A kind of AAV9 specific nano antibody and its application

Abstract

The present invention relates to the field of biological research technology, and discloses an AAV9-specific nano-antibody, which are Nb1E11, 1C10, 1C8 and Nb1H8, respectively, and their performance in many aspects is superior to the commonly used antibody AAVX on the market. The nano-antibody studied in the present invention has extremely high AAV9 specificity, and it only specifically recognizes one serotype of AAV9; the nano-antibody studied in the present invention has extremely high affinity, and its affinity constant for binding to AAV9 is in the nanomolar order; the nano-antibody studied in the present invention has good thermal stability, and its TM value is above 65 degrees Celsius; the nano-antibody studied in the present invention has very good alkali resistance, and after being treated with 0.5 molar NaOH for 15 minutes, it can still efficiently bind to AAV9. These properties show that the AAV9 nano-antibody we studied has great flexibility and application prospects in future product development.

Description

Technical Field

[0001] The present invention relates to the field of biological research technology, and in particular to an AAV9-specific nano antibody and application thereof. Background Art

[0002] Adeno-associated virus (AAV) belongs to the genus Dependoviridae of the family Parvoviridae. It has no envelope and consists of an icosahedral protein capsid with a diameter of about 22 nm and a linear single-stranded DNA genome of about 4.7 kb. Its life cycle depends on helper viruses. AAV can only replicate itself in host cells in the presence of helper viruses such as adenovirus or herpes virus.

[0003] AAV serotype refers to the antiserum type of AAV. At present, researchers have discovered more than 12 human AAV serotypes (AAV1 to AAV12) and more than 100 non-human primate AAV serotypes. Different AAV serotypes have different capsid protein spatial structures, sequences, and tissue specificities, so the cell surface receptors they recognize and bind to are also very different, which also leads to different tissue types, cell types, and infection efficiencies of transfection of different serotypes.

[0004] AAV9 is one of the most commonly used natural serotypes in clinical practice, and is widely used in the central nervous system (CNS) and muscular system. Compared with other AAV serotypes, AAV9 has stronger diffusivity and can cross the blood-brain barrier (BBB) ​​to a certain extent, achieving high gene expression in the brain. At present, some institutions have successfully developed the AAV9-based gene therapy drug Zolgensma for the treatment of spinal muscular atrophy (SMA). The drug carries the SMN1 gene through AAV9 and overexpresses SMN1 on nearby neurons in the spinal cord sheath, allowing people who do not produce normal SMN1 protein to produce normal protein, thereby achieving the purpose of treatment. In addition to Zolgensma, there are currently a large number of gene therapy drugs based on the AAV9 serotype in the clinical or preclinical stage;

[0005] Due to the widespread application of AAV9, the field of gene therapy requires anti-AAV9 specific antibodies with high affinity, strong specificity and good stability for the detection, identification, purification and quality control of AAV9-related gene therapy products;

[0006] Currently, there is a broad-spectrum antibody AAVX on the market that can simultaneously recognize multiple AAV serotypes, but its affinity for these AAV serotypes is generally low. When used for precision detection including SPR or BLI, this low affinity leads to unstable signals; the AAV antibodies currently on the market are all conventional IgG antibodies, which have a long research and development process and high antibody expression and purification costs. Since IgG is a more complex tetrameric molecule, the stability of the antibody itself is insufficient, and its scope of application is limited; therefore, there is currently no anti-AAV9 antibody on the market that can fully meet these needs. Therefore, a person skilled in the art provides an AAV9-specific nanobody and its application to solve the problems raised in the above background technology. Summary of the invention

[0007] In order to solve the problems raised in the above background technology, the present invention provides an AAV9-specific nanobody and its application. The purpose is to provide multiple AAV9 antibodies that are significantly differentiated from the currently commonly used commercial AAV antibody AAVX and have better application prospects. The antibodies of the present invention have a wide range of biological research application value, and their applications involve multiple fields such as serotype determination related to AAV9, clinical treatment using AAV9 as a tool, and production and purification of AAV9.

[0008] The first object of the present invention is to provide an AAV9-specific nanobody, wherein the heavy chain variable region of the nanobody includes complementary determining regions CDR1, CDR2, and CDR3, and the complementary determining regions include any combination of the following or a sequence with a homology of not less than 90% thereto:

[0009] 1) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3;

[0010] 2) CDR1 shown in SEQ ID NO.8, CDR2 shown in SEQ ID NO.9, and CDR3 shown in SEQ ID NO.10;

[0011] 3) CDR1 shown in SEQ ID NO.15, CDR2 shown in SEQ ID NO.16, and CDR3 shown in SEQ ID NO.17;

[0012] 4) CDR1 shown in SEQ ID NO.22, CDR2 shown in SEQ ID NO.23, and CDR3 shown in SEQ ID NO.24.

[0013] Furthermore, the present invention includes those molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, optimally more than 98%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology with the above-mentioned CDR sequences.

[0014] Furthermore, the sequence with a homology of not less than 90% is obtained by amino acid substitution of CDR1, CDR2 and / or CDR3, and the amino acid substitution is selected from one or more of the following:

[0015] 1) Alanine is replaced by valine, leucine or isoleucine,

[0016] 2) Arginine is replaced by lysine, glutamine or asparagine,

[0017] 3) Asparagine is replaced by glutamine, histidine, lysine or arginine,

[0018] 4) Aspartic acid is replaced by glutamic acid,

[0019] 5) Cysteine ​​is replaced by serine,

[0020] 6) Glutamine is replaced by asparagine,

[0021] 7) Glutamic acid replaced by aspartic acid,

[0022] 8) Glycine is replaced by proline or alanine,

[0023] 9) Histidine is replaced by asparagine, glutamine, lysine or arginine,

[0024] 10) isoleucine is replaced by leucine, valine, methionine, alanine or phenylalanine,

[0025] 11) Leucine is replaced by isoleucine, valine, methionine, alanine or phenylalanine,

[0026] 12) Lysine is replaced by arginine, glutamine or asparagine,

[0027] 13) Methionine is replaced by leucine, phenylalanine or isoleucine,

[0028] 14) Phenylalanine is replaced by leucine, valine, isoleucine, alanine or tyrosine,

[0029] 15) Proline is replaced by alanine or glycine,

[0030] 16) Serine is replaced by threonine,

[0031] 17) Threonine is replaced by serine,

[0032] 18) Tryptophan is replaced by tyrosine or phenylalanine,

[0033] 19) Tyrosine is replaced by tryptophan, phenylalanine, threonine or serine,

[0034] 20) Valine is replaced by isoleucine, leucine, methionine, phenylalanine or alanine.

[0035] Furthermore, the variable region of the nanobody includes framework regions FR1, FR2, FR3, and FR4, and a complementary determining region is arranged between two adjacent framework regions, that is, CDR1, CDR2, and CDR3 are separated by framework regions FR1, FR2, FR3, and FR4. Therefore, FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged on the nanobody in sequence.

[0036] Furthermore, the framework region includes one or more of the following combinations:

[0037] 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;

[0038] 2) FR1 shown in SEQ ID NO.11, FR2 shown in SEQ ID NO.12, FR3 shown in SEQ ID NO.13, and FR4 shown in SEQ ID NO.14;

[0039] 3) FR1 shown in SEQ ID NO.18, FR2 shown in SEQ ID NO.19, FR3 shown in SEQ ID NO.20, and FR4 shown in SEQ ID NO.21;

[0040] 4) FR1 shown in SEQ ID NO.25, FR2 shown in SEQ ID NO.26, FR3 shown in SEQ ID NO.27, and FR4 shown in SEQ ID NO.28;

[0041] Furthermore, the nanobody contains a sequence as shown in any one of SEQ ID NO.29-32.

[0042] A second object of the present invention is to provide a polynucleotide molecule encoding the above-mentioned Nanobody.

[0043] The third object of the present invention is to provide an expression vector containing the above polynucleotide molecule.

[0044] Furthermore, the expression vector is selected from DNA, RNA, viral vectors (such as lentivirus, adenovirus, AAV virus, retrovirus or a combination thereof), plasmids, transposons, other gene transfer systems, liposome nanoparticles (LNP, in which DNA or mRNA encoding AAV9 nanobodies is encapsulated), or a combination thereof.

[0045] A fourth object of the present invention is to provide a host cell containing the above-mentioned Nanobody.

[0046] Furthermore, the host cell may be a prokaryotic cell or a eukaryotic cell, such as a plant cell, an animal cell, a microorganism, and the like.

[0047] A fifth object of the present invention is to provide a monovalent antibody, a bivalent antibody or a multivalent antibody comprising the above-mentioned Nanobody.

[0048] A sixth object of the present invention is to provide a recombinant protein or immunoconjugate containing the above-mentioned nanobody.

[0049] Furthermore, the recombinant protein contains

[0050] (a) Anti-AAV9 nanobodies, or the above-mentioned bivalent or multivalent antibodies:

[0051] (b) a tag sequence that facilitates expression and / or purification;

[0052] (c) Specific antibodies or binding ligands that recognize specific cell surface marker proteins or receptor proteins.

[0053] Furthermore, the immunoconjugate contains

[0054] (a) anti-AAV9 nanobody, or the above-mentioned recombinant protein, bivalent or multivalent antibody;

[0055] (b) a conjugated moiety selected from one or more of the following: a detectable label, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

[0056] Preferably, the coupling portion is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or any form of nanoparticles.

[0057] The seventh object of the present invention is to provide the use of the above-mentioned nanobodies, monovalent antibodies, bivalent antibodies, multivalent antibodies, recombinant proteins or immunoconjugates in the preparation of diagnostic, preventive or therapeutic products. The form of the product includes but is not limited to medicaments, reagents, test panels, kits, etc.

[0058] Furthermore, the detection includes flow cytometry, cell immunofluorescence detection, enzyme-linked immunosorbent assay (ELISA) detection, etc.

[0059] The eighth object of the present invention is to provide the use of the above-mentioned nanobodies, monovalent antibodies, bivalent antibodies, multivalent antibodies, recombinant proteins or immunoconjugates in the preparation and purification process of AAV9-related gene therapy products. The form of the product includes but is not limited to agarose microspheres, chromatography columns and other solid support materials coupled with the nanobodies or multivalent antibodies, which are used for affinity purification of AAV9-related gene therapy products.

[0060] Beneficial effects of the present invention:

[0061] The inventors have successfully obtained multiple anti-AAV9 nanobodies through extensive and in-depth research and a large number of screenings. Specifically, the present invention uses purified AAV9 virus particles to immunize camels to obtain a high-quality immune nanoantibody gene library. Then AAV9 is coupled to an ELISA plate, and the immune nanoantibody gene library (camel heavy chain antibody phage display gene library) is screened using phage display technology with this form of antigen, thereby obtaining AAV9-specific nanoantibody genes.

[0062] AAV9-specific nanoantibodies Compared with the prior art, the beneficial effects of the present invention are:

[0063] Specificity: Compared with AAVX, the main AAV antibody on the market, the nanoantibody studied in the present invention has extremely high AAV9 specificity. It only specifically recognizes one serotype of AAV9 and has no binding to other tested AAV serotypes. AAV9 is one of the most widely used AAV serotypes in clinical and pre-clinical applications. There is a great demand for AAV9 antibodies in drug production and clinical testing.

[0064] Affinity: The affinity of the nanoantibodies studied in the present invention for binding to AAV9 is much higher than that for AAV, and the affinity measured by BLI is generally more than 100 times higher than that for AAVX. High affinity is a prerequisite for the success of immunoassays and is also a basic indicator for measuring a high-quality monoclonal antibody. High affinity is very important for the preparation of high-quality antibody drugs, diagnostic reagents, purification tools, etc.

[0065] Thermal stability: AAVX is an IgG antibody with relatively limited stability. The dissolution temperature (TM value) of the Fab region of a conventional IgG antibody is between 55 and 65 degrees, while the dissolution temperature of the nanoantibodies of the present invention is generally above 65 degrees. The high stability gives these nanoantibodies great flexibility in future product development.

[0066] Alkali resistance: Alkali treatment is a key method for regenerating the purification matrix of biological products. Currently, the chromatography matrix based on AAVX is not alkali-resistant, which results in the inability to efficiently reuse such products, increasing the production cost of the gene therapy industry. The multiple nanoantibodies in the present invention have good alkali tolerance and can still bind to AAV9 after being treated with 0.5M NaOH for 15 minutes. This property creates conditions for the development of more stable purification tools for AAV9-related gene therapy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the result of silver staining detection of purified AAV9;

[0068] Figure 2 This is the result of ELISA verification of the binding of enriched monoclonal PPE to AAV9;

[0069] Figure 3 The figure shows the ELISA binding results of AAVX to 5 different AAV serotypes;

[0070] Figure 4 The results of Nb-1H8, Nb-1E11, and Nb-1C8 recognizing five different serotypes of AAV;

[0071] Figure 5 BLI affinity analysis of Nb-1H8, Nb-1E11, Nb-1C8 and AAVX binding to AAV9;

[0072] Figure 6-9 It is a thermal stability analysis (DSF) diagram of each anti-AAV9 nanobody-Fc fusion protein;

[0073] Figure 10-13 The BLI affinity analysis diagram of each anti-AAV9 nanobody binding to AAV9 after alkaline treatment. DETAILED DESCRIPTION

[0074] Example 1

[0075] Preparation and purification of AAV9 adeno-associated virus: The traditional 3-plasmid packaging system was used for packaging. The 3 plasmids were transfected into 293T cells, and the supernatant and cell pellet were collected after 72 hours. All cells were collected and repeatedly frozen and thawed with liquid nitrogen three times to collect the supernatant, which was then digested with Benonase enzyme. The resulting supernatant was purified using a Biomiga brand purification column, and the resulting virus was packaged and stored at -80°C. The AAV titer was determined using international standards for qPCR absolute quantification of ITR. The silver staining results of AAV-9 are shown in Figure 2. Figure 1 As shown, the bands of VP1, VP2, and VP3 are clearly visible.

[0076] Example 2

[0077] Construction of camel immune library and screening, expression and purification of anti-AAV9 adeno-associated virus nanoantibodies: The purified AAV9 adeno-associated virus was mixed with Freund's adjuvant in a 1:1 ratio, and a healthy alpaca was immunized four times by multi-point subcutaneous injection. During the period, a small amount of blood was drawn to separate serum and test the serum titer. 100 mL of peripheral blood was collected 7-10 days after the last immunization, and mononuclear cells (PBMC) were separated using Ficoll lymphocyte separation medium (GE Healthcare 17-1440-03 FICOLL PAQUE PLUS).

[0078] Total mRNA of PBMC was extracted with phenol-chloroform and expressed using SuperScript TM The RNA was converted to cDNA using IVFirst-Strand cDNASynthesis Reaction Kit (Thermo, 18091200). The heavy chain variable region fragment was amplified using nested PCR using cDNA as a template (Q5 High-Fidelity 2X Master Mix NEB, M0492L). The primers used in the two rounds of PCR are shown in Table 1 below:

[0079] Table 1 Primers used for nested PCR amplification of VHH genes

[0080]

[0081] The antibody fragments recovered from the gel of the second round of PCR and the phage vector pADL were digested separately with Not1 and Bstx1 restriction endonucleases, and the digestion products were subjected to agarose electrophoresis and purified, followed by enzyme ligation reaction. The enzyme ligation products were electrotransformed into TG1 competent cells (Weidi Biotechnology, DE1055M) to construct an Escherichia coli library containing nanobody fragments. The clone count showed that the library capacity was 2E8; 40 single clones were randomly selected for sequencing, and the sequencing results showed that the insertion rate of the nanobody gene was about 95%.

[0082] After amplifying the obtained adeno-associated virus immune library, VCSM13 helper phage was added in the logarithmic growth phase to infect bacteria at 37°C for 1 hour; after centrifuging the bacterial culture, the supernatant was discarded and the bacteria were resuspended in 2xYT medium with antibiotics and 0.5% glucose, and the phage was packaged overnight in a constant temperature shaker at 30°C and 225rpm. The next day, phage particles were precipitated with polyethylene glycol / sodium chloride. The library was screened using phage display technology, and three rounds of "adsorption-washing-enrichment" screening processes were performed using AAV-9 and AAV-DJ, respectively, to enrich the expression of nano-antibody phage groups that bind to AAV-9 or AAV-DJ. 96 monoclones randomly selected from the above enriched phage groups were amplified and IPTG-induced expression, and the periplasmic protein extract (PPE) was obtained by bursting the outer wall of the bacteria with a hypotonic solution PPB (phosphate peptone buffer), and the binding of PPE to AAV-9 or AAV-DJ was identified by ELISA. The results showed that 28 of the 96 random clones bound to AAV9 adeno-associated virus; 7 of the 96 random clones bound to AAV-DJ adeno-associated virus ( Figure 2 , clones with ELISA readings more than 5 times the background were defined as positive clones).

[0083] In order to identify whether these clones have specific binding to AAV9 adeno-associated virus, the selected 35 clones were used to interact with insulin-coated ELISA plates. The results showed that none of the 35 clones interacted with insulin, suggesting that the binding of these clones to AAV9 adeno-associated virus was not non-specific binding.

[0084] All 35 clones that specifically bound to adeno-associated virus were sequenced and identified. The 8 non-repetitive VHH coding sequences obtained by sequencing were cloned into the pcDNA3.1-VHH-hFc plasmid with the human Fc domain encoding gene, and Expi-293F cells were transfected to express VHH-hFc fusion protein. After affinity binding with Protein-A magnetic beads and elution with glycine solution, the purified nanobody-Fc fusion protein was obtained.

[0085] Example 3

[0086] ELISA experiment of anti-AAV nanobodies binding to different serotypes of AAV: 50 μL 2.5E7 vg / μL of different AAV serotypes, including AAV1, AAV2, AAV6, AAV9, AAV-DJ, etc., were used to coat the ELISA plate at 4°C overnight; washed 3 times with 0.1% PBST (phosphate-tween buffer), added 150 μL 5% BSA (bovine serum albumin), and incubated at room temperature for 1 hour; washed 3 times with 0.1% PBST; 15 different AAV nanobodies were added at a concentration of 1 μg / mL, and the same concentration of commercial antibody AAVX (MonoRab TM AAVX VP1 / VP2 / VP3 Antibody (5G4), mAb) was used as a control, and the cells were incubated at room temperature for 1 hour; then washed 3 times with 0.1% PBST, and 50 μL of 1:5000 diluted HRP-labeled anti-Fc secondary antibody was added, and the cells were incubated at room temperature for 1 hour; then washed 3 times with 0.1% PBST, and 50 μL of TMB (3,3′,5,5′-tetramethylbenzidine) was added for color development for 8-10 minutes, and the reaction was terminated with 50 μL of stop solution; the absorbance of each well was read at a wavelength of 450 nm. If the nanobody binds to a certain serotype, the reading of the corresponding serotype will increase.

[0087] The results of different serotypes of adeno-associated viruses binding to antibodies are as follows Figure 3-6 As shown. The results show that the commercial antibody AAVX can significantly bind to multiple AAV serotypes including AAV1, AAV2, AAV9 and AAV-DJ, while the 8 nanobodies we developed only bind to AAV9 and not to other AAV serotypes, indicating that compared with AAVX, our antibodies have good AAV9 specificity. We chose to conduct further characterization analysis on these 8 antibodies.

[0088] Table 2 List of Nanobody Sequences

[0089]

[0090]

[0091] Table 3 List of Nanobody Sequences

[0092]

[0093]

[0094] Example 4

[0095] Biolayer interferometry (BLI) was used to determine the affinity of nanobody binding to AAV9. Using the Gator Prime system (Gator Bio) v2.7.3.0728 (https: / / www.gatorbio.com / ), biolayer interferometry was performed to evaluate the binding kinetics of nanobody to AAV9.

[0096] The glass AAV9 probe (Gator Bio) was first immersed in Q buffer (Gator Bio) for about 30 seconds to obtain a baseline signal. The probe was then loaded with 1.23E7 vg / μL AAV9 diluted in Q buffer for 180 seconds, followed by a 30-second Q buffer wash step. Next, the AAV9-bound probe was incubated with 300nM AAV9-VHH, including a no AAV9-VHH control (0nM), for about 180 seconds, followed by a 180-second dissociation step. The binding and dissociation curves were plotted and calculated using GatorPrime (GatorBio) software to obtain the binding kinetic values ​​(dissociation rate constant koff, association rate constant Kon, and binding constant KD). The results are shown in Figure 5 .

[0097] The same method was used to determine the binding kinetics of the control antibody AAVX to adeno-associated virus. The binding kinetic values ​​(koff, Kon and KD) obtained under the same conditions can be used for parallel comparison with the VHH-Fc affinity. The results are shown in Table 4.

[0098] Table 4 Gator determination of the affinity data of various nanobodies (VHH-Fc) and AAV9

[0099]

[0100]

[0101] Example 5

[0102] Differential scanning fluorescence (DSF) was used to detect the melting temperature of anti-adeno-associated virus nanoantibodies: The Tm value of the nanoantibody was determined using a fluorescent quantitative PCR instrument, Quantstudio5 (Thermo Scintific, catalog number: A28575), to evaluate the thermal stability of the nanoantibody.

[0103] Before starting the experiment, preheat the fluorescence quantitative PCR instrument Quantstudio5 for 20 minutes.

[0104] First, prepare the samples to be tested: dilute all samples to be tested with ddH2O to 0.6 mg / ml, 50ul, add 50ul SYPRO orange (Invitrogen, cat. no. S6650) diluted with ddH2O to 10X, so that the final concentration of SYPRO orange is 5X, mix well, and stain at 4℃ for 30-60min. Then add the prepared samples to the PCR plate, 20ul per well, use protein buffer as the blank control group, and set up 3 replicates. Each test group also has 3 replicates. Then rotate the PCR plate (200g, room temperature, 1 minute) to remove bubbles in the solution. Place the PCR plate in the PCR instrument, run the temperature scan from 25℃ to 95℃, 1℃ / min, and select the program type "SYBR green". The experimental data is shown as follows. Figures 6 to 9 Finally, export the experimental data through QuantStudio TM Design & Analysis Software was used to analyze the Tm values ​​of the sample proteins (Table 6).

[0105] Table 6 DSF determination of the melting temperature (TM) data of each nanobody (VHH-Fc)

[0106] Antibody Dissolving temperature(℃) Nb1C8 70.70 Nb1H8 66.76 Nb1C10 76.79 Nb1E11 80.84

[0107] Example 6

[0108] Alkali tolerance test of anti-AAV9 nanobody: The purified AAV9 nanobody was diluted with PBS to a starting concentration of 300nM, 200ul of 1M NaOH storage solution was added to 200ul of nanobody solution and mixed quickly to gradually increase the final concentration of NaOH in the antibody solution to 0.5M, the antibody-NaOH mixture was incubated at room temperature for 15 minutes, and HCL hydrochloric acid solution was added to adjust the pH value to neutral. At this time, the concentration of the nanobody in the solution was about 100nM. The nanobody solutions after alkali treatment were used to determine the ability of these antibodies to bind to AAV9 using the Gatro Primer system using the method in Example 4. The experimental results are shown in Figure 2. Figure 10-13 The affinity constants of each nanobody binding to AAV9 after alkali treatment determined by BLI are shown in Table 5. The results show that the nanobody after strong alkali treatment can still efficiently bind to AAV9.

[0109] Table 5 Gator determination of the affinity data of each nanobody (VHH-Fc) and AAV9 after 15 minutes of 0.5M NaOH treatment

[0110] Antibody Dissociation rate constant (1 / s) Binding rate constant (1 / Ms) Binding constant (M) 1C8 2.62E-004 5.10E+004 5.13E-009 1H8 1.66E-003 2.43E+003 6.82E-007 1C10 2.09E-004 3.40E+003 6.14E-008 1E11 1.54E-003 4.41E+003 3.50E-007

[0111] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0112] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An AAV9-specific nanobody, characterized in that: The heavy chain variable region of the Nanobody includes complementary determining regions CDR1, CDR2, CDR3, and framework regions FR1, FR2, FR3, FR4; the complementary determining regions of the antibody include any combination of the following: 1) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3; 2) CDR1 shown in SEQ ID NO.8, CDR2 shown in SEQ ID NO.9, and CDR3 shown in SEQ ID NO.10; 3) CDR1 shown in SEQ ID NO.15, CDR2 shown in SEQ ID NO.16, and CDR3 shown in SEQ ID NO.17; 4) CDR1 shown in SEQ ID NO.22, CDR2 shown in SEQ ID NO.23, and CDR3 shown in SEQ ID NO.

24.

2. The AAV9-specific nanobody according to claim 1, characterized in that: The framework region of the antibody includes any combination of the following: 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.11, FR2 shown in SEQ ID NO.12, FR3 shown in SEQ ID NO.13, and FR4 shown in SEQ ID NO.14; 3) FR1 shown in SEQ ID NO.18, FR2 shown in SEQ ID NO.19, FR3 shown in SEQ ID NO.20, and FR4 shown in SEQ ID NO.21; 4) FR1 shown in SEQ ID NO.25, FR2 shown in SEQ ID NO.26, FR3 shown in SEQ ID NO.27, and FR4 shown in SEQ ID NO.28.

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