Single-domain antibodies that can bind to different serotypes of aav and uses thereof

Abstract

The application provides a single-domain antibody capable of binding to different serotypes of AAV, and use thereof, the single-domain antibody comprising CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO. 2-4 respectively or mutants thereof. The single-domain antibody can bind to various serotypes of AAV and has high affinity activity, and can be used for affinity purification of AAV.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, and in particular to a single-domain antibody that can bind to different serotypes of AAV and its uses. Background Technology

[0002] Recombination adeno-associated virus (rAAV) vectors are diverse, exhibiting extremely low immunogenicity, high safety, a broad host cell range (capable of infecting both dividing and non-dividing cells), strong dispersibility, and long-term gene expression in vivo. They are currently considered one of the most promising gene therapy vectors for clinical applications. Several AAV-based drugs have already been approved for marketing, and numerous clinical trials are underway.

[0003] In the industrial production of AAV drugs, rapidly, efficiently, and in large quantities purifying packaged AAV particles from cell lysates is a key step in reducing AAV production cycles and costs. Existing AAV purification processes include density gradient centrifugation, ion exchange chromatography, affinity chromatography, and size exclusion chromatography. To obtain clinical-grade AAV products, multiple methods are typically combined to achieve high purity. Among these, affinity chromatography, with its simplicity, speed, efficiency, and mildness, is increasingly being used by AAV drug companies.

[0004] Affinity chromatography utilizes a stationary phase (mostly microspheres) that specifically binds to ligands on intact AAV particles via surface coupling. When cell lysate containing AAV particles passes through the stationary phase, the AAV particles bind to the affinity ligands on the stationary phase, thereby purifying and concentrating AAV. However, currently, the affinity chromatography media used in the industrial production of AAV are all imported products, such as Cytiva's Capto AVB purification packing material, Thermo Fisher's POROS CaptureSelect AAV8, AAV9, and AAVX purification packing material, and Repligen's AVIpure purification packing material. These imported products are expensive and have unstable delivery times, posing a potential risk of supply disruption to domestic AAV pharmaceutical companies. This is a key factor in the industrial production of AAV, and breakthroughs in this field are urgently needed.

[0005] Based on the above problems, the present invention provides a single-domain antibody that has high affinity for various serum AAV particles and has good application prospects in AAV affinity purification. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a single-domain antibody that can bind to different serotypes of AAV and its uses, in order to solve the problem of difficult AAV purification in the prior art.

[0007] To achieve the above and other related objectives, the present invention provides a single-domain antibody comprising CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO: 2, or a mutant having 1-2 amino acid mutations and functionally possessing at least 80%, 85%, 90%, and 95% identity. The amino acid sequence of CDR2 is shown in SEQ ID NO: 3, or a mutant having 1-2 amino acid mutations and functionally possessing at least 80%, 85%, 90%, and 95% identity. The amino acid sequence of CDR3 is shown in SEQ ID NO: 4, or a mutant having 1-2 amino acid mutations and functionally possessing at least 80%, 85%, 90%, and 95% identity.

[0008] The mutant can be one of the following:

[0009] The amino acid sequence of CDR1 is shown in SEQ ID NO: 21; the amino acid sequence of the variable region is shown in SEQ ID NO: 35.

[0010] The amino acid sequence of CDR2 is shown in SEQ ID NO: 30; the amino acid sequence of the variable region is shown in SEQ ID NO: 43.

[0011] The amino acid sequence of CDR3 is shown in SEQ ID NO: 39; the amino acid sequence of the variable region is shown in SEQ ID NO: 51.

[0012] The amino acid sequence of CDR1 is shown in SEQ ID NO: 45; the amino acid sequence of CDR1 is shown in SEQ ID NO: 46; the amino acid sequence of CDR1 is shown in SEQ ID NO: 47; and the amino acid sequence of the variable region is shown in SEQ ID NO: 44.

[0013] Furthermore, the single-domain antibody also includes a frame region FR1 that has at least 80%, 85%, 90%, and 95% identity with SEQ ID NO: 5.

[0014] Furthermore, the single-domain antibody also includes a frame region FR2 that has at least 80%, 85%, 90%, and 95% similarity to SEQ ID NO: 6.

[0015] Furthermore, the single-domain antibody also includes a frame region FR3 that has at least 80%, 85%, 90%, and 95% similarity to SEQ ID NO: 7.

[0016] Furthermore, the single-domain antibody also includes a frame region FR4 that has at least 80%, 85%, 90%, and 95% similarity to SEQ ID NO: 8.

[0017] Furthermore, the amino acid sequence of the single-domain antibody is shown in SEQ ID NO.1.

[0018] Another aspect of the present invention provides a gene encoding the aforementioned single-domain antibody.

[0019] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID NO.16.

[0020] Another aspect of the present invention provides a vector containing or having the aforementioned gene inserted therein.

[0021] Furthermore, the carrier can be pET-22b(+).

[0022] Another aspect of the present invention provides a cell containing the aforementioned carrier.

[0023] Furthermore, the cells may be Escherichia coli.

[0024] Another aspect of the invention provides the use of the single-domain antibody for purifying AAV. The AAV can be common types, such as AAV2, AAV5, AAV6, and AAV8.

[0025] Another aspect of the present invention provides a packing material for purifying AAV, the packing material containing the aforementioned single-domain antibody. The product may be a resin packing material.

[0026] Furthermore, the single-domain antibody is coupled to microspheres. The microspheres may be made of agarose gel. Such microspheres are typically used for affinity purification of AVV. They are commercially available to those skilled in the art.

[0027] As described above, the single-domain antibody of the present invention, which can bind to different serotypes of AAV, has the following beneficial effects:

[0028] It can bind to various serum types of AAV and exhibits high affinity activity. It can be used for affinity purification of AAV. Attached Figure Description

[0029] Figure 1 Agarose gel electrophoresis image of PCR amplification of the inserted gene. Lane M: DNA Marker; Lanes 1-2: PCR amplification products.

[0030] Figure 2 Agarose gel electrophoresis image of bacterial culture for PCR identification. Lane M: DNA Marker; Lanes 1-8: PCR amplification products of bacterial culture.

[0031] Figure 3 Agarose gel electrophoresis image of empty vector digestion. Lane M: DNA Marker; Lane 1: pET-22b(+) empty vector digestion product.

[0032] Figure 4 Recombinant plasmid digested with enzymes, agarose gel electrophoresis image. Lane M: DNA Marker; Lane 1: pET-22b(+) plasmid; Lane 2: pET-22b(+) product digested with ApaI-XhoI enzyme.

[0033] Figure 5 Map of pET-22b(+) plasmid with inserted single-domain antibody gene sequence.

[0034] Figure 6 Sequencing results of pET-22b(+) plasmid with inserted single-domain antibody gene sequence.

[0035] Figure 7 Gel electrophoresis results for expression identification. Lane M: Protein Marker; Lane 1: Uninduced sample; Lanes 2-6: Induced samples.

[0036] Figure 8 Gel electrophoresis results of optimization and solubility analysis. Lane M: Protein Marker; Lane 1: Sample induced with 0.2 mM IPTG at 15℃; Lane 2: Sample induced with 1.0 mM IPTG at 15℃; Lane 3: Sample induced with 0.2 mM IPTG at 37℃; Lane 4: Sample induced with 1.0 mM IPTG at 37℃; Lane 5: Uninduced sample; Lane 6: Precipitated sample after induced with 1.0 mM IPTG at 37℃; Lane 7: Supernatant after induced with 1.0 mM IPTG at 37℃; Lane 8: Precipitated sample after induced with 0.2 mM IPTG at 37℃; Lane 9: Supernatant after induced with 0.2 mM IPTG at 37℃; Lane 10: Precipitated sample after induced with 1.0 mM IPTG at 15℃; Lane 11: Supernatant after induced with 1.0 mM IPTG at 15℃; Lane 12: Sample induced with 0.2 mM IPTG at 15℃. Precipitated sample after IPTG induction; Supernatant sample after IPTG induction in lane 13:15℃ at 0.2mM.

[0037] Figure 9 Affinity purification SDS-PAGE results. Lane M: Protein Marker; Lane 1: Precipitate after disruption; Lane 2: Supernatant after disruption; Lane 3: Eluent; Lane 4: Washing sample; Lane 5: Elution sample.

[0038] Figure 10 Protein dialysis SDS-PAGE results. Lane M: Protein Marker; Lane S: Dialysis sample.

[0039] Figure 11 SDS-PAGE results of protein purification. Lane M: Protein Marker; Lane 1: Reduced sample; Lane 2: Non-reduced sample.

[0040] Figure 12 . Binding activity analysis curve of single-domain antibody with AAV2 ELISA.

[0041] Figure 13 . Binding activity analysis curve of single-domain antibody with AAV5 ELISA.

[0042] Figure 14 . Binding activity analysis curve of single-domain antibody with AAV6 ELISA.

[0043] Figure 15 . Binding activity analysis curve of single-domain antibody with AAV8 ELISA.

[0044] Figure 16 . Binding activity analysis curve of single-domain antibody with AAV2 ELISA.

[0045] Figure 17 . Binding activity analysis curve of single-domain antibody with AAV5 ELISA.

[0046] Figure 18 . Binding activity analysis curve of single-domain antibody with AAV6 ELISA.

[0047] Figure 19 . Binding activity analysis curve of single-domain antibody with AAV8 ELISA.

[0048] Figure 20 Chromatogram of affinity purification of AAV5 dynamic loading by single-domain antibody affinity packing material.

[0049] Figure 21 Silver staining detection of AAV2 dynamic loading after affinity purification using single-domain antibody affinity packing material. Lane 1: Protein Marker; Lane 2: Crude AAV2 extract; Lane 3: Flow-through buffer; Lane 4: Elution buffer; Lane 5: Protein Marker

[0050] Figure 22 Silver staining detection of AAV5 dynamic loading after affinity purification using single-domain antibody affinity packing material. Lane 1: Protein Marker; Lane 2: Crude AAV5 extract; Lane 3: Flow-through buffer; Lane 4: Elution buffer; Lane 5: Protein Marker.

[0051] Figure 23 Silver staining detection of AAV8 dynamic loading after affinity purification using single-domain antibody affinity packing material. Lane 1: Protein Marker; Lane 2: Crude AAV8 extract; Lane 3: Flow-through buffer; Lane 4: Elution buffer; Lane 5: Marker.

[0052] Figure 24 G30A protein purification SDS-PAGE results. Lane M: Protein Marker; Lane 1: Reduced sample; Lane 2: Non-reduced sample.

[0053] Figure 25 SDS-PAGE results of S56T protein purification. Lane M: Protein Marker; Lane 1: Reduced sample; Lane 2: Non-reduced sample.

[0054] Figure 26 SDS-PAGE results of G105A protein purification. Lane M: Protein Marker; Lane 1: Reduced sample; Lane 2: Non-reduced sample.

[0055] Figure 27 SDS-PAGE results of G30A / S56T / G105A protein purification. Lane M: Protein Marker; Lane 1: Reduced sample; Lane 2: Non-reduced sample.

[0056] Figure 28 ELISA binding activity curves of wild-type single-domain antibodies and their mutants with AAV2.

[0057] Figure 29 ELISA binding activity curves of wild-type single-domain antibodies and their mutants with AAV5.

[0058] Figure 30 ELISA binding activity curves of wild-type single-domain antibodies and their mutants with AAV6.

[0059] Figure 31 ELISA binding activity curves of wild-type single-domain antibodies and their mutants with AAV8. Detailed Implementation

[0060] The term "single-domain antibody" has its common meaning in the art, referring to a single heavy-chain variable domain of a naturally occurring antibody type that does not contain a light chain and can be found in camel mammals. The amino acid sequence and structure of a single-domain antibody can be considered to consist of four frame regions, or "FRs," referred to in the prior art and herein as "Frame Region 1" or "FR1"; "Frame Region 2" or "FR2"; "Frame Region 3" or "FR3"; and "Frame Region 4" or "FR4," said frame regions being separated by three complementarity-determining regions, or "CDRs," referred to in the prior art as "Complementarity-determining Region 1" or "CDR1"; "Complementarity-determining Region 2" or "CDR2" and "Complementarity-determining Region 3" or "CDR3," respectively. Therefore, a single-domain antibody can be defined as having the following general amino acid sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to frame regions 1-4, and CDR1 to CDR3 refer to complementarity-determining regions 1-3, respectively. In the context of this invention, the amino acid residues of single-domain antibodies are numbered according to the general coding of the VH domain given by the IMGT numbering system. The results of the Kabat rule numbering are also shown below. However, to avoid ambiguity, the IMGT numbering system is used as the primary system herein.

[0061] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0062] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0063] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0064] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0065] Example 1: Single-domain antibody variable region sequence

[0066] 1. Single-domain antibody variable region sequence:

[0067] QVQLQESGGGLVQAGGSLRLSCAASGRTHGMYAMGWFRQAPGKEREFVAVQDLTASNTHYSSAVKGRFTISRDNAKNTAYLQMNNLKPEDTAVYYCAAGPTIMSGSYNSARDYDYWGQGTQVTVSS (SEQ ID NO. 1).

[0068] 1.1 Wherein the IMGT rule number

[0069] The CDR1 sequence is GRTHGMYA (SEQ ID NO.2).

[0070] The CDR2 sequence is QDLTASNT (SEQ ID NO.3).

[0071] The CDR3 sequence is AAGPTIMSGSYNSARDYDY (SEQ ID NO.4)

[0072] The FR1 sequence is QVQLQESCGCLVGAGGSLRLSCAS (SEQ ID NO.5)

[0073] The FR2 sequence is MGWFRGAPGKEREFVAV (SEQ ID NO.6)

[0074] The FR3 sequence is HYSSAVKGRFTISRDNAKNTAYLIANLXPEDTAVYYC (SEQ ID NO.7)

[0075] The FR4 sequence is WGQGTQVTVSS (SEQ ID NO.8).

[0076] 1.2 Kabat rule number

[0077] The CDR1 sequence is MYAMG (SEQ ID NO.9).

[0078] The CDR2 sequence is VQDLTASNTHYSSAVKG (SEQ ID NO.10).

[0079] The CDR3 sequence is GPTIMSGSYNSARDYDY (SEQ ID NO.11).

[0080] The FR1 sequence is QVQLQESGGGLVQAGGSLRLSCAASGRTHG (SEQ ID NO.12)

[0081] The FR2 sequence is WFRQAPGKEREFVA (SEQ ID NO.13)

[0082] The FR3 sequence is RFTISRDNAKNTAYLQMNNLKPEDTAVYYCAA (SEQ ID NO.14)

[0083] The FR4 sequence is WGQGTQVTVSS (SEQ ID NO.15).

[0084] Example 2: Expression and purification of single-domain antibodies in Escherichia coli

[0085] We expressed and purified the selected single-domain antibodies in eukaryotic and prokaryotic systems.

[0086] 2.1 Plasmid construction, transformation and identification

[0087] 2.1.1 Plasmid Construction

[0088] The nucleotide sequence of the target gene is as follows (SEQ ID NO.16):

[0089] CAGGTGCAGCTGCAGGAAAGCGGCGGTGGTCTGGTTCAGGCAGGTGGTAGTCTGCGCCTGAGTTTGCCGCAAGTGGCCGTACCCATGGTATGTATGCAATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGCGAATTTGTGGCCGTGCAGGATCTGACCGCCAGCAATACCCATTATAGCAGTGCCGTGAAAGG TCGCTTTACCATTAGTCGTGATAATGCCAAAAATACCGCCTATCTGCAGATGAATAATCTGAAACCGGAAGATACCGCCGTTTATTATTGCGCAGCCGGCCCGACCATTATGAGCGGTAGTTATAATAGCGCACGTGATTATGATTATTGGGGCCAGGGTACCCAGGTTACCGTTAGTAGCGCAGCCCCGGGCTGC.

[0090] Primers for the single-domain antibody target gene sequence were used to amplify the fragment PCR product using PCR. The fragment was then recombined into the target vector pET-22b(+) (NdeI, XhoI digested vector) using multi-segment recombination to obtain the full-length construct. The ligation method is as follows.

[0091] The prepared target fragment is linked to the carrier in the reaction system.

[0092] 5ul Purified PCR products 5ul Enzyme digestion vector 10ul Seamless assembly MIX Connect at a constant temperature of 52℃ for 30 minutes

[0093] 2.1.2 Plasmid Transformation

[0094] a. Add 1 μl of plasmid to 100 μl of competent cells, gently shake and rotate to mix, and place on ice for 3 minutes;

[0095] b. 42℃ water bath for 90 seconds;

[0096] c. Place in an ice bath for 3 minutes;

[0097] d. Add 800 μl of LB medium pre-warmed at 37°C to each tube and gently shake at 200 rpm on a shaker at 37°C for 40 minutes.

[0098] 2.1.3 Verification of Recombinants

[0099] a. Prepare agar plates containing the corresponding resistance;

[0100] b. Add 100 μl of bacterial suspension to an agar plate containing the corresponding resistance, gently spread the bacteria on the surface of the plate with a sterile glass spreader, and incubate the plate at 37°C for 15 minutes.

[0101] c. Invert the plates and incubate at 37°C for 12-16 hours;

[0102] d. Pick bacteria from plates, shake at 37℃ and 250 rpm for 14 hours, perform PCR identification using the bacterial culture, and send positive clones for sequencing.

[0103] 2.1.4 Validation of cloned plasmids

[0104] Upstream primer: AATTTTGTTTAACTTTAAGAAGGAGATATACATATGCATCATCATCACCATCAGGTTCAG (SEQ ID NO.17)

[0105] Downstream primer: AGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTAACAACCCGGGGCTGCACTACTCACGGTCACCT (SEQ ID NO.18)

[0106] a. The PCR reaction used a 50 μL system: 2 μL each of primers, 2 μL template, 10 μL polymerase buffer, 1 μL 10 M m dNTPs, 1 μL polymerase, and 32 μL ddH2O. Cycling parameters: 96℃ pre-denaturation for 5 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for approximately 30 s, 23 cycles, and a final extension at 72℃ for 1 min.

[0107] b. Screen positive clones using bacterial culture PCR, extract plasmids from the obtained positive bacterial cultures by shaking at 37°C, and send them for sequencing.

[0108] c. Sequencing and matching the correct plasmids, then performing double digestion with ApaI-XhoI.

[0109] 2.1.5 Plasmid Construction Results

[0110] a. After PCR amplification of the single-domain antibody target gene sequence, agarose gel electrophoresis was performed. The amplification results of the sample are as follows: Figure 1 .

[0111] b. Identification of the gene-producing bacterial culture: After PCR amplification, agarose gel electrophoresis was performed. The amplification results of the samples are shown in the figure. Figure 2 .

[0112] c. Results of empty vector digestion: Agarose gel electrophoresis was performed after digestion of the pET-22b(+) empty vector, and the results are as follows: Figure 3 .

[0113] d. Identification of recombinant cloning plasmids: The target gene obtained by PCR amplification was constructed into the pET-22b(+) vector. The plasmid was extracted, digested with ApaI-XhoI, and then subjected to agarose gel electrophoresis. The results are as follows: Figure 4 .

[0114] e. Plasmid mapping: Results are as follows Figure 5 .

[0115] d. Sequencing results: The sequencing primer was T7ter: TGCTAGTTATTGCTCAGCGG (SEQ ID NO.19), and the results are as follows. Figure 6 .

[0116] 2.2 Expression identification, optimization and solubility analysis

[0117] a. Plasmid pET-22b(+) was transformed into BL21(DE3) competent cells, plated, and incubated overnight at 37°C inverted position. Single clones were selected and cultured in LB medium at 37°C until the bacterial OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.5 mM, and the cells were incubated at 37°C for 4 hours. After centrifugation, the cells were collected, samples were prepared, and analyzed by SDS-PAGE and Western Blot.

[0118] b. After inoculating the culture with the preservative bacteria until the bacterial OD600 is 0.6-0.8, IPTG is added to the culture to the final concentrations of 0.2 mM and 1 mM, respectively. The cultures are then incubated at 37℃ and 15℃ at 220 rpm for 4 hours and 16 hours, respectively, to induce the expression of the fusion protein. Samples under each condition are prepared and analyzed by SDS-PAGE.

[0119] In the previous step, the bacterial culture under each condition was centrifuged to collect the bacterial cells, which were then lysed (using Tris-NaCl buffer). The supernatant precipitate was used to prepare samples for SDS-PAGE analysis.

[0120] 2.3 Scale-up Expression and Purification

[0121] a. The optimal clonal strain was cultured at 37°C on LB medium until OD200 was reached. 600 =0.6~0.8, induce at 15℃ for 16 hours and harvest bacteria;

[0122] b. Centrifuge to collect bacteria, resuspend the bacterial cells, and sonicate to disrupt them;

[0123] c. Centrifuge, and purify the supernatant by nickel column affinity chromatography using the following buffer:

[0124] Equilibration buffer: "PBS-NaCl", pH 7.4

[0125] Washing buffer: "PBS-NaCl", pH 7.4 + 50mM imidazole

[0126] Elution buffer: "PBS-NaCl", pH 7.4 + 500mM imidazole

[0127] Sample preparation and SDS-PAGE analysis.

[0128] 2.4 Protein Dialysis

[0129] The qualified samples were combined and dialyzed into PBS, pH 7.4, and then filtered for sterilization.

[0130] 2.5 Experimental Results

[0131] The results are as follows Figure 1-11 .

[0132] The results showed that the plasmid vector containing the single-domain antibody gene sequence was successfully constructed. Expression of single-domain antibodies in a prokaryotic system is feasible, and sufficient quantities of high-purity single-domain antibodies can be purified from the supernatant after cell lysis.

[0133] Example 3: Expression and purification of single-domain antibodies in a eukaryotic system

[0134] 3.1 Cell transfection

[0135] The VHH-human Fc fusion protein plasmid was constructed, and the plasmid was transfected using the CHO-S expression system transfection reagent. The supernatant was collected and purified.

[0136] 3.2 Protein purification using Mab Select Sure LX gravity column purification

[0137] a. After cell centrifugation, the culture supernatant was directly purified using Mab Select Sure LX. The packing material was then placed in a gravity column tube with the following buffer solution:

[0138] Equilibration buffer: PBS pH 7.4

[0139] Washing buffer: PBS pH 7.4

[0140] Elution buffer: 0.1M glycine, pH 3.5

[0141] b. Equilibrate with 20 column volumes of PBS, add the equilibrated packing material to the culture medium supernatant, and incubate at 4°C by rotation for 1-1.5 hours;

[0142] c. Add the incubation product into a gravity column and collect the flow through;

[0143] d. After loading the sample, continue washing with equilibration buffer for 20 column volumes;

[0144] e. After rinsing, elute with elution buffer and collect;

[0145] f. After elution, adjust the pH to neutral directly with 1M Tris-HCl, pH 7.4;

[0146] g. SDS-PAGE electrophoresis detection, followed by ultrafiltration and PBS replacement to determine concentration and purity.

[0147] Example 4: ELISA verification of the ability of single-domain antibodies to bind to different serotypes of AAV.

[0148] 4.1 ELISA Validation Process

[0149] a. AAV2, AAV5, AAV6, and AAV8 were diluted with coating buffer and incubated overnight at 4°C;

[0150] b. Wash the plate 3 times with 200ul PBST (0.05% Tween-20, pH 7.2);

[0151] c. Add 100ul / well of 5% (v / v) skim milk powder and seal at 37°C for 1 hour;

[0152] d. Wash the plate 3 times, add 100 μL / well of single-domain antibody-human IgG Fc (2 μg / ml) to the experimental group, and add 100 μL / well of PBS (pH 7.2) to the blank control. Incubate at 37°C for 1 hour.

[0153] e. Wash the plate 3 times, add 100 μL / well of anti-human IgG1 Fc antibody, and incubate at 37°C for 1 hour;

[0154] f. Wash the plate 3 times, then add 100ul / well TMB for color development;

[0155] g. Add 100 μL / well stop solution;

[0156] h. The absorbance was measured at a wavelength of 450 nm using an ELISA reader.

[0157] 4.2 The single-domain antibody expressed in the eukaryotic system fused with human IgG1 Fc, as shown in the ELISA results below.

[0158] Table 1. OD450nm readings for validation of binding activity of single-domain antibody with AAV2 ELISA

[0159] AAV2 vg / well Single-domain antibodies control group 1e+010 1.9733 0.0569 3.33e+009 1.467 0.0613 1.11e+009 0.5717 0.0336 3.7e+008 0.1911 0.0438 1.23e+008 0.0535 0.0212

[0160] Table 2. OD450nm readings for validation of binding activity of single-domain antibody with AAV5 ELISA

[0161] AAV5 vg / well Single-domain antibodies control group 1e+010 3.1272 0.2501 3.33e+009 2.4979 0.2931 1.11e+009 1.3628 0.2049 3.7e+008 0.6135 0.1537 1.23e+008 0.2569 0.0904

[0162] Table 3. OD450nm readings for single-domain antibody binding activity verification with AAV6 ELISA

[0163] AAV6 vg / well Single-domain antibodies control group 1e+010 4 0.1649 3.33e+009 2.5534 0.1447 1.11e+009 1.4094 0.1469 3.7e+008 0.5703 0.1433 1.23e+008 0.2084 0.1296

[0164] Table 4. OD450nm readings for single-domain antibody binding activity verification with AAV8 ELISA

[0165] AAV8 vg / well Single-domain antibodies control group 1e+010 4 0.0443 3.33e+009 2.0514 0.0512 1.11e+009 0.894 0.0472 3.7e+008 0.3211 0.05 1.23e+008 0.0842 0.0262

[0166] Combination Figure 12-15 As shown in the table above, single-domain antibodies have a strong binding ability to AAV2, AAV5, AAV6, and AAV8.

[0167] 4.3 A single-domain antibody was expressed in prokaryotes, and its AAV binding activity was verified by ELISA. The antibody used for detection was an anti-single-domain antibody-HRP antibody.

[0168] a. AAV2, AAV5, AAV6, and AAV8 were diluted with coating buffer and incubated overnight at 4°C;

[0169] b. Wash the plate three times with 200ul PBST (0.05% Tween-20, pH 7.2);

[0170] c. Add 100ul / well of 5% (v / v) skim milk powder and seal at 37°C for 1 hour;

[0171] d. Wash the plate 3 times, add 100 μL / well of single-domain antibody (2 μg / ml) to the experimental group, and add 100 μL / well of PBS (pH 7.2) to the blank control, and incubate at 37°C for 1 hour;

[0172] e. Wash the plate 3 times, add 100 μL / well of anti-single-domain antibody-HRP (1:5000), and incubate at 37°C for 1 hour;

[0173] f. Wash the plate 3 times, then add 100ul / well TMB for color development;

[0174] g. Add 100 μL / well stop solution;

[0175] h. The absorbance was measured at a wavelength of 450 nm using an ELISA reader.

[0176] Table 5. OD450nm readings for validation of binding activity of single-domain antibody with AAV2 ELISA

[0177] AAV2 vg / well Single-domain antibodies control group 1e+010 3.6626 0.05299 3.33e+009 1.6421 0.05849 1.11e+009 0.9793 0.035 3.7e+008 0.6114 0.03929 1.23e+008 0.3885 0.0766

[0178] Table 6. OD450nm readings for validation of binding activity of single-domain antibody with AAV5 ELISA

[0179] AAV5 vg / well Single-domain antibodies control group 1e+010 3.161299944 0.08114 3.33e+009 2.325000048 0.063 1.11e+009 1.286200047 0.0422 3.7e+008 0.546199977 0.0385 1.23e+008 0.243499994 0.0595

[0180] Table 7. OD450nm readings for validation of binding activity of single-domain antibody with AAV8 ELISA

[0181] AAV8 vg / well Single-domain antibodies control group 1e+010 2.9156 0.0583 3.33e+009 1.7639 0.0533 1.11e+009 0.7865 0.0484 3.7e+008 0.2412 0.0466 1.23e+008 0.1252 0.0321

[0182] Table 8. OD450nm readings for validation of binding activity of single-domain antibody with AAV6 ELISA

[0183] AAV6 vg / well Single-domain antibodies control group 1e+010 3.536999941 0.0431 3.33e+009 2.668100119 0.0469 1.11e+009 1.697700024 0.0444 3.7e+008 0.668600023 0.0418 1.23e+008 0.227500007 0.0514

[0184] Combination Figure 16-19ELISA results from eukaryotic single-domain antibodies fused with human IgG1 Fc showed that AAV2, AAV5, AAV6, and AAV8 all exhibited strong binding affinity to the single-domain antibodies. Next, we used an anti-single-domain antibody-HRP antibody to detect the binding activity of the single-domain antibodies with different serotypes of AAV. The results indicated that the single-domain antibodies had excellent binding activity with AAV2, AAV5, AAV6, and AAV8.

[0185] Example 5: Purification test of AAV stock solution using purification packing material conjugated with anti-AAV single-domain antibody.

[0186] We also conjugated single-domain antibodies to microspheres to construct an AAV affinity purification packing material and tested its purification capability. The experimental steps and results of AAV purification using the single-domain antibody affinity packing material are as follows:

[0187] 5.1 Preparation of AAV Samples

[0188] 5.1.1 Culture and transfection of the packaging cell line 293T cells

[0189] a. Remove 293T from liquid nitrogen and place it in a 37°C water bath to allow it to dissolve rapidly;

[0190] b. Transfer the cell solution to a 15 mL centrifuge tube, add 1 mL of fresh complete culture medium, mix well by pipetting, centrifuge at 156×g (1000 rpm) for 5 minutes, discard the supernatant, and resuspend the cells in 1 mL of fresh complete culture medium;

[0191] c. Add the cell resuspension to a 100 mm culture dish, add 9 mL of culture medium to each dish in advance, mix well, and then place the culture dish in a 37°C, 5% CO2 incubator for culture.

[0192] 5.1.2 Adeno-associated virus packaging

[0193] a. The adeno-associated virus shuttle plasmid and its auxiliary packaging plasmid were extracted with high purity and free of endotoxin for later use;

[0194] b. When the cell density reaches 70-90%, transfect 293T cells. The plasmid transfection system is as follows:

[0195] ,

[0196] c. Preheat DMEM (containing triple antibodies) to 37°C, allow transfection reagents to return to room temperature, and mix DMEM and pHelper culture medium thoroughly.

[0197] d. Add the reagent from the pHelper tube to the serum type centrifuge tube according to the required amount for each serum type, and then add the corresponding amount of serum type plasmid and mix well;

[0198] e. Add the target plasmid, then add the transfection reagent to the centrifuge tube and mix by pipetting. Let stand for 20 minutes before transferring to a dish.

[0199] 5.1.3 Preparation of crude virus extract

[0200] a. Observe fluorescence after transfection, adding fresh culture medium once during the interval. 72 hours after transfection, digest the cells, centrifuge at 3000 rpm for 10 minutes, discard the supernatant, resuspend in PBS, and repeatedly freeze and thaw the resuspended solution three times in liquid nitrogen and a 37°C water bath, then centrifuge at 3000 rpm for 10 minutes.

[0201] b. Take the supernatant, add 3 μL of Benzonase enzyme (enzyme:PBS=1:100), and incubate at 37°C for 1 hour to remove the cell genome and residual plasmid DNA from the viral solution;

[0202] c. Centrifuge at 3000 rpm, 4℃ for 10 minutes. The resulting crude AAV extract was then used to determine its titer and subsequently for purification and validation experiments of single-domain antibody affinity microspheres.

[0203] 5.2 Purification of AAV using single-domain antibody affinity purification packing material

[0204] a. Pack 1 ml of single-domain antibody affinity packing material into a column, and equilibrate the column with AAV binding buffer (PBS + 0.35 M NaCl + 0.01% F68, pH 7.2-7.4) for 3-5 cycles.

[0205] b. Load the prepared crude AAV extract (treated with totipotent enzyme, centrifuged and filtered at 0.45 μm, 0.01% F68) at a flow rate of 0.8 ml / min.

[0206] c. After sample loading, wash with binding buffer until baseline equilibrium is reached;

[0207] d. Elute with elution buffer (0.1M glycine, pH adjusted to 2.0 with hydrochloric acid, 0.01% F68, 2mM magnesium chloride), and immediately add 1M Tris at a ratio of 1:10 to neutralize the collected eluent to neutral.

[0208] Wash with 5 CV of elution buffer (0.1 M phosphate + 0.01% F68), then wash with PBS (pH 7.2) for another 5 CV, and finally rinse the single-domain antibody with 20% ethanol and store it in 20% ethanol.

[0209] Table 9. Titers of each component after affinity purification of single-domain antibody affinity packing material for dynamic AAV2 loading, as determined by ddPCR.

[0210] AAV2 titer Sample (VG) 5.72E+12 Eluent (VG) 5.19E+12 Recovery rate (%) 90.73

[0211] Table 10. Titers of each component after affinity purification of single-domain antibody affinity packing material for dynamic AAV5 loading, as determined by ddPCR.

[0212] AAV5 titer Sample (VG) 3.36E+13 Eluent (VG) 2.63E+13 Recovery rate (%) 78.12

[0213] Table 11. Titers of each component after affinity purification of single-domain antibody affinity packing material for dynamic AAV8 loading, as determined by ddPCR.

[0214] AAV8 titer Crude extract (VG) for loading 1.33E+13 Eluent (VG) 1.25E+13 Recovery rate (%) 93.67

[0215] Combination Figure 20-23 Tables 11-13 show that agarose gel microspheres coupled with single-domain antibodies can affinity purify adeno-associated viruses AAV2, AAV5, and AAV8, with recoveries of 90.73%, 78.12%, and 93.67%, respectively.

[0216] Example 6 Mutant 1 amino acid sequence

[0217] The variable region sequence G30A of the single-domain antibody mutant 1 (SEQ ID NO.20)

[0218] QVQLQESGGGLVQAGGSLRLSCAASGRTH A MYAMGWFRQAPGKEREFVAVQDLTASNTHYSSAVKGRFTISRDNALNTAYLQMNNLKPEDTAVYYCAAGPTIMSGSYNSARDYDYWGQGTQVTVSS

[0219] Wherein IMGT rule number

[0220] The CDR1 sequence is GRTH A MYA (SEQ ID NO.21)

[0221] The CDR2 sequence is QDLTASNT (SEQ ID NO.22).

[0222] The CDR3 sequence is AAGPTIMSGSYNSARDYDY (SEQ ID NO.23).

[0223] The FR1 sequence is QVQLQESCGCLVGAGGSLRLSCAS (SEQ ID NO.24)

[0224] The FR2 sequence is MGWFRGAPGKEREFVAV (SEQ ID NO.25)

[0225] The FR3 sequence is HYSSAVKGRFTISRDNAKNTAYLIANLXPEDTAVYYC (SEQ ID NO.26)

[0226] The FR4 sequence is WGQGTQVTVSS (SEQ ID NO.27).

[0227] Example 7 Amino acid sequence of mutant 2

[0228] The variable region sequence S56T of the single-domain antibody mutant 2 (SEQ ID NO.28)

[0229] QVQLQESGGGLVQAGGSLLRLSCAASGRTHGMYAMGWFRQAPGKEREFVAVQDLTA T NTHYSSAVKGRFTISRDNALNTAYLQMNNLKPEDTAVYYCAAGPTIMSGSYNSARDYDYWGQGTQVTVSS

[0230] Wherein IMGT rule number

[0231] The CDR1 sequence is GRTHGMYA (SEQ ID NO.29)

[0232] The CDR2 sequence is QDLTA. T NT (SEQ ID NO.30)

[0233] The CDR3 sequence is AAGPTIMSGSYNSARDYDY (SEQ ID NO.31).

[0234] The FR1 sequence is QVQLQESCGCLVGAGGSLRLSCAS (SEQ ID NO.32)

[0235] The FR2 sequence is MGWFRGAPGKEREFVAV (SEQ ID NO.33).

[0236] The FR3 sequence is HYSSAVKGRFTISRDNAKNTAYLIANLXPEDTAVYYC (SEQ ID NO.34)

[0237] The FR4 sequence is WGQGTQVTVSS (SEQ ID NO.35).

[0238] Example 8: Mutant 3-amino acid sequence

[0239] The variable region sequence G105A of the single-domain antibody mutant 3 (SEQ ID NO.36)

[0240] QVQLQESGGGLVQAGGSLRLSCAASGRTHGMYAMGWFRQAPGKEREFVAVQDLTASNTHYSSAVKGRFTISRDNALNTAYLQMNNLKPEDTAVYYCAAGPTIMS A SYNSARDYDYWGQGTQVTVSS

[0241] Wherein IMGT rule number

[0242] The CDR1 sequence is GRTHGMYA (SEQ ID NO.37).

[0243] The CDR2 sequence is QDLTASNT (SEQ ID NO.38).

[0244] The CDR3 sequence is AAGPTIMS A SYNSARDYDY (SEQ ID NO.39)

[0245] The FR1 sequence is QVQLQESCGCLVGAGGSLRLSCAS (SEQ ID NO.40)

[0246] The FR2 sequence is MGWFRGAPGKEREFVAV (SEQ ID NO.41).

[0247] The FR3 sequence is HYSSAVKGRFTISRDNAKNTAYLIANLXPEDTAVYYC (SEQ ID NO.42).

[0248] The FR4 sequence is WGQGTQVTVSS (SEQ ID NO.43).

[0249] Example 9 Mutant 4-amino acid sequence

[0250] The variable region sequence of single-domain antibody mutant 4 is G30A / S56T / G105A (SEQ ID NO.44).

[0251] QVQLQESGGGLVQAGGSLRLSCAASGRTH A MYAMGWFRQAPGKEREFVAVQDLTA T NTHYSSAVKGRFTISRDNALNTAYLQMNNLKPEDTAVYYCAAGPTIMS A SYNSARDYDYWGQGTQVTVSS

[0252] Wherein IMGT rule number

[0253] The CDR1 sequence is GRTH A MYA (SEQ ID NO.45)

[0254] The CDR2 sequence is QDLTA. T NT (SEQ ID NO.46)

[0255] The CDR3 sequence is AAGPTIMS A SYNSARDYDY (SEQ ID NO.47)

[0256] The FR1 sequence is QVQLQESCGCLVGAGGSLRLSCAS (SEQ ID NO.48)

[0257] The FR2 sequence is MGWFRGAPGKEREFVAV (SEQ ID NO.49)

[0258] The FR3 sequence is HYSSAVKGRFTISRDNAKNTAYLIANLXPEDTAVYYC (SEQ ID NO.50).

[0259] The FR4 sequence is WGQGTQVTVSS (SEQ ID NO.51).

[0260] Example 12: Ability of mutants 1-4 to bind to different serotypes of AAV

[0261] To verify the binding ability of different mutants to AAV of different serotypes, we constructed and expressed mutants 1-4, and performed experiments identical to those for the single-domain antibody in Example 1, using the same procedures as described above. The specific results are as follows:

[0262] The quality control results of the purified single-domain antibody via SDS-PAGE electrophoresis are as follows: Figure 1-6 As shown:

[0263] Figure 24 G30A protein purification SDS-PAGE results. Lane M: Protein Marker; Lane 1: Reduced sample; Lane 2: Non-reduced sample.

[0264] Figure 25 SDS-PAGE results of S56T protein purification. Lane M: Protein Marker; Lane 1: Reduced sample; Lane 2: Non-reduced sample.

[0265] Figure 26SDS-PAGE results of G105A protein purification. Lane M: Protein Marker; Lane 1: Reduced sample; Lane 2: Non-reduced sample.

[0266] Figure 27 SDS-PAGE results of G30A / S56T / G105A protein purification. Lane M: Protein Marker; Lane 1: Reduced sample; Lane 2: Non-reduced sample.

[0267] It is evident that all mutants were successfully expressed.

[0268] The ELISA results of the binding ability of mutant single-domain antibodies to different serotypes of AAV are shown in the following figure and table:

[0269] Figure 28 ELISA binding activity curves of wild-type single-domain antibodies and their mutants with AAV2.

[0270] Figure 29 ELISA binding activity curves of wild-type single-domain antibodies and their mutants with AAV5.

[0271] Figure 30 ELISA binding activity curves of wild-type single-domain antibodies and their mutants with AAV6.

[0272] Figure 31 ELISA binding activity curves of wild-type single-domain antibodies and their mutants with AAV8.

[0273] Table 12. OD450nm readings for validating the binding activity of single-domain antibody wild-type and its mutants with AAV2 ELISA

[0274] lg(vg / well) Single-domain antibody wild type Single-domain antibody mutant 1 Single-domain antibody mutant 2 Single-domain antibody mutant 3 Single-domain antibody mutant 4 control group 1E+10 3.7994 3.9994 3.9214 3.4697 3.7989 0.0137 3.33E+09 3.1128 3.0504 2.4724 2.4509 2.9109 0.0136 1.11E+09 2.0303 1.5527 1.3346 1.2212 1.405 0.0098 3.70E+08 0.7922 0.5188 0.5149 0.239 0.4949 0.0099 1.23E+08 0.2286 0.1923 0.1466 0.0971 0.1519 0.0143

[0275] Table 13. OD450nm readings for validating the binding activity of single-domain antibody wild-type and its mutants with AAV5 ELISA

[0276] lg(vg / well) Single-domain antibody wild type Single-domain antibody mutant 1 Single-domain antibody mutant 2 Single-domain antibody mutant 3 Single-domain antibody mutant 4 control group 1E+10 3.587 3.2707 3.5555 3.6232 3.9985 0.0085 3.33E+09 1.8126 2.5356 2.7899 2.2836 2.6612 0.0053 1.11E+09 1.147 1.609 1.6995 1.8137 1.7695 0.0066 3.70E+08 0.5075 0.748 0.8048 0.9696 0.8268 0.0051 1.23E+08 0.218 0.2855 0.3241 0.4124 0.3871 0.0043

[0277] Table 14. OD450nm readings for the binding activity verification of single-domain antibody wild-type and its mutants with AAV6 ELISA

[0278] lg(vg / well) Single-domain antibody wild type Single-domain antibody mutant 1 Single-domain antibody mutant 2 Single-domain antibody mutant 3 Single-domain antibody mutant 4 control group 1E+10 3.9226 3.9169 3.9987 3.9976 3.8558 0.0508 3.33E+09 3.3238 3.2402 3.3618 3.3231 2.8528 0.0371 1.11E+09 2.3172 1.48 1.7172 1.4073 1.7082 0.0253 370000000 0.7064 0.3929 0.501 0.4256 0.5742 0.0175 123000000 0.316 0.1779 0.2015 0.1771 0.2676 0.0207

[0279] Table 15. OD450nm readings for validating the binding activity of single-domain antibody wild-type and its mutants with AAV8 ELISA

[0280] lg(vg / well) Single-domain antibody wild type Single-domain antibody mutant 1 Single-domain antibody mutant 2 Single-domain antibody mutant 3 Single-domain antibody mutant 4 control group 1E+10 3.7446 3.4981 3.6608 3.0697 3.4241 0.0259 3.33E+09 2.6998 2.9481 2.9794 2.2406 2.4365 0.0232 1.11E+09 1.3577 1.3146 1.2971 0.9936 1.5583 0.0224 370000000 0.4589 0.3489 0.4061 0.2675 0.3997 0.0146 123000000 0.2189 0.172 0.1607 0.1116 0.172 0.0127

[0281] The ELISA results show that single-domain antibodies and their mutants exhibited considerable activity in binding to AAV2, AAV5, and AAV6.

[0282] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A single-domain antibody, characterized in that, The single-domain antibody can bind to AAV2, AAV5, AAV6, and AAV8, and the single-domain antibody includes CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO: 2; The amino acid sequence of CDR2 is shown in SEQ ID NO: 3; The amino acid sequence of CDR3 is shown in SEQ ID NO:

4.

2. The single-domain antibody according to claim 1, characterized in that: The single-domain antibody also includes one or more of the following technical features: The single-domain antibody also includes a frame region FR1 that has at least 80% identity with SEQ ID NO: 5; The single-domain antibody also includes a frame region FR2 that has at least 80% identity with SEQ ID NO: 6; The single-domain antibody also includes a frame region FR3 that has at least 80% identity with SEQ ID NO: 7; The single-domain antibody also includes a frame region FR4 that has at least 80% identity with SEQ ID NO:

8.

3. The single-domain antibody according to claim 1, characterized in that: The amino acid sequence of the single-domain antibody is shown in SEQ ID NO.

1.

4. A single-domain antibody, characterized in that, The single-domain antibody can bind to AAV2, AAV5, AAV6, and AAV8, and the single-domain antibody includes CDR1, CDR2, and CDR3. The single-domain antibody is selected from any one of the following antibodies: a. The amino acid sequence of CDR1 is shown in SEQ ID NO: 21; the amino acid sequence of CDR2 is shown in SEQ ID NO: 22; the amino acid sequence of CDR3 is shown in SEQ ID NO: 23; b. The amino acid sequence of CDR1 is shown in SEQ ID NO: 29; the amino acid sequence of CDR2 is shown in SEQ ID NO: 30; the amino acid sequence of CDR1 is shown in SEQ ID NO: 31; c. The amino acid sequence of CDR1 is shown in SEQ ID NO: 37; the amino acid sequence of CDR1 is shown in SEQ ID NO: 38; the amino acid sequence of CDR3 is shown in SEQ ID NO: 39; d. The amino acid sequence of CDR1 is shown in SEQ ID NO: 45, the amino acid sequence of CDR2 is shown in SEQ ID NO: 46, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

47.

5. A gene encoding a single-domain antibody as described in claim 1.

6. The gene according to claim 5, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

16.

7. A carrier, characterized in that: The vector contains or has inserted the gene as described in claim 5.

8. A cell, characterized in that, The cell contains the carrier as described in claim 7.

9. Use of the single-domain antibody as described in any one of claims 1-4 for purifying AAV.

10. A packing material for purifying AAV, characterized in that: The packing material contains a single-domain antibody as described in any one of claims 1-4.

11. The packing material according to claim 10, characterized in that: The single-domain antibody is coupled to the microspheres.

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