lines were screened to express the AQP4 ScFV antibody. Specifically:

[0130] The gene fragment of the AQP4 ScFV antibody was directly artificially synthesized and directionally subcloned into the EcoRI and NotI cleavage sites of the expression plasmid pATX2. Transfection method for CHO cells: Discard the original medium of CHO cells, add about 5 mL of fresh medium, then add 100 μl of the DNA / DOTAP mixture, gently shake the culture flask to make it evenly distributed in the medium, and place it in a 37 °C cell culture incubator for 10 h, then change to fresh complete 1640 medium. After 48 h, add G418 (800 μg / mL) for screening. Untransfected cells and empty vector cells were set as negative controls in the experiment and cultured in serum-free 1640 medium. The supernatant of each experimental group was collected every 24 h, filtered through a 0.45 μm filter, and the supernatant was stored at -20 °C; The cell culture supernatant was collected for purification to screen high-expression mammalian cell lines.

[0131] (2) Fermentation, high-expression and purification of the AQP4 ScFV antibody mammalian cell line:

[0132] Take the cell culture supernatant of the highly expressing cell line, slowly add saturated ammonium sulfate at a ratio of 1:1, mix well, place at 4°C for more than 10 hours, centrifuge at 10,000 g for 30 min at 4°C to collect the precipitate. Dissolve the precipitate in 100 ml of equilibration buffer and dialyze against 5000 ml of equilibration buffer for more than 24 hours, changing the buffer 5 times in the middle. The sample is then loaded onto an anion exchange column equilibrated with equilibration buffer or stored at -20°C for later use. Elute with equilibration buffers containing 0.1 mol / L, 0.2 mol / L, 0.5 mol / L, and 1 mol / L NaCl respectively, collect the elution peaks, identify by SDS-PAGE, and take the fraction containing the protein with a molecular weight of 26.0 kD. Equilibrate the Separose 12 chromatography column with equilibration buffer. Load the concentrated 26.0 kD protein fraction onto the column at a ratio of 2 - 5% of the bed volume, with a flow rate of 1.5 ml / min. Collect in fractions. After identification by SDS-PAGE electrophoresis, collect the fraction with a high purity of the protein with a molecular weight of 26.0 kD, which is the AQP4 ScFV antibody.

[0133] (3) Use SDS-PAGE to check the purity of the AQP4 ScFV antibody raw material prepared as above. The result shows that the purity of the AQP4 ScFV antibody can reach over 95% ( Figure 1 ).

[0134] The relevant solutions involved in the examples include:

[0135] Serum-free 1640 medium.

[0136] Saturated ammonium sulfate:

[0137] Prepare in the following proportions per 1000 ml:

[0138] Ammonium sulfate above 1000 g.

[0139] Equilibration buffer:

[0140] 20 mmol PB buffer (pH 7.2), prepare in the following proportions per 1000 ml:

[0141] Sodium dihydrogen phosphate·2H2O 3.12 g;

[0142] Disodium hydrogen phosphate·12H2O 7.16 g;

[0143] EDTA 0.37 g.

[0144] Binding activity of AQP4 ScFV antibody and AQP4 antigen in Example 3

[0145] The indirect ELISA method was used to detect the binding activity of the AQP4 ScFV antibody (prepared in Example 2) with the AQP4 antigen. Specific steps: An indirect ELISA method was established using the polypeptide AQP4-1, the polypeptide AQP4-2, and the AQP4 ScFV antibody (prepared in Example 2). The checkerboard titration method was adopted.

[0146] (1) The polypeptide AQP4-1 and the polypeptide AQP4-2 antigens were serially diluted with the coating buffer at pH 9.6 to coat the ELISA plate, 100 μl per well, and incubated overnight at 4°C.

[0147] (2) After washing three times with PBS for 5 min each time, the affinity-purified antibody (primary antibody) of M40146-AQP4 mouse ascites (1:500) was taken, 100 μl per well, incubated at 37°C for 1 h, and then washed three times with PBS for 5 min each time.

[0148] (3) The Anti-His rabbit monoclonal antibody (secondary antibody) (1:1000) was serially diluted and added longitudinally, 100 μl per well, and incubated at 37°C for 1 h. Then washed three times with PBS for 5 min each time.

[0149] (4) After washing, the HRP-labeled goat anti-rabbit IgG antibody (tertiary antibody) (1:5000) was added, 100 μl per well, and incubated at 37°C for 1 h.

[0150] (5) TMB was used for color development in the dark at room temperature for 10 min, and the optical density (OD) value was read at 450 nm. At the same time, a blank control was set up. The results showed that the polypeptide AQP4-1 reacted with the purified antibody (see Tables 3 to 4 in the attached table).

[0151] Table 3 Coating amount of AQP4 antigen

[0152] Plate 1 AQP4-1(0.70mg / ml) A 10 B 5 C 2.5 D 1.25 E 0.625 F 0.3125 G 0.15625 H 0

[0153] Table 4 OD450nm - OD620nm

[0154] Plate 1 OD A 1.619 B 1.276 C 0.848 D 0.439 E 0.318 F 0.164 G 0.123 H 0.094

[0155] Example 4 Binding activity of AQP4 ScFV antibody with nerve cells

[0156] PC-12 cells are a commonly used nerve cell line, derived from rat adrenal medulla pheochromocytoma. The IFA method was used to identify the binding activity of the AQP4 ScFV antibody with these cells.

[0157] (1) According to the cell culture method, they were dispensed into a culture plate with cover slips and cultured into a monolayer of cells in an incubator at 37°C.

[0158] (2) After removing the cover glass from the culture plate, rinse it twice with 0.01 mol / L PBS, place it on filter paper, dry it, and then fix it in pre-cooled acetone in a refrigerator at -30 to -28 °C for 30 min. Wash it three times with PBS for 5 min each time.

[0159] (3) After air-drying, add the AQP4 ScFV antibody (primary antibody, obtained from Example 2) (about 250 μL dropped on the slide), and repeatedly aspirate and blow it with a pipette tip to make it uniform. Incubate at 37 °C for 1 hour, wash it three times with PBS for 5 min each time, and air-dry it.

[0160] (4) Add Anti-His rabbit monoclonal antibody (secondary antibody) (1:200) (about 250 μL dropped on the slide), and repeatedly aspirate and blow it with a pipette tip to make it uniform. Incubate at 37 °C for 45 min in the dark; wash it three times with PBS for 5 min each time in the dark, and air-dry it.

[0161] (5) Add goat anti-rabbit IgG-FITC (tertiary antibody) (1:200) (about 250 μL dropped on the slide), and repeatedly aspirate and blow it with a pipette tip to make it uniform. Incubate at 37 °C for 45 min in the dark;

[0162] (6) Wash it three times with PBS for 5 min each time in the dark, and air-dry it; drop a drop of 50% glycerol (prepared with PBS) (50 μL) on the glass slide; place the cover glass under a fluorescence microscope to observe the results ( Figure 2 ). At the same time, set up a blank control. The results show that the AQP4 ScFV antibody can bind to nerve cells.

[0163] Example 5 Blocking effect of AQP4 ScFV antibody on intact AQP4 antibody

[0164] (1) The indirect ELISA method was used to detect the blocking effect of the AQP4 ScFV antibody (prepared in Example 2) on the intact AQP4 antibody. Specific steps: An indirect ELISA method was established using the polypeptide AQP4-1 and the AQP4 ScFV antibody. By the checkerboard titration method, the polypeptide AQP4-1 was serially diluted with coating buffer at pH 9.6 to coat the ELISA plate, 100 μl per well, and coated overnight at 4 °C. Take the AQP4 ScFV antibody (blocking antibody) (1:500), 100 μl / well, and incubate at 37 °C for 1 h. Take the mouse ascites affinity-purified antibody (primary antibody) (1:500) of M40146-AQP4 (derived from cell line 1-A11-E2), 100 μl / well, and incubate at 37 °C for 1 h. After washing, add the HRP-labeled goat anti-mouse IgG antibody (secondary antibody) (1:5000) 100 μl / well, and incubate at 37 °C for 1 h. After washing, develop the color with TMB at room temperature in the dark for 10 min, and read the optical density (OD) value at 450 nm. At the same time, set up a blank control. The results show that the AQP4 ScFV antibody has a blocking effect on the intact AQP4 antibody, as shown in Table 5:

[0165] Table 5 OD450nm

[0166] Plate 1 OD450nm A 0.215 B 0.235 C 0.218 D 0.224 E 0.208 F 0.198 G 0.139 H 0.057

[0167] (2) Identification of the blocking effect of AQP4 ScFV antibody on the complete AQP4 antibody by the IFA method. According to the cell culture method, it was aliquoted into a culture plate with a cover glass and cultured in an incubator at 37 °C to form a monolayer of cells. After removing the cover glass from the culture plate, it was rinsed twice with 0.01 mol / L PBS, placed on filter paper, dried, and then fixed in pre-cooled acetone in a refrigerator at -30 to -28 °C for 30 min. It was washed three times with PBS, 5 min each time. After air-drying, AQP4 ScFV antibody (blocking antibody) (about 250 μL was dropped on the slide and evenly aspirated and blown with a pipette tip) was added and incubated at 37 °C for 1 hour. It was washed three times with PBS, 5 min each time, and air-dried; M40146-AQP4 mouse ascites affinity-purified antibody (primary antibody) (1:200) (about 250 μL was dropped on the slide and evenly aspirated and blown with a pipette tip) was added and incubated at 37 °C for 45 min in the dark; it was washed three times with PBS in the dark, 5 min each time, and air-dried; Evan's blue dye solution and goat anti-mouse IgG-FITC (secondary antibody) (1:200) (about 250 μL was dropped on the slide and evenly aspirated and blown with a pipette tip) were added and incubated at 37 °C for 45 min in the dark; it was washed three times with PBS in the dark, 5 min each time, and air-dried; a drop of 50% glycerol (prepared with PBS) (50 μL) was dropped on the glass slide; the cover glass was placed under a fluorescence microscope to observe the results ( Figure 3 ). The results showed that the AQP4 ScFV antibody had a blocking effect on the complete AQP4 antibody.

[0168] (3) Identification of the blocking effect of AQP4 ScFV antibody on the complete AQP4 antibody by SDS-PAGE electrophoresis and Western blot: The polypeptide AQP4-1 and polypeptide AQP4-2 samples were subjected to SDS-PAGE electrophoresis, and Coomassie brilliant blue staining and Western blot analysis were performed respectively. The proteins in the SDS-PAGE gel were transferred to a nitrocellulose membrane (NC membrane) by a transfer instrument at a voltage of 90 V for 60 min. After the transfer was completed, the NC membrane was removed and blocked in 5% skim milk for 1 h, washed 4 times with PBST, 10 min each time; AQP4 ScFV antibody (blocking antibody) (1:1000) was used at room temperature for 1 h, washed 4 times with PBST, 10 min each time; M40146-AQP4 mouse ascites affinity-purified antibody (primary antibody) (1:1000) was used at room temperature for 1 h, washed 4 times with PBST, 10 min each time; HRP-labeled goat anti-mouse IgG (1:5000) was used on a shaker at room temperature for 1 h, washed 4 times with PBST; DAB chromogenic solution was used for color development. ( Figure 4), The results showed that the AQP4 ScFV antibody blocked the intact AQP4 antibody.

[0169] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. AQP4 antibody or antigen binding molecule, Its heavy chain CDR1 to 3 have the amino acid sequences shown in SEQ ID NOs: 1 to 3 in sequence, or have a sequence in which one or more amino acids are substituted, deleted and / or added based on the amino acid sequences shown in SEQ ID NOs: 1 to 3; or have a sequence with an identity of more than 80% with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3; Its light chain CDR1 to 3 have the amino acid sequences shown in SEQ ID NOs: 4 to 6 in sequence, or have a sequence in which one or more amino acids are substituted, deleted and / or added based on the amino acid sequences shown in SEQ ID NOs: 4 to 6; or have a sequence with more than 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6.

2. The antibody or antigen-binding molecule according to claim 1, characterized in that Its heavy chain FR1 to 4 have the amino acid sequences shown in SEQ ID NOs: 7 to 10 in sequence, or have a sequence in which one or more amino acids are substituted, deleted and / or added based on the amino acid sequences shown in SEQ ID NOs: 7 to 10; or have a sequence with an identity of more than 80% with the amino acid sequence shown in any one of SEQ ID NOs: 7 to 10; Its light chain FR1 to 4 have the amino acid sequences shown in SEQ ID NOs: 11 to 14 in sequence, or have a sequence in which one or more amino acids are substituted, deleted and / or added based on the amino acid sequences shown in SEQ ID NOs: 11 to 14; or have a sequence with more than 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 11 to 14.

3. The antibody or antigen-binding molecule according to claim 1, characterized in that Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO:15; The light chain variable region thereof has the amino acid sequence shown in SEQ ID NO:

16.

4. The antibody or antigen-binding molecule according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO:

17.

5. A nucleic acid encoding the AQP4 antibody or antigen-binding molecule according to any one of claims 1 to 4.

6. The nucleic acid according to claim 5, characterized in that The nucleic acid encoding the heavy chain variable region has the nucleic acid sequence shown in SEQ ID NO: 18; The nucleic acid encoding the light chain variable region has the nucleic acid sequence shown in SEQ ID NO:

19.

7. The nucleic acid according to claim 6, characterized in that The nucleic acid encoding the AQP4 antibody or antigen-binding molecule of any one of claims 1 to 4 has the nucleic acid sequence shown in SEQ ID NO:

20.

8. A plasmid vector comprising the nucleic acid according to any one of claims 5 to 7.

9. A host cell, the genome of which is integrated with the nucleic acid of any one of claims 5 to 7, or is transformed or transfected with the plasmid vector of claim 8.

10. A method for preparing the antibody or antigen-binding molecule according to any one of claims 1 to 4, comprising: Cultivate the host cell of claim 9 to obtain a culture containing the antibody or antigen-binding molecule.

11. A labeled antibody comprising a label and the AQP4 antibody or antigen-binding molecule according to any one of claims 1 to 4, wherein the label is a chemical label or a biological label.

12. The labeled antibody according to claim 11, characterized in that The chemical marker is a fluorescent indicator, a chemiluminescent indicator, a radioactive marker and / or a colloid indicator; The biological marker is biotin, avidin or enzyme marker.

13. The labeled antibody according to claim 12, characterized in that The enzyme label is selected from one or more of horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-anti-peroxidase bridge, alkaline phosphatase-anti-alkaline phosphatase bridge, β-galactosidase-anti-β-galactosidase bridge; The fluorescent indicator is selected from one or more of fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, rhodamine B isothiocyanate, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, phycoerythrin, propidium iodide, 4,6-diamidino-2-phenylindole, acridine orange, Texas Red, luciferase, green fluorescent protein or red fluorescent protein; The chemiluminescent indicator is selected from one or more of luminol or its derivatives, isoluminol or its derivatives, acridinium esters, acridinium sulfonamides, acridinium carboxylates, lucigenin, o-phenylenediamine, 4,5-diaminophthalhydrazide or aminobutylethyl o-benzohydrazide; The isotope is selected from one or more of iodine-125, iodine-131, iodine-123, phosphorus-32, sulfur-35, tritium, carbon-14, gallium-67, gallium-68, technetium-99m, indium-111, thallium-201, fluorine-18, copper-64, copper-67, zirconium-89, yttrium-90, bismuth-213, radium-223, or actinium-225; The colloid indicator is selected from one or more of colloidal gold, colloidal carbon, colloidal selenium, magnetic colloid or quantum dot colloid.

14. A conjugate comprising a matrix material and the AQP4 antibody or antigen-binding molecule according to any one of claims 1 to 4.

15. The combination according to claim 14, characterized in that The matrix material is an ELISA plate, a culture plate, a glass slide, magnetic beads or latex microspheres.

16. Use of the AQP4 antibody or antigen-binding molecule according to any one of claims 1 to 4, the nucleic acid according to any one of claims 5 to 7, the plasmid vector according to claim 8, the host cell according to claim 9, the labeled antibody according to any one of claims 11 to 13, or the conjugate according to claim 14 or 15 in at least one of the following: 1) preparing a detection reagent for AQP4 protein; II), preparing imaging reagents for neural cells; III), preparing a blocking reagent for blocking the binding between AQP4 protein and AQP4 intact antibody; IV) Preparation of drugs for preventing and treating neuromyelitis optica spectrum disorders.

17. The use according to claim 16, characterized in that The neuromyelitis optica spectrum disorders include optic neuritis, acute myelitis, area postrema syndrome, acute brainstem syndrome, acute diencephalic syndrome and / or cerebral syndrome.

18. A detection reagent comprising the AQP4 antibody or antigen-binding molecule according to any one of claims 1 to 4, the labeled antibody according to any one of claims 11 to 13, and / or the conjugate according to claim 14 or 15.

19. A method for detecting AQP4 protein or optic nerve cells expressing AQP4 protein, comprising detecting a sample with the reagent according to claim 18.

20. A drug comprising the AQP4 antibody or antigen-binding molecule of any one of claims 1 to 4, the nucleic acid of any one of claims 5 to 7, the plasmid vector of claim 8, the host cell of claim 9, or the culture obtained by the preparation method of claim 10.

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