Anti-AAV9 nano antibody and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to an anti-AAV9 nano antibody and application thereof.The nano antibody Nb64 obtained through screening has extremely high specificity and only recognizes the single serotype of AAV9; the nano antibody shows excellent stability, the activity is not affected after the nano antibody is treated for 18 hours in a strong acid or strong alkali environment or heated for 70 minutes at the high temperature of 95 DEG C, and the nano antibody can still be efficiently combined with AAV9. The characteristics show that the Nb64 has a good application prospect in the field of developing AAV9 affinity purification reagents.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to an anti-AAV9 nanobody. Background Art

[0002] Adeno-associated virus (AAV) is a small, non-enveloped, single-stranded DNA virus with a small genome size of approximately 4.7 kb. Numerous AAV serotypes exist, and differences in the amino acid sequence and structure of their capsid proteins, as well as their interactions with host cellular factors, result in varying efficiencies of infection in different tissues and cells. Currently, 12 human AAV serotypes (AAV1 to AAV12) and over 100 non-human primate AAV serotypes have been identified.

[0003] AAV9, a serotype in the AAV family, is highly efficient at transducing a variety of tissues, including the heart, skeletal muscle, liver, pancreas, and eyes. It can also cross the blood-brain barrier (BBB) to transduce both neurons and non-neuronal cells in the central nervous system (CNS), making it one of the most commonly used natural serotypes in clinical practice. Due to its unique advantages, AAV9 has excelled in gene therapy, particularly in the treatment of neurological and cardiovascular diseases. Due to its widespread application, anti-AAV9 antibodies with high affinity, strong specificity, and excellent stability are required for the purification of AAV9-related gene therapy products.

[0004] Nanobodies are a class of heavy-chain antibodies found in camelids that are naturally devoid of light chains. Nanobodies offer the advantages of high specificity, high affinity, and high stability. Several AAV9-specific nanobodies have been reported, such as patent publication number CN 118638214 A, which discloses an AAV9-specific nanobody with a TM value above 65°C. However, these antibodies still suffer from poor specificity and / or low stability, making them insufficient for large-scale commercial applications. Therefore, there is an urgent need to develop new AAV9 nanobodies with high stability and specificity. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention proposes an anti-AAV9 nanobody and its application.

[0006] The technical solution of the present invention is achieved as follows: In one aspect, the present application provides an anti-AAV9 nanobody, wherein the heavy chain variable region of the anti-AAV9 nanobody comprises any one of the following combinations: (1) CDR1 with the sequence shown in SEQ ID No. 6, CDR2 with the sequence shown in SEQ ID No. 7, and CDR3 with the sequence shown in SEQ ID No. 8; (2) CDR1 with the sequence shown in SEQ ID No. 9, CDR2 with the sequence shown in SEQ ID No. 10, and CDR3 with the sequence shown in SEQ ID No. 11; (3) CDR1 with the sequence shown in SEQ ID No. 12, CDR2 with the sequence shown in SEQ ID No. 13, and CDR3 with the sequence shown in SEQ ID No. 14; (4) CDR1 with the sequence shown in SEQ ID No. 15, CDR2 with the sequence shown in SEQ ID No. 16, and CDR3 with the sequence shown in SEQ ID No. 17; (5) CDR1 with the sequence shown in SEQ ID No. 18, CDR2 with the sequence shown in SEQ ID No. 19, and CDR3 with the sequence shown in SEQ ID No. 20; (6) CDR1 with the sequence shown in SEQ ID No. 21, CDR2 with the sequence shown in SEQ ID No. 22, and CDR3 with the sequence shown in SEQ ID No. 23; (7) CDR1 with the sequence shown in SEQ ID No. 24, CDR2 with the sequence shown in SEQ ID No. 25, and CDR3 with the sequence shown in SEQ ID No. 26; (8) CDR1 having a sequence as shown in SEQ ID No. 27, CDR2 having a sequence as shown in SEQ ID No. 28, and CDR3 having a sequence as shown in SEQ ID No. 29.

[0007] The anti-AAV9 nanobody also includes a monovalent antibody, a bivalent antibody, a multivalent antibody, a recombinant protein or an immunoconjugate of the anti-AAV9 nanobody according to claim 1 or 2.

[0008] Preferably, the amino acid sequence of the anti-AAV9 nanobody is shown as SEQ ID No.1 or SEQ ID No.3-SEQID No.5.

[0009] In a second aspect, a gene fragment encoding the above-mentioned anti-AAV9 nanobody is also provided, for example, the nucleotide sequence of the gene fragment of the anti-AAV9 nanobody whose amino acid sequence is shown in SEQ ID No.1 is shown in SEQ ID No.2.

[0010] In a third aspect, the present application also provides an expression vector containing the above-mentioned gene fragment.

[0011] In a fourth aspect, a host cell is provided, wherein the host cell contains the above-mentioned expression vector, or the above-mentioned gene fragment is integrated into its genome.

[0012] In a fifth aspect, the present application provides a method for preparing anti-AAV9 nanoantibodies, comprising the following steps: transfecting the above-mentioned expression vector into competent cells, and culturing the cells to obtain anti-AAV9 nanoantibodies.

[0013] In a sixth aspect, a monovalent antibody, a bivalent antibody or a multivalent antibody comprising the above-mentioned nanobody is provided.

[0014] In a seventh aspect, a recombinant protein or immunoconjugate containing the above-mentioned nanobody is provided.

[0015] Furthermore, the recombinant protein contains (a) Anti-AAV9 nanobodies, or bivalent or multivalent antibodies as described above (b) a tag sequence to facilitate expression and / or purification; (c) Specific antibodies or binding ligands that recognize specific cell surface marker proteins or receptor proteins.

[0016] Furthermore, the immunoconjugate contains (a) anti-AAV9 nanobody, or the above-mentioned recombinant protein, bivalent or multivalent antibody; (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.

[0017] Preferably, the coupling portion is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized 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.

[0018] An eighth object of the present invention is to provide the use of the aforementioned Nanobodies, monovalent antibodies, bivalent antibodies, multivalent antibodies, recombinant proteins, or immunoconjugates in the preparation of diagnostic, preventive, or therapeutic products. The products may be in the form of, but are not limited to, pharmaceutical agents, reagents, test panels, and test kits.

[0019] Furthermore, the detection includes flow cytometry, cell immunofluorescence detection, enzyme-linked immunosorbent assay (ELISA) detection, etc.

[0020] A ninth object of the present invention is to provide the use of the aforementioned nanobodies, monovalent antibodies, bivalent antibodies, multivalent antibodies, recombinant proteins, or immunoconjugates in the preparation and purification of AAV9-related gene therapy products. The products may be in the form of, but not limited to, agarose microspheres, chromatography columns, or other solid support materials coupled with the nanobodies or multivalent antibodies, for affinity purification of AAV9-related gene therapy products.

[0021] The present invention has the following beneficial effects: 1. After extensive and in-depth research, the present invention has screened for the first time the preparation and application of highly stable AAV9 nanobodies. Experiments have demonstrated that the nanobodies of the present invention can bind to AAV9 with high affinity and specificity. In addition, the AAV9 nanobodies of the present invention have excellent acid-base tolerance and high temperature tolerance, making them suitable for industrial production.

[0022] 2. The nanobody Nb64 of the present invention has extremely high specificity, recognizing only a single serotype of AAV9. It also exhibits excellent stability, with its activity remaining unaffected after treatment with strong acid or alkaline environments for 18 hours, or heating at 95°C for 70 minutes, and it still efficiently binds to AAV9. These properties suggest that Nb64 has promising application prospects in the development of AAV9 affinity purification reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only 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.

[0024] Figure 1 A is a gel run of total RNA extracted from peripheral blood lymphocytes separated after blood was taken from alpacas after five immunizations. Lanes 1 and 2 are two tubes of extracted RNA samples; B is the electrophoresis diagram of the VHH gene before and after enzyme digestion. Lanes 1 and 3 are IgG2c and IgG2b subtype antibody gene fragments, respectively, and lanes 2 and 4 are IgG2c and IgG2b subtype antibody gene fragments after enzyme digestion, respectively; C is the plate for determining the number of transformants in the library; D is the result of the VHH amino acid sequence alignment of the monoclonal bacterial liquid.

[0025] Figure 2 This is the SDS-PAGE run of four different nanobodies expressed in prokaryotes.

[0026] Figure 3 The results of affinity test of four nanoantibodies to AAV9.

[0027] Figure 4 The results of affinity test of four nanoantibodies to AAV8.

[0028] Figure 5 The results of affinity test of four nanoantibodies to AAV6.

[0029] Figure 6 The results of affinity test of four nanoantibodies to AAV2.

[0030] Figure 7 The results of affinity test between Nb64 and AAV9 after strong acid and strong base treatment.

[0031] Figure 8 The affinity test results of Nb64 and AAV9 after treatment at different temperatures for 10 min.

[0032] Figure 9 The results of affinity test between Nb64 and AAV9 after treatment at 75℃ and 95℃ for different time periods. DETAILED DESCRIPTION

[0033] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0035] Example 1: Construction of AAV9 Nanobody Library (1) High-purity AAV9 (purchased from Saiye Biotechnology Co., Ltd.) was mixed with adjuvant at a ratio of 1:1 and emulsified overnight at 4°C. Complete Freund's adjuvant (purchased from Sigma, USA) was used for the primary immunization, and incomplete Freund's adjuvant was used for the booster immunization. Two-year-old male alpacas (purchased from China Pet Animal Husbandry Co., Ltd.) were injected subcutaneously in the neck. Each immunization was performed 15 days apart, for a total of 4 immunizations. (2) One week after the fourth immunization, 100 mL of blood was collected from the jugular vein of the alpaca, peripheral blood lymphocytes were separated, and total RNA was extracted. Figure 1 A, reverse transcription into cDNA; (3) Nested PCR was used to amplify the variable domain of heavy chain of heavy-chain antibody (VHH) gene of alpaca heavy chain antibody; (4) 10 μg of pComb3XSS phagemid vector and 4 μg of VHH were digested with restriction endonuclease SfiI (purchased from Beijing NEB Company) and ligated with T4 ligase (purchased from Beijing NEB Company) to construct phagemid. Figure 1 B; (5) The ligation product was electroporated into ER2738 competent cells (purchased from Lucigen, USA), and plated after gradient dilution. The reservoir capacity was measured to be 5.8×10 8 pfu / ml, 48 clones were randomly selected for colony PCR detection, and the nanobody gene insertion rate was 100%. The library was infected with helper phage and cultured overnight. After PEG precipitation, the phage-displayed AAV9 nanobody library was obtained. Figure 1 C.

[0036] Example 2: Screening and identification of AAV9 nanobodies 1. Antibody Screening: (1) Dilute AAV9 to 5 E10 Titer-coated ELISA plates (purchased from Corning, USA) were prepared with 100 μL per well and placed at 4°C overnight; (2) Wash 4 times with PBST (PBS + 0.05% Tween-20), add 300 μL of 5% skim milk powder, and block at 37°C for 1 h; (3) After washing, add 100 μL of phage library to each well and shake at room temperature for 2 h; (4) Wash the plate 10 times with PBST to remove non-specifically bound phages; (5) Add 100 μL of Gly-HCl (0.2 M, pH 2.2) elution buffer to the ELISA wells, place the ELISA plate on a horizontal shaker, and shake slowly at room temperature for 8 min. Collect the eluate and add the neutralization buffer Tris-HCl (2 M, pH 8.5), which is set as the output of this round. Infect ER2738 Escherichia coli with the helper phage, incubate overnight on a shaker, and set the PEG-precipitated phage as the input of the next round. The same screening process was repeated for a total of 4 rounds, during which the screening pressure was continuously increased. The AAV9 coating titer and library binding time in the second, third, and fourth rounds gradually decreased, and the number of PBST washes gradually increased.

[0037] 2. Identification of positive phage clones: (1) A total of 192 monoclonal colonies were picked from the four rounds of output titer plates and inoculated into 1 mL of 2YT medium containing 100 μg / mL ampicillin. The culture was shaken at 37°C and 250 rpm until the OD 600The concentration of the phage was about 0.6. After static infection with the helper phage, kanamycin was added and cultured overnight in a shaker. The next day, the phage was centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was used for phage enzyme-linked immunosorbent assay. (2) Coat AAV9 and BSA proteins overnight at 4°C, add 100 μL of phage monoclonal supernatant, wash the plate, add 100 μL of phage secondary antibody (purchased from Chengdu Apec Company), and react at 37°C for 30 min. After washing, add 100 μL of TMB colorimetric solution, develop at room temperature for 10 min, add 50 μL of 2 M HCl, and read at 450 nm; (3) When the OD value of the AAV9 well is more than 15 times greater than the OD value of the BSA well, the monoclonal colony is determined to be a positive clone. All positive clones were sequenced and analyzed, and a total of 4 different families of AAV9 nanobodies were obtained, numbered Nb1, Nb7, Nb22 and Nb64; the amino acid sequence of Nb1 is shown in SEQ ID No. 3, the amino acid sequence of Nb7 is shown in SEQ ID No. 4, and the amino acid sequence of Nb22 is shown in SEQ ID No. 5.

[0038] Table 1 Heavy chain variable region of Nb1 antibody Table 2 Heavy chain variable region of Nb7 antibody Table 3 Heavy chain variable region of Nb22 antibody Table 4 Heavy chain variable region of Nb64 antibody Example 3: Expression and purification of AAV9 nanobodies (1) Streak four bacterial clones onto a 2YT culture plate containing 100 μg / mL ampicillin and incubate inverted at 37°C overnight; (2) Select a single colony and inoculate it into 3 mL of 2YT culture medium containing 100 μg / mL ampicillin and 0.05% glucose, and shake at 37°C and 250 rpm overnight; (3) Inoculate 1 mL of overnight culture into 500 mL of 2YT culture medium containing 100 μg / mL ampicillin and 0.05% glucose, and culture until the OD 600 When the value reached 0.6-0.8, IPTG (purchased from Solebao Biotechnology Co., Ltd.) was added and cultured overnight at 30°C and 220 rpm in a shaking incubator; (4) Collect the bacteria by centrifugation and use ultrasonic method to break the bacteria to obtain the crude antibody extract; (5) 20 mg of Nb1, Nb7, Nb22 and Nb64 proteins with a purity of more than 90% were obtained by nickel column purification (see SDS-PAGE gel image). Figure 2 ).

[0039] Example 4: Binding activity and specificity of AAV9 nanobodies (1) AAV2, AAV6, AAV8 (purchased from Saiye Biotechnology Co., Ltd.) and AAV9 were coated onto the ELISA plate, 100 μL per well, and incubated at 4°C overnight; (2) Wash the plate three times with PBST (PBS + 0.05% Tween-20), add 300 μL of 5% skim milk powder, and block at 37°C for 1 h; after washing, add 100 μL / well of four nanoantibodies at different concentrations and incubate at 37°C for 30 min; after washing, add 100 μL of His-tagged secondary antibody and react at 37°C for 30 min; after washing, add 100 μL of TMB color development solution and develop at 37°C for 10 min, then add 50 μL of 2 M HCl to terminate the reaction and read at 450 nm; (3) Import the raw data into Graph-prism and calculate EC 50 Affinity.

[0040] Table 5 EC values of antibodies against AAV2, AAV6, AAV8, and AAV9 50 Affinity The results are as follows Figure 3 , Figure 4 , Figure 5 and Figure 6 As shown in Table 5, AAV9 nanobody Nb64 has the highest affinity and the strongest specificity.

[0041] Example 5: Nb64 acid and alkali tolerance test (1) Nb64 was added to pH 10.0 and pH 3.0 buffers to a final concentration of 500 μg / mL, respectively. After treatment for 4 and 18 hours, the pH was adjusted to neutral and diluted to different concentrations using PBS. (2) Use AAV9 to coat the ELISA plate, add 100 μL per well, and place at 4°C overnight; wash three times with PBST (PBS + 0.05% Tween-20), add 300 μL 5% skim milk powder, and block at 37°C for 1 hour; after washing, add 100 μL / well of differently treated Nb64 and incubate at 37°C for 30 minutes; after washing, add 100 μL of His-tagged secondary antibody and react at 37°C for 30 minutes; after washing, add 100 μL TMB color development solution and develop at 37°C for 5 minutes, add 50 μL 2 M HCl to stop the reaction, and read at 450 nm; (3) Import the raw data into Graph-prism and calculate EC 50 Affinity.

[0042] Table 6 EC of antibody Nb64 under different pH conditions 50 Affinity The results are as follows Figure 7 As shown in Table 6, the AAV9 binding activity of Nb64 was essentially unaffected after treatment under extreme acid and alkaline conditions for up to 18 hours, indicating its excellent acid and alkaline tolerance.

[0043] Example 6: Nb64 high temperature tolerance test (1) Nb64 was heated in metal baths at 25°C, 35°C, 45°C, 55°C, 65°C, 75°C, 85°C and 95°C for 10 min, respectively; Nb64 was heated in metal baths at 75°C and 95°C for 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min and 70 min, respectively; (2) Use AAV9 to coat the ELISA plate, add 100 μL per well, and place at 4°C overnight; wash three times with PBST (PBS + 0.05% Tween-20), add 300 μL 5% skim milk powder, and block at 37°C for 1 hour; after washing, add 100 μL / well of differently treated Nb64 and incubate at 37°C for 30 minutes; after washing, add 100 μL of His-tagged secondary antibody and react at 37°C for 30 minutes; after washing, add 100 μL TMB color development solution, develop at 37°C for 5 minutes, add 50 μL 2 M HCl to stop, and read at 450 nm; (3) Import the raw data into Graph-prism for analysis.

[0044] The results are as follows Figure 8 and Figure 9As shown, the AAV9 binding activity of Nb64 was not affected after heating at 95°C for up to 70 minutes, demonstrating its excellent heat resistance and its applicability for later industrial filler applications.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An anti-AAV9 nanobody, characterized in that: The heavy chain variable region of the anti-AAV9 nanobody includes any one of the following combinations: (1) CDR1 with the sequence shown in SEQ ID No. 6, CDR2 with the sequence shown in SEQ ID No. 7, and CDR3 with the sequence shown in SEQ ID No. 8; (2) CDR1 with the sequence shown in SEQ ID No. 9, CDR2 with the sequence shown in SEQ ID No. 10, and CDR3 with the sequence shown in SEQ ID No. 11; (3) CDR1 with the sequence shown in SEQ ID No. 12, CDR2 with the sequence shown in SEQ ID No. 13, and CDR3 with the sequence shown in SEQ ID No. 14; (4) CDR1 with the sequence shown in SEQ ID No. 15, CDR2 with the sequence shown in SEQ ID No. 16, and CDR3 with the sequence shown in SEQ ID No. 17; (5) CDR1 with the sequence shown in SEQ ID No. 18, CDR2 with the sequence shown in SEQ ID No. 19, and CDR3 with the sequence shown in SEQ ID No. 20; (6) CDR1 with the sequence shown in SEQ ID No. 21, CDR2 with the sequence shown in SEQ ID No. 22, and CDR3 with the sequence shown in SEQ ID No. 23; (7) CDR1 with the sequence shown in SEQ ID No. 24, CDR2 with the sequence shown in SEQ ID No. 25, and CDR3 with the sequence shown in SEQ ID No. 26; (8) CDR1 having a sequence as shown in SEQ ID No. 27, CDR2 having a sequence as shown in SEQ ID No. 28, and CDR3 having a sequence as shown in SEQ ID No.

29.

2. The anti-AAV9 nanobody according to claim 1, characterized in that: The amino acid sequence of the anti-AAV9 nanobody is shown as SEQ ID No.1 or SEQ ID No.3-SEQ ID No.

5.

3. A gene fragment encoding the anti-AAV9 nanobody according to claim 1 or 2.

4. An expression vector containing the gene fragment according to claim 3.

5. A host cell, characterized in that: The host cell contains the expression vector according to claim 4, or the gene fragment according to claim 3 is integrated into its genome.

6. A method for preparing an anti-AAV9 nanobody, characterized in that: The steps are: transfecting the expression vector described in claim 4 into competent cells, and culturing them to obtain anti-AAV9 nanoantibodies.

7. The anti-AAV9 nanobody according to claim 1 or 2, characterized in that: The anti-AAV9 nanobody also includes a monovalent antibody, a bivalent antibody, a multivalent antibody, a recombinant protein or an immunoconjugate of the anti-AAV9 nanobody according to claim 1 or 2.

8. The anti-AAV9 nanobody according to claim 1 or 2, characterized in that: The use of the anti-AAV9 nanobody in the preparation of diagnostic, preventive or related gene therapy products.

9. The anti-AAV9 nanobody according to claim 1 or 2, characterized in that: Application in the detection, identification, purification and quality control of related gene therapy products.

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