Nanobodies specifically binding aav9 and uses thereof

Abstract

The application belongs to the technical field of nanobodies, and more particularly relates to a nanobody specifically binding to AAV9 and application thereof. The nanobody provided by the application can specifically bind to AAV9 adeno-associated virus, and does not bind to other AAV serotypes such as AAV2, AAV5 and AAV8, so that the AAV9 adeno-associated virus can be detected with high sensitivity and high specificity. In actual application, the nanobody provided by the application is coupled with a solid-phase carrier as an affinity filler, so that the AAV9 adeno-associated virus can be effectively captured and eluted, the virus purification and recovery effect is effectively improved, and the nanobody is suitable for high-purity recovery of the AAV9 adeno-associated virus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanobodies, and more particularly relates to a nanobody specifically binding to AAV9 and application thereof. BACKGROUND

[0002] Adeno-associated virus (AAV) belongs to the Parvoviridae family, is a single-stranded DNA virus that cannot replicate autonomously and is non-enveloped, and has infectivity to both dividing and non-dividing cells. AAV virus can infect a large variety of tissue cells, has no or low pathogenicity and low immunogenicity in the host cell proliferation process, and can stably express the carried gene for a long time, and is suitable for use as a vector for in vivo gene transfer. At present, a variety of human AAV serotypes have been found, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12, and mutants thereof. Different AAV serotypes have different capsid protein spatial structures, sequences and tissue specificities, and there are obvious differences in the cell surface receptors they recognize and bind, so the tissue types, cell types and infection efficiencies of different AAV serotypes are different.

[0003] AAV9 is one of the most clinically used natural serotypes at present, and is widely used in the central nervous system and muscle system. Compared with other AAV serotypes, AAV9 is one of the few serotypes that can cross the blood-brain barrier (BBB) after intravenous injection, making it the preferred vector for nervous system diseases (such as SMA and ALS). AAV9 has high affinity for liver, heart, skeletal muscle and peripheral nerves, and is suitable for systemic diseases (such as DMD and Pompe disease). AAV9 vector is used as a carrier for gene engineering drugs to deliver drugs, and has wide clinical applications. For example, Zolgensma (onasemnogene abeparvovec) is a drug that uses AAV9 vector to carry SMN1 gene to treat spinal muscular atrophy disease. In addition, AAV9 vector is used for drug delivery in genetic diseases such as Duchenne muscular dystrophy (DMD), Pompe disease, Fabry disease, amyotrophic lateral sclerosis (ALS), and hereditary transthyretin amyloidosis (hATTR). It is of great significance to provide an antibody with high affinity, good stability and specific binding to AAV9 for the enrichment, purification, detection and quality control of the viral vector AAV9 for gene therapy. However, the current broad-spectrum antibody that can recognize multiple AAV serotypes has low affinity for specific AAV9 serotypes, poor purification effect, and complex antibody preparation process, long preparation cycle, and is not suitable for mass production, which limits the clinical application of AAV9. SUMMARY

[0004] In view of the defects of the prior art, the purpose of the present application is to provide a nanobody specifically binding to AAV9 and application thereof, aiming to solve the problems of low affinity of existing nanobody to AAV9 serotype, poor purification effect, and complex preparation process of nanobody, long preparation period, and high cost.

[0005] To achieve the above-mentioned purpose, in a first aspect, the present application provides a nanobody specifically binding to AAV9, wherein the heavy chain variable region of the nanobody comprises complementarity determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the complementarity determining regions of the nanobody are selected from any one of (a) to (h):

[0006] (a): CDR1 as shown in SEQ ID NO. 1, CDR2 as shown in SEQ ID NO. 2 and CDR3 as shown in SEQ ID NO. 3;

[0007] (b): CDR1 as shown in SEQ ID NO. 8, CDR2 as shown in SEQ ID NO. 9 and CDR3 as shown in SEQ ID NO. 10;

[0008] (c): CDR1 as shown in SEQ ID NO. 13, CDR2 as shown in SEQ ID NO. 14 and CDR3 as shown in SEQ ID NO. 15;

[0009] (d): CDR1 as shown in SEQ ID NO. 20, CDR2 as shown in SEQ ID NO. 21 and CDR3 as shown in SEQ ID NO. 22;

[0010] (e): CDR1 as shown in SEQ ID NO. 26, CDR2 as shown in SEQ ID NO. 27 and CDR3 as shown in SEQ ID NO. 28;

[0011] (f): CDR1 as shown in SEQ ID NO. 32, CDR2 as shown in SEQ ID NO. 33 and CDR3 as shown in SEQ ID NO. 34;

[0012] (g): an amino acid sequence having at least 90% sequence homology with (a), (b), (c), (d), (e) or (f);

[0013] (h): a functional conservative variant of the amino acid sequence as shown in (a), (b), (c), (d), (e) or (f) by one or more of substitution, deletion, insertion of one or more amino acids.

[0014] Preferably, the heavy chain variable region of the above-mentioned nanobody further comprises a framework region FR1, FR2, FR3, FR4.

[0015] Preferably, the amino acid sequence of the framework region of the above-mentioned nanobody is selected from any one of (1)-(8):

[0016] (1): FR1 as shown in SEQ ID NO. 4, FR2 as shown in SEQ ID NO. 5, FR3 as shown in SEQ ID NO. 6, and FR4 as shown in SEQ ID NO. 7;

[0017] (2): FR1 as shown in SEQ ID NO. 4, FR2 as shown in SEQ ID NO. 11, FR3 as shown in SEQ ID NO. 12, and FR4 as shown in SEQ ID NO. 7;

[0018] (3): FR1 as shown in SEQ ID NO. 16, FR2 as shown in SEQ ID NO. 17, FR3 as shown in SEQ ID NO. 18, and FR4 as shown in SEQ ID NO. 19;

[0019] (4): FR1 as shown in SEQ ID NO. 23, FR2 as shown in SEQ ID NO. 24, FR3 as shown in SEQ ID NO. 25, and FR4 as shown in SEQ ID NO. 7;

[0020] (5): FR1 as shown in SEQ ID NO. 29, FR2 as shown in SEQ ID NO. 30, FR3 as shown in SEQ ID NO. 31, and FR4 as shown in SEQ ID NO. 7;

[0021] (6): FR1 as shown in SEQ ID NO. 35, FR2 as shown in SEQ ID NO. 36, FR3 as shown in SEQ ID NO. 37, and FR4 as shown in SEQ ID NO. 7;

[0022] (7): an amino acid sequence having at least 90% sequence homology with (1), (2), (3), (4), (5), or (6);

[0023] (8): a functional conservative variant of the amino acid sequence as shown in (1), (2), (3), (4), (5), or (6) with one or several substitutions, deletions, insertions of one or more amino acids.

[0024] Preferably, the amino acid sequence of the heavy chain variable region of the above-mentioned nanobody is selected from any one of (I)-(III):

[0025] (I): any one of SEQ ID NO. 38 ~ SEQ ID NO. 43;

[0026] (II): an amino acid sequence having at least 90% sequence homology with (I);

[0027] (III): a function-conservative variant of the amino acid sequence shown in (I) by one or more of substitution, deletion, insertion of one or several amino acids.

[0028] In a second aspect, the present application provides a multivalent construct comprising at least one of the above-mentioned nanobodies, or a combination thereof.

[0029] In a third aspect, the present application provides a fusion protein comprising one or more antigen binding functional moieties; wherein the antigen binding functional moiety comprises the above-mentioned nanobody.

[0030] In a fourth aspect, the present application provides a nucleic acid comprising a nucleic acid sequence encoding the above-mentioned nanobody.

[0031] In a fifth aspect, the present application provides an expression vector comprising the above-mentioned nucleic acid.

[0032] In a sixth aspect, the present application provides a host cell comprising the above-mentioned nucleic acid or the above-mentioned expression vector.

[0033] In a seventh aspect, the present application provides the above-mentioned nanobody, the above-mentioned multivalent construct or the above-mentioned fusion protein for use in the preparation of an AAV9 adeno-associated virus detection kit or a purification kit.

[0034] In an eighth aspect, the present application provides an AAV9 adeno-associated virus detection kit comprising the above-mentioned nanobody, the above-mentioned multivalent construct or the above-mentioned fusion protein.

[0035] In a ninth aspect, the present application provides an AAV9 adeno-associated virus purification kit comprising the above-mentioned nanobody, the above-mentioned multivalent construct or the above-mentioned fusion protein.

[0036] Overall, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages:

[0037] (1) The nanobody provided by the present application can specifically bind to AAV9 adeno-associated virus, but not to other AAV serotypes such as AAV2, AAV5, AAV8, thereby achieving high sensitivity and high specificity in detecting AAV9 adeno-associated virus. In actual application, the nanobody provided by the present application is coupled to a solid-phase carrier as an affinity filler, which can effectively capture AAV9 adeno-associated virus and elute it gently, thereby effectively improving the virus purification and recovery effect, and being suitable for high-purity recovery of AAV9 adeno-associated virus.

[0038] (2) The preparation process of the nanobody provided in the application is short, wherein the incubation combination time of the phage library containing the nanobody and the antigen (AAV9 adeno-associated virus) is only 10 min, which is obviously shorter than the conventional preparation process (the incubation combination time of the phage library containing the nanobody and the antigen is 30 min). In actual production, the production cycle can be greatly shortened, the production cost can be greatly reduced, and batch production is facilitated. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The SDS-PAGE silver staining results of the affinity filler purified AAV9 virus prepared by the embodiment of the application and coupled with 6 nanobodies respectively. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the application clearer, the application will be further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the application and should not be used to limit the application.

[0041] In the description of the embodiments of the application, the words such as "exemplary" or "for example" are used to represent an example, illustration or description. Any embodiment or design scheme described as "exemplary" or "for example" in the embodiments of the application should not be interpreted as more preferred or more advantageous than other embodiments or design schemes. Rather, the words such as "exemplary" or "for example" are intended to present the relevant concept in a specific manner.

[0042] In the description of the embodiments of the application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0043] The term "antibody" as used in the present application is used in its broadest sense to include immunoglobulins or other types of molecules comprising one or more antigen binding domains that specifically bind to an antigen, a protein or polypeptide that exhibits binding specificity to a particular antigen. Specific examples of antibodies can include intact antibodies (e.g., classical four-chain antibody molecules), single chain antibodies, nanobodies, multispecific antibodies, and the like. A classical antibody molecule is typically a tetramer composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds. Based on the conserved differences in amino acid sequences, the heavy and light chains are divided into variable regions (V) at the amino terminal and constant regions (C) at the carboxy terminal. The variable regions are responsible for recognizing and binding antigens, and the constant regions (e.g., Fc fragment) are responsible for initiating downstream effects, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Within the variable regions of the heavy and light chains, there are three local regions of amino acid composition and arrangement that have a higher degree of variability, which are the key positions for antibody-antigen binding, and thus are also called complementarity determining regions (CDRs). The amino acid sequences of the CDRs can be determined using art-recognized numbering schemes, such as using Kabat, Chothia, IMGT, AbM, or Contact. The three complementarity determining regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, and the three complementarity determining regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3. Each heavy chain variable region (VH) and light chain variable region (VL) can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0044] The term "nanobody" is also referred to as "single domain antibody (sdAb)" or "V H H antibody" refers to an antibody molecule having antigen binding ability comprising a heavy chain variable region without a light chain.

[0045] The term "specifically binds" refers to the reaction of a nanobody with a particular target antigen more frequently, more rapidly, for a longer duration, and / or with greater affinity than with other target antigens.

[0046] The term "sequence identity" is also referred to as "sequence homology" and refers to the degree of identity between amino acid sequences or between nucleotide sequences, typically expressed as a percentage.

[0047] In the present application, "functionally conservative variant" or "variant" refers to an amino acid sequence which is based on the parent amino acid sequence and has one or more substitutions, deletions, insertions of amino acids without changing the desired properties (such as antigen affinity and / or antigen specificity). It can be understood that, based on the specific nanobody sequences provided in the present application, corresponding variants of the nanobody provided in the present application can be obtained by replacing, deleting, adding a small number of amino acids and verifying or screening the binding ability or biological activity of the obtained product to the corresponding antigen, so that these variants should also be included in the scope of the present application.

[0048] The term "multivalent construct" refers to a construct having at least two antigen binding sites / binding units. The multivalent construct can be, but is not limited to, a bivalent construct, a trivalent construct or a tetravalent construct.

[0049] The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" can be used interchangeably and refer to a nucleotide polymer. Such nucleotide polymers can contain natural and / or unnatural nucleotides. "Nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0050] The term "fusion protein" refers to a protein molecule artificially generated (for example, by genetic engineering technology) by at least two different peptide segments. These peptide segments do not exist in nature or do not exist in the same protein molecule. Common fusion proteins including antibody fragments include but are not limited to multispecific antibodies, enzyme-labeled antibodies for immunodetection, etc.

[0051] The term "vector" refers to a nucleic acid molecule that can be engineered to contain a polynucleotide of interest (e.g., a sequence encoding a polypeptide of interest) or a nucleic acid molecule that can replicate in a host cell (e.g., a nucleic acid, a plasmid, or a virus, etc.). The vector can include one or more of the following components: an origin of replication, one or more regulatory sequences that regulate the expression of the polynucleotide of interest (such as a promoter and / or an enhancer), and / or one or more selectable marker genes (such as an antibiotic resistance gene and a gene that can be used in colorimetric analysis, e.g., beta-galactosidase). The term "expression vector" refers to a vector for expressing a polypeptide of interest in a host cell.

[0052] The present application provides a nanobody specifically binding to AAV9, the heavy chain variable region of which comprises complementarity determining regions CDR1, CDR2, CDR3, the amino acid sequences of the complementarity determining regions of the above-mentioned nanobody being selected from any one of (a) to (h):

[0053] (a): CDR1 as shown in SEQ ID NO. 1, CDR2 as shown in SEQ ID NO. 2 and CDR3 as shown in SEQ ID NO. 3;

[0054] (b): CDR1 as shown in SEQ ID NO. 8, CDR2 as shown in SEQ ID NO. 9, and CDR3 as shown in SEQ ID NO. 10;

[0055] (c): CDR1 as shown in SEQ ID NO. 13, CDR2 as shown in SEQ ID NO. 14, and CDR3 as shown in SEQ ID NO. 15;

[0056] (d): CDR1 as shown in SEQ ID NO. 20, CDR2 as shown in SEQ ID NO. 21, and CDR3 as shown in SEQ ID NO. 22;

[0057] (e): CDR1 as shown in SEQ ID NO. 26, CDR2 as shown in SEQ ID NO. 27, and CDR3 as shown in SEQ ID NO. 28;

[0058] (f): CDR1 as shown in SEQ ID NO. 32, CDR2 as shown in SEQ ID NO. 33, and CDR3 as shown in SEQ ID NO. 34;

[0059] (g): an amino acid sequence having at least 90% sequence identity to (a), (b), (c), (d), (e), or (f);

[0060] (h): a functional conservative variant of the amino acid sequence as shown in (a), (b), (c), (d), (e), or (f) with one or more of substitution, deletion, insertion of one or several amino acids.

[0061] The nanobody provided in the present application can specifically bind to AAV9 adeno-associated virus, but not to other AAV serotypes such as AAV2, AAV5, AAV8, and can be used for purifying AAV9 virus. In the purification process, AAV9 virus can be captured and eluted very gently, and the purified AAV9 virus has high purity and high yield.

[0062] In some embodiments, the heavy chain variable region of the above-mentioned nanobody further comprises a framework region FR1, FR2, FR3, FR4.

[0063] In some embodiments, the amino acid sequence of the framework region of the above-mentioned nanobody is selected from any one of (1)-(8):

[0064] (1): FR1 as shown in SEQ ID NO. 4, FR2 as shown in SEQ ID NO. 5, FR3 as shown in SEQ ID NO. 6, and FR4 as shown in SEQ ID NO. 7;

[0065] (2): FR1 shown in SEQ ID NO. 4, FR2 shown in SEQ ID NO. 11, FR3 shown in SEQ ID NO. 12, and FR4 shown in SEQ ID NO. 7;

[0066] (3): FR1 shown in SEQ ID NO. 16, FR2 shown in SEQ ID NO. 17, FR3 shown in SEQ ID NO. 18, and FR4 shown in SEQ ID NO. 19;

[0067] (4): FR1 shown in SEQ ID NO. 23, FR2 shown in SEQ ID NO. 24, FR3 shown in SEQ ID NO. 25, and FR4 shown in SEQ ID NO. 7;

[0068] (5): FR1 shown in SEQ ID NO. 29, FR2 shown in SEQ ID NO. 30, FR3 shown in SEQ ID NO. 31, and FR4 shown in SEQ ID NO. 7;

[0069] (6): FR1 shown in SEQ ID NO. 35, FR2 shown in SEQ ID NO. 36, FR3 shown in SEQ ID NO. 37, and FR4 shown in SEQ ID NO. 7;

[0070] (7): an amino acid sequence having at least 90% sequence identity to any one of (1), (2), (3), (4), (5), or (6);

[0071] (8): a function-conservative variant of the amino acid sequence shown in any one of (1), (2), (3), (4), (5), or (6) by one or more of substitution, deletion, insertion of one or several amino acids.

[0072] In some embodiments, the amino acid sequence of the heavy chain variable region of the above-mentioned nanobody is selected from any one of (I) - (III):

[0073] (I): any one of SEQ ID NO. 38 ~ SEQ ID NO. 43;

[0074] (II): an amino acid sequence having at least 90% sequence identity to (I);

[0075] (III): a function-conservative variant of the amino acid sequence shown in (I) by one or more of substitution, deletion, insertion of one or several amino acids.

[0076] The nanobodies are easily converted into multivalent formats, such as bivalent, trivalent, tetravalent or other multispecific constructs. Based on this, the present application provides a multivalent construct comprising at least one of the above-mentioned nanobodies, or a combination thereof.

[0077] In some embodiments, the above-mentioned multivalent construct is a bivalent construct or a trivalent construct.

[0078] In some embodiments, the above-mentioned nanobodies can be linked to a detectable label to form a fusion protein. The detectable label is beneficial for detecting the nanobodies, or beneficial for detecting the immune complex formed by the nanobodies and the corresponding antigen (AAV or VP protein thereof), which can be used to indicate the presence or content of AAV or VP protein thereof in the sample. The present application does not have special restrictions on the detectable label, including but not limited to enzymes commonly used in immunoassays (such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), etc.); and fluorescent proteins (such as GFP). In other embodiments, the above-mentioned nanobodies can also be linked to a purifiable label to form a fusion protein. The purifiable label includes but is not limited to His6 tag, Flag tag, MBP tag, GST tag, SUMO tag, etc., which is beneficial for separating the above-mentioned nanobodies from the sample. Based on this, the present application provides a fusion protein comprising one or more antigen binding functional moieties, wherein the antigen binding functional moiety comprises the above-mentioned nanobodies.

[0079] The present application also provides a nucleic acid comprising a nucleic acid sequence encoding the above-mentioned nanobodies.

[0080] It is well known to those skilled in the art that the nucleic acid sequence encoding the nanobodies is operably linked to a vector. Based on this, the present application provides an expression vector comprising the above-mentioned nucleic acid molecule. It can be understood that the above-mentioned vector refers to any genetic element, including but not limited to plasmid, bacteriophage, transposon, cosmid, chromosome, virus (such as retrovirus, lentivirus), virion, etc., which can replicate when combined with appropriate control elements, and can transfer gene sequences to cells.

[0081] In some embodiments, the above-mentioned expression vector can further comprise other genes, such as marker genes that allow selection of the expression vector in appropriate host cells and under appropriate conditions. In addition, the above-mentioned expression vector can further comprise expression control elements that allow the coding region to be correctly expressed in appropriate hosts. The above-mentioned expression control elements include but are not limited to promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, etc.

[0082] The present application also provides a host cell comprising the nucleic acid molecule or the expression vector, which can be used to prepare the nanobody. The host cell can be a prokaryotic cell or a eukaryotic cell. The eukaryotic cell includes mammalian cells (such as primate or non-primate cells), fungal cells (such as yeast), plant cells, and insect cells. It also includes, but is not limited to, CHO cells, HEK-293 cells, BHK cells, or PER-C6 cells, and their derivative cells, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells.

[0083] The present application also provides a solid phase carrier for isolation and / or purification of AAV9 adeno-associated virus, which is coupled with the nanobody. The nanobody is immobilized on the surface of the solid phase carrier by binding with the functional groups on the surface of the solid phase carrier, such as hydroxyl, carboxyl, amino, etc. The solid phase carrier includes, but is not limited to, polymer microspheres, agarose gel, dextran, cellulose, polycarbonate, digestible fiber, silica, or magnetic microspheres, etc.

[0084] The nanobody, multivalent construct, or fusion protein provided by the present application can be used for isolation and / or purification of AAV9 adeno-associated virus. In some embodiments, the isolation and / or purification process comprises the following steps: coupling the nanobody to the solid phase carrier, contacting the sample comprising AAV9 adeno-associated virus with the solid phase carrier, and then separating the solid phase carrier from the sample and eluting the adsorbed AAV9 adeno-associated virus.

[0085] The present application also provides the use of the nanobody, the multivalent construct, or the fusion protein in the preparation of a kit for detecting or purifying AAV9 adeno-associated virus. In the art, the methods for detecting antigens using antibodies include, but are not limited to, antigen-antibody precipitation reaction, immunodiffusion experiment, immune turbidity, immunoelectrophoresis (including immunoblotting), immunofluorescence technology, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), etc. The ELISA method can also be divided into direct ELISA, indirect ELISA, sandwich ELISA, etc. These detection methods can be used to detect AAV9 adeno-associated virus using the nanobody provided by the present application.

[0086] In another aspect, the present application also provides a kit for detecting AAV9 adeno-associated virus, which comprises the nanobody, the multivalent construct, or the fusion protein. It can be understood that when the fusion protein is used for immunodetection, various enzymes (such as HRP) or fluorescent proteins (GFP) can be used to facilitate the detection of AAV9 adeno-associated virus.

[0087] The application also provides an AAV9 adeno-associated virus purification kit, which comprises the above-mentioned nanobody, the above-mentioned multivalent construct or the above-mentioned fusion protein, and further comprises the above-mentioned solid carrier.

[0088] It should be understood that materials identical or similar to the types, models, qualities, properties or functions of the reagents and instruments used in the following examples can be used to implement the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0089] The main reagents and materials used in the following examples are as follows:

[0090] Lymphocyte separation medium was purchased from Solabio Company, TRIzol™ Reagent kit was purchased from Thermo Fisher Company, Taq enzyme was purchased from TAKARA Company, rabbit anti-goat secondary antibody was purchased from Jinser Biological Technology Co., Ltd., NHS-activated 4FF affinity filler, SC-Ni NTA FF affinity column were purchased from Shangchun Biological Technology (Wuhan) Co., Ltd., iTaq Universal SYBR Green Supermix was purchased from Bio-Rad Company, and POROSTM CaptureSelectTM AAV9 affinity resin was purchased from Thermo Company.

[0091] The following are examples:

[0092] Example 1 Screening of Anti-AAV9 Adeno-Associated Virus Nanobody

[0093] 1. Experimental materials

[0094] AAV9 virus, lymphocyte separation medium, TRIzol™ Reagent kit, Taq enzyme, E. coli TG1 competent cells

[0095] 2. Experimental steps

[0096] (1) An adult healthy alpaca was immunized with purified AAV9 adeno-associated virus, and the immunization dose was 1×10 11 virus particles per animal, and a total of three rounds of immunization were performed with an immunization interval of two weeks. After immunization, 50 mL of alpaca peripheral blood was collected, and B lymphocytes were obtained by using lymphocyte separation medium (item number P8900).

[0097] (2) The total RNA of B lymphocytes was extracted by using TRIzol™ Reagent kit (item number 15596018), and cDNA was generated by reverse transcription. The VHH gene was obtained by nest PCR amplification using the cDNA as a template. The first round of primers used in the nest PCR amplification included primer F1 and primer R1, wherein the nucleotide sequence of primer F1 was 5'-CTTGGTGGTCCTGGCTGC-3'; and the nucleotide sequence of primer R1 was 5'-GGTACGTGCTGTTGAACTGTTCC-3'. The second round of primers used in the nest PCR amplification included primer F2, primer R2 and primer R3, wherein the nucleotide sequence of primer F2 was 5'-CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGT GGAGTC-3'; the nucleotide sequence of primer R2 was 5'-CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGG TCTTCGCTGTGGTGCG-3'; and the nucleotide sequence of primer R3 was 5'-CATGCCATGACTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGT GTCTTGGG-3'. The DNA polymerase used in the nest PCR amplification was Taq enzyme (item number R001A), and the amplification program was set according to the instruction of Taq.

[0098] (3) The amplified VHH gene fragment and phagemid display vector pComb 3XSS were respectively digested by Sfil restriction endonuclease, and then the enzyme-digested VHH gene fragment and linearized vector pComb 3XSS were connected by using T4 DNA ligase. The ligation product was recovered, and the enzyme ligation product was electrotransformed into E. coli TG1 competent cells to construct a nanobody phage display library. The plate gradient dilution method was used to determine that the library capacity was 1.83x10 9 The cloning efficiency of the nanobody gene inserted into the vector was 98%.

[0099] (4) According to the requirement of immunoaffinity purification, the nanobody with the "fast on and fast off" binding characteristics was expected to be obtained. Unlike the conventional screening strategy (the incubation time of the nanobody phage display library and the antigen was 30 min), the incubation time of the library and the antigen was greatly shortened, and a mild elution method was used to screen the nanobody against AAV9 adeno-associated virus. The specific screening steps were as follows:

[0100] The first round of screening: the microplate was coated with AAV9 adeno-associated virus according to 100 μL / well, and the virus coating dose was 1x10 11VP / mL, 4°C overnight, the next day, 1% bovine serum albumin (BSA) was used to block the microplate, and the microplate was washed with PBST; 100 μL of the constructed nanobody phage display library was added, and the microplate was incubated at room temperature for 10 min, and then washed with PBST and PBS, respectively; 100 μL of citrate buffer (50 mM, pH 3.0) was added to the microplate, and after gentle shaking for 2 min, the eluate was collected; the eluate was used to infect E. coli TG1 in the logarithmic growth phase, and a small amount of the infected E. coli TG1 was used to determine the titer of the eluate by plate gradient dilution method, and the remaining was used to obtain an anti- AAV9 specific phage enrichment library by amplification.

[0101] The second round of screening: the same operation as in the "first round of screening" was used, except that 1% ovalbumin (OVA) was used to block the microplate, the library incubation time was 5 min, and 100 μL of the enrichment library obtained by the first round of screening was used to bind the microplate coated with AAV9 adeno-associated virus. After elution and library expansion, an anti- AAV9 specific phage enrichment library was obtained.

[0102] The third round of screening: the same operation as in the "first round of screening" was used, except that the library incubation time was 5 min, and 100 μL of the enrichment library obtained by the second round of screening was used to bind the microplate coated with AAV9 adeno-associated virus.

[0103] The fourth round of screening: the same operation as in the "first round of screening" was used, except that 1% ovalbumin (OVA) was used to block the microplate, the library incubation time was 5 min, and 100 μL of the enrichment library obtained by the third round of screening was used to bind the microplate coated with AAV9 adeno-associated virus.

[0104] The phage eluate obtained by the fourth round of screening was used to infect E. coli TG1, and single colonies were randomly picked from the culture dish for titer determination, inoculated into a 2 mL deep well plate, and cultured at 30°C until the OD 600 About 0.5, and the nanobody-containing bacterial culture supernatant was collected by centrifugation. The specificity of the nanobody to AAV9 adeno-associated virus was detected by enzyme-linked immunosorbent assay (ELISA), and the specific steps were as follows: 1 × 10 11The AAV9 adeno-associated virus at 1 x 106vp / mL was blocked with 5% skim milk, then the bacterial culture supernatant containing the nanobody was added at an appropriate dilution, and binding was performed at 37°C for 1 h. Unbound antibodies were washed away using PBST for 3 times, then a 1:5000 dilution of HRP-conjugated rabbit anti-goat secondary antibody was added, and binding was performed at 37°C for 1 h. After washing with PBST for 3 times, TMB (3,3',5,5'-tetramethylbenzidine) substrate solution was added for color development for 15 min, and color development was terminated with 2M H2SO4. The absorbance value of each well was read at a wavelength of 450 nm. A negative control (containing only blocking agent skim milk, no bacterial culture supernatant containing nanobody) and a blank well (buffer) were set.

[0105] 3. Experimental results

[0106] OD of the test well 450 If the value is more than 3 times that of the negative control, it is determined to be positive, and the results are shown in Table 1.

[0107] Table 1. Detection results of positive clones screened in the application

[0108]

[0109] As can be seen from Table 1, 6 positive clones screened in the application have affinity for AAV9 adeno-associated virus.

[0110] Example 2. Preparation and large-scale production of anti-AAV9 adeno-associated virus nanobody

[0111] 1. Experimental materials

[0112] Positive wells screened in Example 1, SC-Ni NTA FF affinity column

[0113] 2. Experimental steps

[0114] (1) Preparation of anti-AAV9 adeno-associated virus nanobody: the positive well corresponding clone was preserved and the vector was sequenced to obtain the VHH coding gene sequence. After the DNA sequence was translated into an amino acid sequence and compared, the clones with the same CDR1, CDR2 and CDR3 sequences were regarded as the same antibody strain, and the clones with different CDR sequences were regarded as different antibody strains.

[0115] (2) Large-scale production of anti-AAV9 adeno-associated virus nanobody: a VHH coding gene sequence with a 6xHis tag at the N-terminus is synthesized by whole gene synthesis, wherein the VHH coding gene can be any one of nanobodies A9-1 to A9-6, and then the fusion gene is inserted into the NcoI and NheI enzyme cutting sites of the PET25b vector. The obtained expression vector PET25b is transformed into E. coli Rosetta (DE3) competent cells to obtain a VHH expression recombinant strain. The recombinant strain is inoculated into LB medium containing 50 μg / mL ampicillin (Amp) at an inoculation amount of 1%, and cultured at 37°C with shaking until the OD 600 is 0.8, and 0.5 mM IPTG is added to a final concentration. The culture is induced at 22°C and 200 rpm for 16 h. The supernatant is discarded by centrifugation, and the bacterial pellet is resuspended in 20 mL of PBS (20 mM, pH 7.4) buffer. The washed bacterial pellet is resuspended in 20 mL of PBS and sonicated for 3-5 min to break the bacterial suspension. The solution is clarified, and the supernatant is separated by centrifugation at 10,000 rpm for 20 min. The broken cell supernatant is purified by a nickel column (SC-Ni NTA FF affinity column, item number SCB0E005B). The column is washed with 10 column volumes (CV) of a wash buffer (PBS + 10 mM imidazole) to remove impurities, and the target protein is eluted with 2 CV of an elution buffer (PBS + 250 mM imidazole). The eluted sample is dialyzed in PBS buffer at 4°C for 16 h, and then concentrated using a 3KDa ultrafiltration tube to obtain the purified nanobody. The protein concentration is determined by the BCA method, and the purity is detected by SDS-PAGE.

[0116] 3. Experimental results

[0117] Sequencing obtained anti-AAV9 adeno-associated virus nanobodies A9-1 to A9-6, and the amino acid sequence information is shown in Tables 2-7. CDR1-3 represents the complementarity determining region CDR1-3, FR1-4 represents the complementarity determining region FR1-4, and the amino acid sequence is divided based on the IMGT numbering rule.

[0118] The SDS-PAGE detection results show that the purity of anti-AAV9 adeno-associated virus nanobodies A9-1 to A9-6 is greater than 95%.

[0119] Table 2 Sequence information of nanobody A9-1 screened in the application

[0120]

[0121] Table 3 Sequence information of nanobody A9-2 screened in the application

[0122]

[0123] Table 4 Sequence information of the nanobody A9-3 screened in the present application

[0124]

[0125] Table 5 Sequence information of the nanobody A9-4 screened in the present application

[0126]

[0127] Table 6 Sequence information of the nanobody A9-5 screened in the present application

[0128]

[0129] Table 7 Sequence information of the nanobody A9-6 screened in the present application

[0130]

[0131] Example 3 Specificity of the anti-AAV9 adeno-associated virus nanobodies A9-1 to A9-6

[0132] 1. Experimental materials

[0133] Nanobodies A9-1 to A9-6, rabbit anti-goat secondary antibody

[0134] 2. Experimental procedure

[0135] 1 x 10 11 vp / mL of AAV9, AAV2, AAV5, AAV8 adeno-associated virus were coated, blocked with 5% skim milk, and different nanobody solutions (nanobodies A9-1 to A9-6) were added, followed by incubation at 37°C for 1 h. The unbound antibodies were washed away by PBST for 3 times, and then 1:5000 diluted HRP-conjugated rabbit anti-goat secondary antibody was added, followed by incubation at 37°C for 1 h. The secondary antibody was washed away by PBST for 3 times, and then TMB (3,3',5,5'-tetramethylbenzidine) substrate solution was added for color development for 15 min. The color development was terminated by 2M H2SO4. The absorbance value of each well was read at 450 nm wavelength.

[0136] 3. Experimental results

[0137] Table 8 Specificity of the nanobodies A9-1 to A9-6

[0138]

[0139] As shown in Table 8, it can be seen that the nanobodies A9-1 to A9-6 provided by the application only bind to AAV9, but not to other serotypes of AAV viruses such as AAV2, AAV5, AAV8, indicating that the nanobodies A9-1 to A9-6 provided by the application can specifically bind to AAV9 adeno-associated virus.

[0140] Example 4 Preparation of nanobody-coupled affinity packing and purification of AAV9 adeno-associated virus using the same

[0141] 1. Experimental materials

[0142] Nanobodies A9-1 to A9-6, NHS-activated 4FF affinity packing, 3KDa ultrafiltration membrane, Tween 20, citric acid, phosphoric acid, guanidine hydrochloride, iTaq Universal SYBR Green Supermix

[0143] 2. Experimental steps

[0144] (1) Pre-activation of affinity packing: take 2g of NHS-activated 4FF affinity packing (item number SCB0E009), and wash with pre-cooled 1mM hydrochloric acid (0°C~4°C) for three times, each time for 10min.

[0145] (2) Preparation of nanobody solution: the purified nanobody (nanobodies A9-1 to A9-6) solution is exchanged into coupling buffer (sodium bicarbonate buffer, pH 8.5) using 3KDa ultrafiltration membrane, and the protein concentration is determined by BCA protein quantification method.

[0146] (3) Nanobody-coupled affinity packing: 2g of NHS-activated 4FF affinity packing is added to 6mL of nanobody solution, the mass ratio of affinity packing to nanobody is 1g:10mg, and the reaction is carried out at 28°C, 250rpm for 3.5h, after the reaction is completed, 1M ethanolamine (pH 8.0) is used for overnight blocking, and the nanobody-coupled affinity packing is obtained.

[0147] (4) Preparation of AAV virus loading solution: the AAV virus (AAV9, AAV2, AAV5, AAV8 adeno-associated virus) harvest liquid obtained after fermentation is lysed using Tween 20, and nuclease is added for incubation, the fermented liquid after incubation is filtered through a 0.22μm filter membrane to obtain AAV supernatant. Then the AAV supernatant is concentrated and exchanged by ultrafiltration membrane, so that the exchanged AAV sample is consistent with the equilibrium liquid of the affinity packing, and the exchanged sample is filtered through a 0.22μm filter membrane, and the filtrate is collected as the AAV virus loading solution for the affinity packing.

[0148] (5) Affinity packing material coupled with nanobodies purifies AAV Adeno-associated virus: 1 mL of the affinity packing material coupled with nanobodies prepared in step (3) is loaded into a chromatographic column, and the AAV9 adeno-associated virus sample solution prepared in step (4) is loaded to detect the purification effect. Before purification, the chromatographic column is first balanced with PBS balancing solution, and after loading is completed, the chromatographic column is eluted with citric acid (pH 2.5), phosphoric acid (pH 1.9), and 4M guanidine hydrochloride, respectively. The viral genome titer in the eluted sample is detected by qPCR method. The specific operation is as follows: after the sample is diluted by 10-100 times with purified water, 200 μL of the diluted sample is treated by nuclease and proteinase K double enzyme method, and then the treated sample is diluted by a proper multiple, and the qPCR system is configured, and the detection is performed according to the qPCR reaction program. The qPCR system includes: 0.3 μL of upstream primer (10 μM), 0.3 μL of downstream primer (10 μM), 5 μL of iTaq Universal SYBR Green Supermix, 2 μL of sample to be detected, and finally ultrapure water is added to 8 μL. The qPCR reaction program is as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, wherein the denaturation and annealing and extension are sequentially performed for 40 cycles. The primers used in qPCR include primer Q-F and primer Q-R, wherein the nucleotide sequence of primer Q-F is: 5'-CCGTTGTCAGGCAACGTG-3'; and the nucleotide sequence of primer Q-R is: 5'-AGCTGACAGGTGGTGGCAAT-3'. The AAV purification yield is calculated according to the viral genome titer before and after sample purification, and the recovery rate calculation method is: (viral genome titer in purified sample / viral genome titer in sample solution) x 100%. At the same time, the purity of the eluted sample is detected by SDS-PAGE silver staining.

[0149] 3. Experimental results

[0150] The qPCR detection results of AAV9, AAV2, AAV5 and AAV8 adeno-associated viruses purified by the affinity packing material coupled with six nanobodies prepared in the present application are shown in Table 9, wherein AC-Load represents the sample containing adeno-associated virus before purification, which is clarified and filtered and has been changed to PBS balancing solution in the purification step; AC-FT represents the flow-through liquid in the purification step; and AC-E represents the virus eluent in the purification step, most of the purified virus is eluted in the citric acid (pH 2.5) eluent. Figure 1 The SDS-PAGE silver staining detection results of AAV9 virus purified by the affinity packing material coupled with six nanobodies prepared in the present application are shown in Figure 3, wherein lane Marker represents a molecular marker.

[0151] The control group is imported filler POROS™ CaptureSelect™ AAV9 affinity resin.

[0152] Table 9 qPCR detection results of affinity filler purified AAV9 adeno-associated virus of coupled nanobodies

[0153]

[0154] As shown in Table 9, Figure 1 It can be seen that, compared with the imported filler, the six nanobodies prepared in the present application can be used for purifying AAV9 virus after being coupled with the affinity filler, and the AAV9 virus can be captured and eluted very gently during the purification process; and the purity of the recovered AAV9 virus is high.

[0155] In summary, the nanobodies A9-1 to A9-6 prepared in the present application can specifically bind to AAV9 adeno-associated virus, and have good effect of affinity purification of AAV9 virus. In addition, the preparation process of the nanobodies provided in the present application is short, and the incubation combination time of the phage library containing the nanobodies and the antigen (AAV9 adeno-associated virus) is only 10 min, which greatly reduces the production cost and is convenient for mass production.

[0156] Those skilled in the art will readily understand that the above description is only a preferred embodiment of the present application, and is not intended to limit the present application, and any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A nanobody that specifically binds to AAV9, wherein the heavy chain variable region of the nanobody includes complementarity-determining regions CDR1, CDR2, and CDR3, characterized in that, The amino acid sequence of the complementarity-determining region of the nanobody is selected from any one of (a)-(f): (a): CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3; (b): CDR1 shown in SEQ ID NO.8, CDR2 shown in SEQ ID NO.9, and CDR3 shown in SEQ ID NO.10; (c): CDR1 shown in SEQ ID NO.13, CDR2 shown in SEQ ID NO.14, and CDR3 shown in SEQ ID NO.15; (d): CDR1 shown in SEQ ID NO.20, CDR2 shown in SEQ ID NO.21, and CDR3 shown in SEQ ID NO.22; (e): CDR1 shown in SEQ ID NO.26, CDR2 shown in SEQ ID NO.27, and CDR3 shown in SEQ ID NO.28; (f): CDR1 shown in SEQ ID NO.32, CDR2 shown in SEQ ID NO.33 and CDR3 shown in SEQ ID NO.

34.

2. The nanobody according to claim 1, characterized in that, The heavy chain variable region of the nanobody also includes framework regions FR1, FR2, FR3, and FR4; The amino acid sequence of the framework region of the nanobody is selected from an amino acid sequence that has at least 90% sequence homology with one of the amino acid sequences (1), (2), (3), (4), (5) or (6); The sequence information of the amino acid sequences (1)-(6) is as follows: (1): FR1 shown in SEQ ID NO.4, FR2 shown in SEQ ID NO.5, FR3 shown in SEQ ID NO.6 and FR4 shown in SEQ ID NO.7; (2): FR1 shown in SEQ ID NO.4, FR2 shown in SEQ ID NO.11, FR3 shown in SEQ ID NO.12 and FR4 shown in SEQ ID NO.7; (3): FR1 shown in SEQ ID NO.16, FR2 shown in SEQ ID NO.17, FR3 shown in SEQ ID NO.18 and FR4 shown in SEQ ID NO.19; (4): FR1 shown in SEQ ID NO.23, FR2 shown in SEQ ID NO.24, FR3 shown in SEQ ID NO.25 and FR4 shown in SEQ ID NO.7; (5): FR1 shown in SEQ ID NO.29, FR2 shown in SEQ ID NO.30, FR3 shown in SEQ ID NO.31 and FR4 shown in SEQ ID NO.7; (6): FR1 shown in SEQ ID NO.35, FR2 shown in SEQ ID NO.36, FR3 shown in SEQ ID NO.37 and FR4 shown in SEQ ID NO.

7.

3. The nanobody according to claim 1, characterized in that, The heavy chain variable region of the nanobody also includes framework regions FR1, FR2, FR3, and FR4; The amino acid sequence of the framework region of the nanobody is selected from one or more functionally conserved variants of the amino acid sequence shown in amino acid sequences (1), (2), (3), (4), (5) or (6) by substitution, deletion or insertion of one or more amino acids. The sequence information of the amino acid sequences (1)-(6) is as follows: (1): FR1 shown in SEQ ID NO.4, FR2 shown in SEQ ID NO.5, FR3 shown in SEQ ID NO.6 and FR4 shown in SEQ ID NO.7; (2): FR1 shown in SEQ ID NO.4, FR2 shown in SEQ ID NO.11, FR3 shown in SEQ ID NO.12 and FR4 shown in SEQ ID NO.7; (3): FR1 shown in SEQ ID NO.16, FR2 shown in SEQ ID NO.17, FR3 shown in SEQ ID NO.18 and FR4 shown in SEQ ID NO.19; (4): FR1 shown in SEQ ID NO.23, FR2 shown in SEQ ID NO.24, FR3 shown in SEQ ID NO.25 and FR4 shown in SEQ ID NO.7; (5): FR1 shown in SEQ ID NO.29, FR2 shown in SEQ ID NO.30, FR3 shown in SEQ ID NO.31 and FR4 shown in SEQ ID NO.7; (6): FR1 shown in SEQ ID NO.35, FR2 shown in SEQ ID NO.36, FR3 shown in SEQ ID NO.37 and FR4 shown in SEQ ID NO.

7.

4. The nanobody according to claim 2, characterized in that, The amino acid sequence of the framework region of the nanobody is selected from any one of the amino acid sequences (1)-(6).

5. The nanobody according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the nanobody is selected from an amino acid sequence that has at least 90% sequence homology with amino acid sequence (I); The sequence information of the amino acid sequence (Ⅰ) is as follows: (I): Any one of SEQ ID NO.38 to SEQ ID NO.

43.

6. The nanobody according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the nanobody is selected from one or more functionally conserved variants of the amino acid sequence shown in amino acid sequence (I) by substitution, deletion, or insertion of one or more amino acids. The sequence information of the amino acid sequence (Ⅰ) is as follows: (I): Any one of SEQ ID NO.38 to SEQ ID NO.

43.

7. The nanobody according to claim 5, characterized in that, The amino acid sequence of the heavy chain variable region of the nanobody is selected from the amino acid sequence (I).

8. A fusion protein, characterized in that, The nanobody as described in any one of claims 1 to 7 is linked to a tag to form a fusion protein; The label is a detectable label or a purifiable label.

9. A nucleic acid, characterized in that, It includes a nucleic acid sequence encoding a nanobody as described in any one of claims 1 to 7.

10. An expression vector comprising the nucleic acid as described in claim 9.

11. A host cell, characterized in that, It includes the nucleic acid as described in claim 9 or the expression vector as described in claim 10.

12. The use of a nanobody as described in any one of claims 1 to 7 or a fusion protein as described in claim 8 in the preparation of an AAV9 adeno-associated virus detection kit or purification kit.

13. An AAV9 adeno-associated virus detection kit or purification kit, characterized in that, It includes the nanobody as described in any one of claims 1 to 7 or the fusion protein as described in claim 8.

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