Monoclonal antibody against AAV8 as well as preparation method and application thereof

Abstract

The invention belongs to the field of virus detection and diagnosis, and relates to an anti-AAV8 monoclonal antibody as well as a preparation method and application thereof. The invention provides an anti-AAV8 rabbit monoclonal antibody, and amino acid sequences of a heavy chain variable region and a light chain variable region of the anti-AAV8 rabbit monoclonal antibody. The anti-AAV8 rabbit monoclonal antibody provided by the invention can be specifically combined with VLP of AAV8, and can be used for detecting an AAV virus antigen. The anti-AAV8 monoclonal antibody provided by the invention provides an effective detection tool for AAV8 detection in a gene therapy vector research and development process and AAV8 detection in basic scientific research.

Description

Anti-AAV8 monoclonal antibodies and preparation methods and uses thereof

[0001] Cross-reference information

[0002] This application claims priority to Chinese patent application No. 202211675599.X filed on December 26, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of virus detection and diagnosis, and relates to an anti-AAV8 monoclonal antibody. The present invention also relates to a preparation method and use of the anti-AAV8 monoclonal antibody. Background Art

[0004] Adeno-associated virus (AAV) belongs to the Parvoviridae family and is a non-enveloped, single-stranded, linear DNA virus. Its genome is approximately 4.7 kb long and primarily consists of two genes: Rep and Cap. The Cap gene encodes the three viral capsid proteins: VP1, VP2, and VP3. The C-terminal sequences of VP1 and VP2 proteins are identical to those of VP3. VP3 is the most abundant protein in the viral capsid, while the concentrations of VP1 and VP2 are each only one-tenth of that of VP3.

[0005] AAV virus is a non-pathogenic virus that can infect humans and a variety of other vertebrates. There are many serotypes of AAV, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVDJ and AAVrh10. Due to its low immunogenicity and the different targeting and expression efficiencies of different serotypes of AAV for different tissues and organs, AAV has become a commonly used gene manipulation tool and plays a key role in both active immunotherapy by delivering antigens and passive immunotherapy by delivering antibodies. The CRISPR-Cas9 system carried by AAV virus also provides a new means for gene editing in organs and tissues such as the brain, cochlea and muscle.

[0006] Due to the large number of AAV virus serotypes, newly discovered and developed AAV vectors are constantly emerging. Monoclonal antibodies that specifically recognize AAV8 are convenient detection tools in the development of AAV8 gene therapy vectors.

[0007] Summary of the Invention

[0008] In one aspect, the present invention provides an anti-AAV8 monoclonal antibody or a functional fragment thereof, comprising a heavy chain CDR and a light chain CDR, wherein the heavy chain CDR and the light chain CDR are selected from the group consisting of CDRs of the amino acid sequences shown in SEQ ID NO: 12 to SEQ ID NO: 35 and variants thereof each comprising up to three amino acid mutations (e.g., one, two, or three), and the combination of the group is defined by one or more of a to d:

[0009] In some embodiments, the heavy chain CDR and light chain CDR are selected from the group consisting of CDRs of the amino acid sequences shown in SEQ ID NO: 12 to SEQ ID NO: 35.

[0010] In some embodiments, the heavy chain CDRs and light chain CDRs are selected from the following sequences:

[0011] a. The amino acid sequences of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 12, 13, 14, 15, 16 and 17, respectively;

[0012] b. The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 18, 19, 20, 21, 22 and 23, respectively;

[0013] c. the amino acid sequences of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 24, 25, 26, 27, 28 and 29, respectively; or

[0014] d. The amino acid sequences of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 30, 31, 32, 33, 34 and 35, respectively.

[0015] In some embodiments, the anti-AAV8 monoclonal antibody or a functional fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of variable regions of the amino acid sequences shown in SEQ ID NO: 4 to SEQ ID NO: 11 and variants thereof each comprising an amino acid sequence having at least 80% identity, and the combination of the group is defined by one or more groups A to D:

[0016] In some embodiments, the heavy chain variable region sequence comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:4, 6, 8 or 10; and the light chain variable region sequence comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:5, 7, 9 or 11. In some embodiments, the heavy chain variable region sequence comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:4, 6, 8 or 10; and the light chain variable region sequence comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:5, 7, 9 or 11.

[0017] In some embodiments, the heavy chain variable region and light chain variable region are selected from the following sequences:

[0018] (A) the heavy chain variable region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:4, and the light chain variable region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:5;

[0019] (B) the heavy chain variable region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:6, and the light chain variable region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:7;

[0020] (C) the heavy chain variable region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:8, and the light chain variable region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:9;

[0021] (D) the heavy chain variable region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 10, and the light chain variable region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 11.

[0022] In some embodiments, the heavy chain variable region and light chain variable region are selected from the group consisting of variable regions with amino acid sequences shown in SEQ ID NO:4 to SEQ ID NO:11.

[0023] In some embodiments, the heavy chain variable region and light chain variable region are selected from the following sequences:

[0024] A. the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5;

[0025] B. the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7;

[0026] C. the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9; or

[0027] D. The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11.

[0028] In some embodiments, the heavy chain variable region and the light chain variable region are selected from the following sequences:

[0029] A. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5;

[0030] B. the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7;

[0031] C. the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9; or

[0032] D. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 11.

[0033] In some embodiments, the anti-AAV8 monoclonal antibodies or functional fragments thereof are paired antibody compositions or combination products, comprising antibodies from two or three different epitopes for combined use, such as the above-mentioned combination ab, ac, ad, bc, bd, cd; or the above-mentioned combination AB, AC, AD, BC, BD, CD. In some embodiments, the composition or combination product contains at least bd or BD.

[0034] In some embodiments, the antibody has a constant region, the heavy chain constant region sequence is selected from any one of the constant region sequences of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; and the light chain constant region is a κ or λ chain.

[0035] In some embodiments, the anti-AAV8 monoclonal antibody or a functional fragment thereof is a rabbit antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0036] "Rabbit-derived antibodies" refer to antibodies whose variable and constant regions (if present) are derived from rabbit immunoglobulin sequences. Rabbit antibodies can be conveniently obtained by immunizing rabbits with the corresponding antigen and isolating the target antibody therefrom. Alternatively, after immunizing rabbits with the corresponding antigen, cells expressing the target antibody (such as B cells) are isolated and cultured to obtain the target antibody. Alternatively, after immunizing rabbits with the corresponding antigen, cells expressing the target antibody are isolated and cultured, and these cells are fused with immortalized cells such as myeloma cells to obtain hybridoma cells. Culturing hybridoma cells can then produce the target antibody (such as a monoclonal antibody) over a long period of time and in large quantities.

[0037] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a first animal-derived antibody with the constant region of a second animal-derived antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes a specific monoclonal antibody of the first animal origin, then clone the variable region gene from the hybridoma cell, and then clone the constant region gene of the second animal-derived antibody as needed. The first animal-derived variable region gene and the second animal-derived constant region gene are connected to form a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present invention, the first animal origin is rabbit origin, and the second animal origin is preferably human origin, which can reduce the immune response induced by the first animal-derived antibody. The antibody light chain of the chimeric antibody further comprises a light chain constant region of a human κ, λ chain or a variant thereof. The antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0038] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the CDR sequence of a first animal origin into the antibody variable region framework of a human, i.e., different types of human germline antibody framework sequences. The heterologous reaction induced by chimeric antibodies due to carrying a large amount of rabbit protein components can be overcome. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be obtained in the "VBase" human germline sequence database (www.mrccpe.com.ac.uk / vbase), and can be found in Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid a decrease in immunogenicity and a decrease in activity caused by the decrease, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0039] The humanized antibodies of the present invention, when their degree of humanization is further improved, also include humanized antibodies (human antibodies) after further affinity maturation of CDRs by phage display. In a preferred embodiment of the present invention, the first animal source is rabbit source. The human antibody variable region framework is designed and selected, for example, wherein the heavy chain FR region sequence on the antibody heavy chain variable region is derived from the combination sequence of human germline heavy chain IGHV1-18*01 and hjh6.1, or the combination sequence of human germline heavy chain IGHV1-3*01 and hjh6.1; wherein the light chain FR region sequence on the antibody light chain variable region is derived from the combination sequence of human germline heavy chain IGKV1-39*01 and hjk4.1. In order to avoid a decrease in immunogenicity and a decrease in activity caused by the decrease, the human antibody variable region can be subjected to minimal reverse mutation to maintain activity.

[0040] In some embodiments, the anti-AAV8 monoclonal antibody or a functional fragment thereof has a detectable label.

[0041] Detectable labels can be selected from any one or more of a chromophore, a digoxigenin-labeled probe, an electron-dense substance, colloidal gold, or an enzyme. The following non-limiting list of these labels is:

[0042] Enzymes that generate a detectable signal, such as by colorimetry, fluorescence, and luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose-6-phosphate dehydrogenase.

[0043] Chromophores, such as fluorophores, quantum dots, fluorescent microspheres, luminescent compounds, and dyes.

[0044] Groups having an electron density that can be detected by electron microscopy or by their electrical properties, such as conductivity, current analysis, voltage measurement, and resistance.

[0045] A detectable group, such as a molecule of sufficient size to induce a detectable modification in its physical and / or chemical properties; such detection can be achieved by optical methods (such as diffraction, surface plasmon resonance, surface variation and contact variation angle) or physical methods (such as atomic force spectroscopy and tunneling).

[0046] Electron-dense substances, such as radioactive molecules (e.g. 32 P, 35 S or 125 I).

[0047] In some embodiments, the detectable label is selected from one or more of alkaline phosphatase, acridinium ester, horseradish peroxidase, terpyridine ruthenium, isoluminol or rare earth elements, preferably alkaline phosphatase, acridinium ester or horseradish peroxidase.

[0048] In another aspect, the present invention provides an isolated polynucleotide encoding the above-mentioned anti-AAV8 monoclonal antibody or a functional fragment thereof.

[0049] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding the heavy chain variable region of the above-mentioned anti-AAV8 monoclonal antibody or a functional fragment thereof, and a nucleotide sequence encoding the light chain variable region of the anti-AAV8 monoclonal antibody or a functional fragment thereof.

[0050] In another aspect, the present invention provides an expression vector comprising the polynucleotide.

[0051] The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40).

[0052] In another aspect, the present invention provides a host cell or a cell-free expression system comprising the expression vector.

[0053] Suitable host cells or cell lines for expressing the antigen-binding proteins of the present invention include mammalian cells such as NSO, Sp2 / 0, CHO, COS, HEK, fibroblasts, and myeloma cells. Human cells can be used, thereby allowing the molecules to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines can be employed. The selection of suitable mammalian host cells, as well as methods for transformation, culture, amplification, screening, and product production and purification, are known in the art.

[0054] Bacterial cells can be used as host cells that are suitable for expressing recombinant Fab or other embodiments of the present invention. However, since proteins expressed in bacterial cells tend to be in an unfolded or improperly folded or non-glycosylated form, any recombinant Fab produced in bacterial cells must be screened to retain antigen binding capacity. If the bacterial cell-expressed molecule is produced in a properly folded form, the bacterial cell will be the desired host, or, in an alternative embodiment, the molecule can be expressed in a bacterial host and subsequently refolded. For example, various strains of E. coli used for expression are well-known host cells in the field of biotechnology. Various strains of Bacillus subtilis, Streptomyces, other Bacillus species, etc. can also be used in this method.

[0055] Yeast cell strains known to those skilled in the art, as well as insect cells, such as Drosophila and Lepidoptera insects, and viral expression systems, can also be used as host cells if desired.

[0056] In some embodiments, the nucleic acid is inserted into the cell genome and can be stably expressed.

[0057] The insertion method can be selected from the vectors described above, or the nucleic acid can be directly transferred into the cell without being linked to a vector (eg, liposome-mediated transfection technology).

[0058] In another aspect, the present invention provides a method for preparing an anti-AAV8 monoclonal antibody or a functional fragment thereof, comprising:

[0059] Cultivating the host cell as described above under appropriate culture conditions; and

[0060] The antibodies or antigen-binding fragments thereof thus produced are recovered from the culture medium or from the cultured cells.

[0061] The culture method of the present invention is typically a serum-free culture method, typically using serum-free suspension culture of cells. Similarly, once the antibodies of the present invention are produced, they can be purified from the cell culture contents according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. Such techniques are within the skill of the art and do not limit the present invention. Another method for expressing antibodies can utilize expression in animals (particularly transgenic animals or nude mice). This involves an expression system utilizing an animal casein promoter, which, when transgenically incorporated into a mammal, allows the female animal to produce the desired recombinant protein in its milk. The culture medium containing the secreted antibodies can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column containing an adjusted buffer. Nonspecifically bound components are washed away. Bound antibodies are then eluted using a pH gradient method, and antibody fragments are detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, for example, at -70°C, or lyophilized.

[0062] In another aspect, the present invention provides a kit for detecting AAV8 virus, wherein the kit comprises the monoclonal antibody or a functional fragment thereof as described above.

[0063] The present invention also relates to a chromatography medium for separating AAV8 virus, wherein the chromatography medium comprises a matrix support and the monoclonal antibody or a functional fragment thereof as described above immobilized on the matrix support.

[0064] In some embodiments, the matrix support comprises any one of agar, agarose, agarose derivatives, magnetic beads, silica, titanium dioxide, alginate, cellulose, cellulose derivatives, dextran, starch, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum ghatti, tragacanth gum, karaya gum, locust bean gum, xanthan gum, pectin, mucin, heparin, gelatin, silicon, ceramic, glass (e.g., borosilicate glass), polyurethane, polystyrene, polystyrene divinylbenzene, polymethyl methacrylate, polyacrylamide, polyethylene terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinyl pyrrolidone, or copolymers of any of them.

[0065] The matrix support may be of any shape, such as substantially spherical, substantially cubic, etc.

[0066] The present invention also relates to a chromatographic separation device comprising the chromatographic medium described above.

[0067] There are many types of chromatographic separation devices, including but not limited to the following: SPE solid phase extraction columns, centrifuge tubes with separation membranes, magnetic beads, separation membranes, rapid detection bio-chips, fiber bundle columns, and the chromatographic separation device is preferably columnar, such as a monolithic column and a conventional analytical or preparative grade chromatography column.

[0068] In another aspect, the present invention provides use of the above-described antibody or antigen-binding fragment thereof in detecting and / or purifying AAV8. Beneficial effects:

[0069] Different AAV serotypes have different tropisms for different tissues and organs. Current research on AAV viral delivery vectors lacks efficient and sensitive serological detection methods specific for different serotypes. The anti-AAV8 monoclonal antibody developed in this invention specifically reacts with AAV8 virus-like particles (VLPs). When paired with a sandwich ELISA, it can detect pg-level AAV8 VLPs, providing a tool for functional validation of AAV8 viral delivery vectors and quantitative detection of the virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0071] FIG1 is a diagram showing the titer test results of rabbit serum after immunization;

[0072] FIG2 shows the ELISA reaction results of monoclonal antibodies with different serotypes of AAV;

[0073] Figure 3 shows the EC50 reaction results of monoclonal antibodies and AAV8 VLPs;

[0074] FIG4 shows the pairing results of monoclonal antibodies. DETAILED DESCRIPTION

[0075] The present invention relates to a monoclonal antibody against AAV8. The embodiments of the present invention are described in detail below with reference to the examples. Unless otherwise specified, the technical and scientific terms used in the present invention have the same meanings as those commonly understood by ordinary technicians in the field to which the present invention belongs.

[0076] The term "adeno-associated virus" (AAV) is a commonly used gene manipulation tool. It belongs to the Parvoviridae family and is a non-enveloped single-stranded linear DNA virus. The genome is about 4.7kb long, and the Cap gene encodes three viral capsid proteins: VP1, VP2, and VP3. There are many serotypes of AAV, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVDJ, and AAVrh10. AAV8 is a viral vector that is susceptible to multiple tissues and organs in the human body, including the nervous system, muscles, and liver.

[0077] The term "virus-like particle" or "VLP" refers to non-infectious particles that contain no viral genomic nucleic acid and are assembled solely from viral structural proteins. VLPs generally resemble mature viral particles and are highly immunogenic. VLPs can be expressed in a variety of expression systems, including mammalian and baculovirus systems. The VLPs used in the present invention are self-assembled from the VP1, VP2, and VP3 proteins of AAV expressed in 293T cells.

[0078] The term "antibody" is intended to refer to immunoglobulin molecules composed of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds (i.e., "complete antibody molecules"), as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain is composed of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (consisting of domains CHI, CH2, and CH3). Each light chain is composed of a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). The amino acid sequence of the CDRs can be readily determined using numbering schemes recognized in the art, such as Kabat, Chothia, IMGT, AbM, or Contact. In a specific embodiment, the present invention has determined the CDRs of the antibodies described herein according to the Kabat numbering scheme. Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to hydroxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the present invention, the FRs of the antibodies (or antigen-binding fragments thereof) may be identical to human germline sequences or may be naturally or artificially modified.

[0079] The term "monoclonal antibody" refers to a homogeneous antibody directed against only a specific antigenic epitope. In contrast to typical polyclonal antibody preparations that include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen. The modifier "monoclonal" indicates the homogeneous character of the antibody and is not to be construed as requiring the antibody to be produced by any particular method. The monoclonal antibodies of the present invention are preferably produced by recombinant DNA methods or obtained by screening methods described elsewhere herein.

[0080] The term "mutation" refers to a monoclonal antibody or a functional fragment thereof containing an alteration of one or more (several) amino acid residues at one or more (several) positions, i.e., a substitution, insertion, and / or deletion. Substitution refers to the replacement of the amino acid occupying a position with a different amino acid; deletion refers to the removal of the amino acid occupying a position; and insertion refers to the addition of 1-3 amino acids adjacent to and following the amino acid occupying a position.

[0081] The term "isolated polynucleotide" refers to a polynucleotide that is not naturally present in nature, including polynucleotides isolated from nature (including organisms) by biological techniques, and also includes artificially synthesized polynucleotides. The isolated polynucleotide can be genomic DNA, cDNA, mRNA or other synthetic RNA, or a combination thereof. It should be noted that those skilled in the art can, based on the amino acid sequences of the heavy chain variable region and the light chain variable region provided herein, design nucleotide sequences that are not completely identical to the provided nucleotide sequences, but all encode the same amino acid sequence, based on codon degeneracy. These modified nucleotide sequences are also included within the scope of the present invention.

[0082] When referring to polynucleotides, the term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer nucleotide encoding information into a host cell. The term "expression vector" or "expression cassette" refers to a vector suitable for expressing a gene of interest (nucleotide sequence to be expressed) in a host cell, and generally includes components such as the gene of interest, a promoter, a terminator, and a marker gene.

[0083] The term "host cell" refers to a cell that has been or is capable of being transformed with a nucleic acid sequence to express a selected gene of interest. The term includes the progeny of a parent cell, regardless of whether the progeny is identical in morphology or genetic makeup to the original parent cell, as long as the gene of interest is present in the progeny. Commonly used host cells include bacteria, yeast, and mammalian cells.

[0084] The term "functional antibody fragment" means an antigen-binding fragment of an antibody and an antibody analog, which generally includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of a parental antibody. The antibody fragment retains at least some of the binding specificity of the parental antibody. For example, antibody fragments capable of binding to an AAV viral capsid protein or a portion thereof include, but are not limited to, sdAb (single domain antibody), Fab (e.g., an antibody obtained by papain digestion), F(ab')2 (e.g., obtained by pepsin digestion), Fv, or scFv (e.g., obtained by molecular biology techniques).

[0085] The term "amino acid substitution" refers to replacing an existing amino acid residue with a different amino acid residue in a predetermined (original) amino acid sequence. In general, it is recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th ed., 1987)). Such exemplary substitutions are preferably performed according to the following substitutions:

[0086] Table 1 Exemplary conservative amino acid substitutions

[0087] "Percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the sequences being compared.

[0088] Unless otherwise specifically stated, the use of the singular includes the plural. Unless otherwise specifically stated, the word "a" or "an" means "at least one". Unless otherwise specified, the use of "or" means "and / or". The phrase "at least one" has the same meaning as the phrase "one or more". In addition, the use of the term "including" and other forms such as "includes" and "included" is not restrictive. In addition, unless otherwise specifically stated, terms such as "element" or "component" include elements or components comprising one unit and elements and components comprising more than one unit.

[0089] Unless otherwise stated, the methods and materials of the embodiments described below are conventional products that can be purchased on the market. Those skilled in the art will understand that the methods and materials described below are merely exemplary and should not be considered as limiting the scope of the present invention.

[0090] Example 1 Immunization of Animals with AAV8 Virus-like Particles

[0091] New Zealand white rabbits were immunized with 100 μg of recombinant AAV8 virus-like particles (VLP, capsid protein VP1, VP2 and VP3 protein sequences are shown in SEQ ID NO: 1, 2 and 3). Subsequently, the immunization was repeated every other week to boost the experimental rabbits for a total of 3 times. The serum titer of both rabbits reached 10 after 3 immunizations. 5 Seven days after the last immunization, sterile blood was collected from the rabbit numbered R06835 for subsequent antibody discovery.

[0092] AAV8 capsid protein VP1 amino acid sequence (SEQ ID NO: 1):

[0093] AAV8 capsid protein VP2 amino acid sequence (SEQ ID NO: 2):

[0094] AAV8 capsid protein VP3 amino acid sequence (SEQ ID NO: 3):

[0095] Example 2 Obtaining B cells and screening of monoclonal antibodies

[0096] 1) Isolation of antigen-positive B cells

[0097] PBMCs were isolated from 15 mL of anticoagulated rabbit blood aseptically, and antigen-positive cells were then enriched using antigen. The enriched cells were plated at a density of 10 cells per well in 96-well cell culture plates pre-seeded with feeder cells. The plates were incubated at 37°C in 5% CO2. On day 7, the supernatant from the overnight culture was collected and assayed for antibodies against AAV8 VLPs using an indirect ELISA as described below.

[0098] 2) Indirect ELISA

[0099] An indirect ELISA was used to assess the binding ability of antibodies in the supernatant to VLPs. VLPs were diluted to 1 μg / mL in PBS, and 100 μL was applied to each well of a 96-well microtiter plate overnight at 4°C. After washing with PBST (0.05% Tween), the plates were blocked with 150 μL / well of 1% BSA in PBST at 37°C for 1 hour. The blocking buffer was then discarded, and 100 μL of B cell culture supernatant was added to each plate, followed by incubation at 37°C for 1 hour. The supernatant was discarded, and the plates were washed three times with PBST. The plates were then incubated with 100 μL / well of horseradish peroxidase-conjugated goat anti-rabbit IgG (Fc-specific) secondary antibody (GenScript, A01856) at 37°C for 0.5 hour. The plates were washed five times with PBST, and then TMB colorimetric solution was added and allowed to react at room temperature in the dark for 13 minutes. Finally, 50 μL of 1 M HCl stop solution (Sinopharm, 10011018) was added to terminate color development. The plate was read at 450 nm using a microplate reader. Cells from positive wells with an absorbance greater than 1.0 were selected for subsequent experiments.

[0100] Example 3 Variable region sequencing of monoclonal antibodies

[0101] TRIzol (Life Technology, 15596-026) was used to extract RNA from total cells in wells with OD values ​​greater than 1.0, and universal primers (Prime Script TM 1 st The cDNA was reverse transcribed into cDNA using the Strand cDNA Synthesis Kit (Takara). Rabbit immunoglobulin heavy and light chain V-region fragments were subsequently amplified by RACE PCR. The amplified fragments were homologously recombined into the pCDNA3.4 vector, and the inserts were sequenced using vector-specific primers. Ultimately, the unique V-region protein amino acid sequences and plasmids of clones 24F5-1, 72E8-1, 75D3-1, and 75F2-1 were obtained.

[0102] 24F5-1 heavy chain variable region amino acid sequence (SEQ ID NO: 4):

[0103] 24F5-1 light chain variable region amino acid sequence (SEQ ID NO: 5):

[0104] 72E8-1 heavy chain variable region amino acid sequence (SEQ ID NO: 6):

[0105] 72E8-1 light chain variable region amino acid sequence (SEQ ID NO: 7):

[0106] 75D3-1 heavy chain variable region amino acid sequence (SEQ ID NO: 8):

[0107] 75D3-1 light chain variable region amino acid sequence (SEQ ID NO: 9):

[0108] 75F2-1 heavy chain variable region amino acid sequence (SEQ ID NO: 10):

[0109] 75F2-1 light chain variable region amino acid sequence (SEQ ID NO: 11):

[0110] Table 2 CDR region sequences of antibodies

[0111] Example 4 Production of monoclonal antibodies based on recombinant expression

[0112] The plasmids containing the antibody heavy chain and light chain were co-transfected into ExpiCHO-S TM Cells (Gibco, A29133) cells were cultured in a shake flask at 37°C for 6 days, and the supernatant was collected for antibody purification. Antibody purification steps: The protein A column was balanced with a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0). The harvested cell culture supernatant was then diluted 1:1 with 2× the above buffer and sterilized by filtration. The filtered supernatant and protein A column were incubated at room temperature for 2 hours. After washing the column with 1× the above buffer, the IgG was eluted with sterile 0.1M sodium citrate (pH 3.5), and the eluate was collected and neutralized with one-ninth volume of sterile 1M Tris-HCl (pH 9.0). Under sterile conditions, the product buffer was exchanged with PBS (pH 7.4) to remove residual elution buffer, and the antibody was quantified by OD280nm using an extinction coefficient Ec (0.1%) of 1.43.

[0113] Example 5 Cross-reactivity of monoclonal antibodies to various serotypes of AAV virus VLPs

[0114] Indirect ELISA was used to evaluate the binding ability of purified antibodies to VLPs of AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVDJ, and AAV8. 100 μL of 1 μg / mL VLP diluted in PBS was added to each well of a 96-well microtiter plate and coated overnight at 4°C. After washing with PBS-T (0.05% Tween), each well was blocked with 150 μL of 1% BSA in PBST at 37°C for 1 hour. The blocking buffer was then discarded, and 100 μL of recombinantly expressed antibody at a concentration of 1 μg / mL was added to each well, followed by incubation at 37°C for 1 hour. After washing three times with PBST, 100 μL of horseradish peroxidase-conjugated goat anti-rabbit IgG (Fc-specific) secondary antibody (GenScript, A01856) was added to each well and incubated at 37°C for 0.5 hour. After washing five times with PBST, TMB colorimetric solution was added and incubated at room temperature in the dark for 13 minutes. Finally, 50 μL of 1 M HCl stop solution (Sinopharm, 10011018) was added to stop color development. The plate was read at 450 nm using a microplate reader. The results are shown in Figure 2.

[0115] Example 6 Monoclonal Antibody to AAV8 VLP Binding EC 50 test

[0116] The binding ability of the purified antibodies to AAV8 VLPs was assessed by indirect ELISA. A 96-well microtiter plate was coated with 1 μg / mL AAV8 VLPs overnight at 4°C. After washing with PBS-T (0.05% Tween), 250 μL of 1% BSA in PBST was added to each well and blocked at 37°C for 2 hours. The blocking buffer was then discarded, and 100 μL of the purified antibody at 1 μg / mL was added to the first well. The antibody was diluted three-fold to a total of 11 test concentrations, plus a control well containing only PBST. The plates were then incubated at 37°C for 1 hour. After three washes with PBST, 100 μL of horseradish peroxidase-conjugated goat anti-rabbit IgG (Fc-specific) secondary antibody (GenScript, A01856) was added to each well and incubated at 37°C for 0.5 hours. After four washes with PBST, TMB colorimetric solution (GenScript) was added and incubated at room temperature in the dark for 15 minutes. Finally, 50 μL of 1 M HCl stop solution (Sinopharm, 10011018) was added to stop the color development. The plate was read at 450 nm using an enzyme reader. The EC values ​​of each monoclonal antibody were 50 The detection curve is shown in Figure 3, and the EC50 values ​​are shown in Table 3.

[0117] Table 3 EC of antibodies binding to AAV8 VLP 50 value

[0118] Example 7: Paired detection of monoclonal antibodies

[0119] 96-well microtiter plates were coated with 2.5 μg / mL of unlabeled purified antibody (e.g., 75F2-1) overnight at 4°C. The plates were washed with PBST (0.05% Tween) and blocked with 250 μL / well of PBST containing 1% BSA at 37°C for 2 hours. The blocking buffer was then discarded and 100 μL of AAV8 VLPs (pre-well concentration: 100 ng / mL, 2-fold dilution, and 0 ng / mL) were added, respectively, and incubated at 37°C for 1 hour. After washing four times with PBST, 0.5 μg / mL of biotin-labeled antibody (e.g., Biotin-72E8-1) was added to the plates, with 100 μL per well added, and incubated at 37°C for 1 hour. The plates were washed four times with PBST and incubated with 100 μL / well of streptavidin-HRP (SA-HRP, GenScript) at 37°C for 15 minutes. After washing 4 times with PBST, TMB colorimetric solution (GenScript) was added to each well and reacted in the dark at room temperature for 15 minutes. The reaction was terminated by adding 50 μL of 1 M HCl stop solution (Sinopharm, 10011018). The plate was read at 450 nm using a microplate reader. The specific OD values ​​are shown in Table 4 and Figure 4. The well values ​​with an antigen concentration of 0 were lower than 0.1, and the values ​​of the detection antibody had a good linear relationship with the decrease of the antigen gradient (R 2 value greater than 0.99), we determined that these two antibodies were a relatively good pairing antibody and could be used for the development of an ELISA kit for double-antibody sandwich detection of antigens.

[0120] Table 4 Antibody pairing results

[0121] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. An anti-AAV8 monoclonal antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises a heavy chain CDR and a light chain CDR, wherein the heavy chain CDR and the light chain CDR are selected from the group consisting of CDRs of the amino acid sequences shown in SEQ ID NO: 12 to SEQ ID NO: 35 and variants thereof each comprising at most three amino acid mutations, and the combination of the group is defined by one or more of the groups a to d:

2. The anti-AAV8 monoclonal antibody or a functional fragment thereof according to claim 1, wherein the heavy chain CDR and the light chain CDR are selected from the following sequences: a. The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 12, 13, 14, 15, 16 and 17, respectively; b. The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 18, 19, 20, 21, 22 and 23, respectively; c. the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 24, 25, 26, 27, 28 and 29, respectively; or d. Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and The amino acid sequences of the light chain CDR3 are shown in SEQ ID NOs: 30, 31, 32, 33, 34 and 35, respectively.

3. The anti-AAV8 monoclonal antibody or functional fragment thereof according to claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of variable regions of the amino acid sequences shown in SEQ ID NO: 4 to SEQ ID NO: 11 and variants thereof each comprising an amino acid sequence with at least 80% identity, and the combination of the group is defined by one or more of A to D:

4. The anti-AAV8 monoclonal antibody or functional fragment thereof according to claim 3, wherein the heavy chain variable region and the light chain variable region are selected from the following sequences: A. the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:5; B. the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:6, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7; C. the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9; or D. The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

11.

5. The anti-AAV8 monoclonal antibody or a functional fragment thereof according to any one of claims 1, 2, and 4, which is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

6. The anti-AAV8 monoclonal antibody or functional fragment thereof according to any one of claims 1, 2, and 4, which has a detectable label. 7 . An isolated polynucleotide encoding the anti-AAV8 monoclonal antibody or a functional fragment thereof according to any one of claims 1 to 6 .

8. The polynucleotide according to claim 7, characterized in that The polynucleotide comprises a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody or a functional fragment thereof, and a nucleotide sequence encoding the light chain variable region of the monoclonal antibody or a functional fragment thereof.

9. An expression vector comprising the polynucleotide according to claim 7 or 8.

10. A host cell or a cell-free expression system comprising the expression vector according to claim 9.

11. A method for preparing an anti-AAV8 monoclonal antibody or a functional fragment thereof, comprising: Cultivating the host cell according to claim 10 under suitable culture conditions; as well as The antibodies or antigen-binding fragments thereof thus produced are recovered from the culture medium or from the cultured cells.

12. A kit for detecting AAV8 virus, comprising the monoclonal antibody or a functional fragment thereof according to any one of claims 1 to 6.

13. A chromatographic medium for separating AAV8 virus, the chromatographic medium comprising a matrix support and the monoclonal antibody or a functional fragment thereof according to any one of claims 1 to 6 immobilized on the matrix support.

14. A chromatographic separation device comprising the chromatographic medium according to claim 13.

15. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in detecting and / or purifying AAV8.

Images