WO2022166949A1 - Anti-aav2 monoclonal antibody, and preparation method therefor and use thereof - Google Patents

Abstract

Provided are an anti-AAV2 monoclonal antibody, and a preparation method therefor and the use thereof. The anti-AAV2 monoclonal antibody can specifically bind to AAV2, and the binding capacity is significantly better than that of commercially-available antibodies. Both possibilities and convenience are provided for ELISA and WB detection of AAV2.

Description

Anti-AAV2 monoclonal antibody and preparation method and application thereof

claim of priority

This application claims priority to Chinese Patent Application No. 202110174898.4 filed on February 7, 2021, which is incorporated herein by reference in its entirety.

technical field

The present application belongs to the field of downstream detection of monoclonal antibodies, and relates to a monoclonal antibody for anti-AAV2 ELISA and Western Blot (WB) applications. The present application also relates to the preparation method and use of the anti-AAV2 monoclonal antibody.

Background technique

AAV (Adeno-associated virus), namely adeno-associated virus, belongs to the Parvoviridae family. The diameter is about 20-25nM, the genome is single-stranded DNA, the length is about 4.7kb, and the two ends of the genome are composed of two ITRs (inverted terminal repeats). The core elements of AAV are mainly composed of four non-structural proteins (Rep78, Rep68, Rep52 and Rep40) and three structural proteins (VP1, VP2 and VP3). VP1, VP2, and VP3 interact and assemble approximately 1:1:10 to form an approximately icosahedral structure.

At present, AAV has been widely used in gene therapy, and a number of AAV drugs have been launched. As a vector for gene delivery, large-scale production of AAV has been supported in several ways, including early stage process development and GMP-grade production. However, due to the specificity of AAV, about 40%-70% of individuals have been infected with AAV, which leads to a certain amount of AAV antibodies in the human body. The presence of these antibodies has a major impact on the durable efficacy of AAV.

In order to better understand the relationship between AAV and antibodies and identify potential antigens, we must obtain some antibodies that can recognize AAV for early AAV-related research. At the same time, with the deepening of gene therapy research, more and more scientists invest in the field of gene therapy, and the demand for high-quality AAV also increases, so the significance of developing its detection antibody is very prominent. AAV has a variety of serotypes, and at least 10 different serotypes of AAV have been found, including AAV1-9 and AAV-DJ. The main difference between different serotypes of AAV is the difference in their capsid proteins, which also leads to different AAVs' infectivity to different tissues and cell types. AAV2 has very low immunogenicity, is not easily rejected by the body's immune system, and has the ability to infect dividing and non-dividing cells. It has been reported that it has a more permanent expression of heterologous genes, therefore, AAV2 is the most widely studied. At present, the transformation of AAV vectors mainly focuses on the coat protein. In the transformation process, both the detection of the wild type and the characterization of new AAV vectors are required, which requires AAV with very good specificity and affinity. Antibody.

SUMMARY OF THE INVENTION

In one aspect, the present application provides an anti-AAV2 monoclonal antibody or a functional fragment thereof, the antibody or functional fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain can be The variable region comprises HCDR1, HCDR2 and HCDR3 comprising an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 3 or a variant of the amino acid sequence shown comprising up to three (eg, one, two or three) amino acid mutations The HCDR2 comprises a variant of the amino acid sequence shown in SEQ ID NO: 4 or a variant of the amino acid sequence comprising at most three amino acid mutations; the HCDR3 comprises an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5; and (b) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the LCDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 6 or the Said amino acid sequence comprises a variant of at most three amino acid mutations; said LCDR2 sequence comprises a variant selected from the amino acid sequence shown in SEQ ID NO: 7 or a variant of said amino acid sequence comprising at most three amino acid mutations; said LCDR3 sequence comprises is selected from the amino acid sequence shown in SEQ ID NO: 8 or a variant of the shown amino acid sequence comprising up to three amino acid mutations.

In some embodiments, the HCDR1 sequence comprises an amino acid sequence selected from SEQ ID NO: 3, the HCDR2 sequence comprises an amino acid sequence selected from SEQ ID NO: 4, and the HCDR3 sequence comprises SEQ ID NO: 4 The amino acid sequence shown in ID NO: 5; and the LCDR1 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, the LCDR2 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, the LCDR3 The sequence comprises an amino acid sequence selected from those shown in SEQ ID NO:8.

In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the following sequences: the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are set forth in SEQ ID NOs: 3, 4, and 5, respectively, and LCDR1, The amino acid sequences of LCDR2 and LCDR3 are shown in SEQ ID NOs: 6, 7 and 8, respectively.

In some embodiments, the heavy chain variable region sequence comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 1; and the light chain variable region sequence comprises the same as SEQ ID NO: The amino acid sequences shown in 2 have amino acid sequences that are at least 80% identical. In some embodiments, the heavy chain variable region sequence comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% of the amino acid sequence set forth in SEQ ID NO: 1 , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences; the light chain variable region sequence Comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 with the amino acid sequence shown in SEQ ID NO: 2 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences.

In some embodiments, the heavy chain variable region sequence comprises the amino acid sequence set forth in SEQ ID NO:1; the light chain variable region sequence comprises the amino acid sequence set forth in SEQ ID NO:2.

In a specific embodiment, the heavy chain variable region and the light chain variable region are selected from the following sequences: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, the light chain variable region The amino acid sequence of the region is shown in SEQ ID NO:2.

In another aspect, the present application provides isolated polynucleotides encoding the above-described anti-AAV2 monoclonal antibodies or functional fragments thereof.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding the heavy chain variable region of the above-mentioned anti-AAV2 monoclonal antibody or a functional fragment thereof, and a nucleotide sequence encoding the anti-AAV2 monoclonal antibody or a functional fragment thereof Nucleotide sequence of light chain variable region.

In another aspect, the application provides an expression vector comprising the polynucleotide.

In another aspect, the present application provides a host cell or cell-free expression system comprising the expression vector.

In another aspect, the present application provides an antibody for ELISA and WB detection, the detection antibody comprising the monoclonal antibody or a functional fragment thereof.

On the other hand, the present application provides the application of the anti-AAV2 monoclonal antibody or its functional fragment in detecting AAV2 by ELISA and WB detection methods.

In some embodiments, the AAV2 virus is recombinantly expressed AAV2 after purification.

In another aspect, the present application provides a method for preparing an anti-AAV2 monoclonal antibody or a functional fragment thereof, comprising: (1) immunizing an animal with purified AAV2, and generating an immune response against AAV2 in the animal; (2) ) Take the hybridoma cells obtained by fusing the spleen cells of the animal with myeloma cells, and screen them to obtain a positive clone that specifically recognizes AAV2; (3) subcloning the positive parent clone to obtain a stable hybrid tumor cell line; (4) performing gene sequencing on the hybridoma cell line to obtain the variable region coding sequences of the anti-AAV2 heavy chain and light chain; (5) using the stable hybridoma cell line for antibody production or The coding sequence of the variable region was used for recombinant antibody production to obtain a functional anti-AAV2 monoclonal antibody.

In some embodiments, the monoclonal antibody is murine, chimeric, humanized or fully human.

Description of drawings

The present application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These examples are not limiting, and in these examples, the same numbers refer to the same structures, wherein:

Fig. 1 is the result graph of the detection result of mouse serum titer after immunization shown in some embodiments of the present application;

Fig. 2 is a graph showing the results of SDS-PAGE identification after purification of monoclonal antibodies shown in some embodiments of the present application;

Fig. 3 is according to the WB detection result diagram of monoclonal antibody shown in some embodiments of the present application and different titers of AAV2;

FIG. 4 is a graph showing the results of WB detection of monoclonal antibodies and different serotypes of AAV according to some embodiments of the present application.

Detailed ways

In order to illustrate the technical solutions of the embodiments of the present application more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments. Obviously, the accompanying drawings in the following description are only some examples or embodiments of the present application. For those of ordinary skill in the art, without any creative effort, the present application can also be applied to the present application according to these drawings. other similar situations. Unless obvious from the locale or otherwise specified, the same reference numbers in the figures represent the same structure or operation.

As shown in this application and in the claims, unless the context clearly dictates otherwise, the words "a", "an", "an" and/or "the" are not intended to be specific in the singular and may include the plural. Generally speaking, the terms "comprising" and "comprising" only imply that the clearly identified steps and elements are included, and these steps and elements do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

The embodiments of the present application will be described in detail below with reference to the examples. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The term "AAV (Adeno-associated virus)", namely adeno-associated virus, belongs to the Parvoviridae family. The diameter is about 20-25nM, the genome is single-stranded DNA, the length is about 4.7kb, and the two ends of the genome are composed of two ITRs (inverted terminal repeats).

The term "antibody" is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains (wherein two heavy (H) and two light (L) chains are connected to each other by disulfide bonds (ie "complete antibody molecule")) , and multimers thereof (eg, IgM) or antigen-binding fragments thereof. Each heavy chain consists of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (consisting of the domains CH1, CH2 and CH3). Each light chain consists of a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) with more conserved regions interposed therebetween called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to hydroxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the application, the FRs of the antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified.

The term "monoclonal antibody" refers to a uniform antibody directed against only a particular epitope. Each monoclonal antibody is directed against a single epitope on an antigen, in contrast to typical polyclonal antibody preparations that include different antibodies directed against different antigenic determinants (epitopes). The modifier "monoclonal" denotes a uniform characteristic of an antibody and is not to be construed as requiring that the antibody be produced by any particular method. The monoclonal antibodies of the present application are preferably produced by recombinant DNA methods, or obtained by screening methods described elsewhere in this application.

The term "mutation" refers to a monoclonal antibody or functional fragment thereof comprising an alteration of one or more (several) amino acid residues at one or more (several) positions, ie, a substitution, insertion and/or deletion of a polypeptide. A substitution refers to replacing an amino acid occupying a position with a different amino acid; a deletion refers to removing an amino acid occupying a position; and an insertion refers to adding 1-3 amino acids adjacent to and after the amino acid occupying a position.

The term "isolated polynucleotide" refers to a polynucleotide that is not in a naturally occurring state in nature, including polynucleotides isolated from nature (including in vivo) by biological techniques, as well as artificially synthesized polynucleotides. An isolated polynucleotide can be genomic DNA, cDNA, mRNA, or other synthetic RNA, or a combination thereof. It should be pointed out that those skilled in the art can design the provided nucleotide sequences with different nucleotide sequences according to the amino acid sequences of the heavy chain variable region and light chain variable region provided herein and based on the degeneracy of codons. nucleotide sequences, but both encode the same amino acid sequence. These altered nucleotide sequences are also included within the scope of this application.

The term "vector" when referring to a polynucleotide refers to any molecule (eg, nucleic acid, plasmid or virus, etc.) used to transfer information encoded by a nucleotide into a host cell. The term "expression vector" or "expression cassette" refers to a vector suitable for expressing a gene of interest (nucleotide sequence to be expressed) in a host cell, usually including parts of the gene of interest, promoter, terminator, marker gene and the like.

The term "hybridoma cell" refers to a cell that can sustainably expand and stably secrete monoclonal antibodies.

The term "antibody functional fragment" means antigen-binding fragments and antibody analogs of antibodies, which typically include at least a portion of the antigen-binding or variable regions (eg, one or more CDRs) of the parental antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. For example, antibody fragments capable of binding AAV2 or a portion thereof include, but are not limited to, sdAbs (single domain antibodies), Fab (eg, antibodies obtained by papain digestion), F(ab')2 (eg, obtained by pepsin digestion) ), Fv or scFv (eg obtained by molecular biology techniques).

The term "amino acid substitution" refers to the replacement of existing amino acid residues with different amino acid residues in a predetermined (original) amino acid sequence. In general, it is recognized by those skilled in the art that single amino acid substitutions in non-essential regions of polypeptides do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (Fourth Edition, 1987)). Such exemplary substitutions are preferably made in accordance with the substitutions shown in Table 1:

Table 1 Exemplary conservative amino acid substitutions

original residue	conservative substitution
Ala(A)	Gly; Ser
Arg(R)	Lys; His
Asn(N)	Gln; His
Asp(D)	Glu; Asn
Cys(C)	Ser; Ala
Gln(Q)	Asn
Glu(E)	Asp;Gln
Gly(G)	Ala

His(H)	Asn; Gln
Ile(I)	Leu; Val
Leu(L)	Ile; Val
Lys(K)	Arg; His
Met(M)	Leu; Ile; Tyr
Phe(F)	Tyr; Met; Leu

"Percent (%) amino acid sequence identity" with respect to antibody sequences is defined as comparing sequences and introducing gaps where necessary to obtain maximum percent sequence identity, and without considering any conservative substitutions as part of sequence identity, among candidate sequences The percentage of amino acid residues that are identical to amino acid residues in a particular peptide or polypeptide sequence. Alignment of sequences to determine percent amino acid sequence identity can be performed in a variety of ways that are within the skill in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to obtain maximal alignment over the full length of the sequences being compared.

Unless otherwise indicated, the methods and materials of the examples described below are conventional products that are commercially available. Those skilled in the art to which the present application pertains will appreciate that the methods and materials described below are exemplary only and should not be considered as limiting the scope of the present application.

Example 1: Recombinant AAV2 Packaging

1.1 Cell preparation: One day before packaging, seed about 2.8×10 ⁷ Lenti-X 293T (TKARA, Cat: 632180) cells in a T225 culture flask, add DMEM (Gibco, Cat: 10569-010) + 10% FBS (Gibco , Cat: 10099-141C) medium into a culture flask with a final volume of 50 mL/T225. The cells were cultured for about 24h to about 80% confluence, and then transfected.

1.2 Transfection: Use PEI MAX for transfection, and prepare according to 5×T225.

a. Mix the plasmid (TAKARA Cat: 6230) with Opti-MEM (Gibco, Cat: 31985-070), and the mixing system is shown in Table 2 below:

Table 2 Plasmid and Opti-MEM mixed system

reagent	concentration	volume
pAAV–MCS	1μg/μL	293μL
pAAV-RC	1μg/μL	293μL
pHelper Vector	1μg/μL	293μL
Opti-MEM	Not marked	8.25mL

b. Mix PEI-MAX (Polyplus, Cat: 24765-2) with Opti-MEM, and the mixed system is shown in Table 3 below:

Table 3 Mixed system of PEI-MAX and Opti-MEM

reagent	concentration	volume
PEI-MAX	2.5mg/mL	1.76mL
Opti-MEM	Not marked	8.2mL

c. Mix a and b and place at room temperature for 10min;

d. Add 3.5 mL of the c mixture dropwise to a 1×T225 culture flask, and then place the cells in a 37°C, 5% CO ₂ incubator to continue culturing.

1.3 Change the medium: at least 6h after transfection, change the medium from DMEM+10%FBS to fresh DMEM+5%FBS.

1.4 Virus harvesting: Virus harvesting was performed after culturing for 72 h, and 1/80 (V/V) 0.5M EDTA (pH 8) was added to the medium supernatant and mixed. Place at room temperature for 10min. Collect the digested cells into a 50 mL centrifuge tube, centrifuge at 1750 g for 10 min at 4°C. Collect the centrifuged supernatant into a new centrifuge tube. The centrifuge tube containing cells was re-centrifuged at 1750g and 4°C for about 1-2 min, and the supernatant was collected. At this point, the supernatant and cells have been collected separately for the next experiment.

Example 2: Recombinant AAV2 virus purification

The virus supernatant obtained in Example 1 was purified according to the kit instructions (TAKARA Cat: 6232), and the specific steps were as follows:

2.1 Transfer the supernatant of Example 1 to a new 50 mL centrifuge tube, and add 1/100 volume of Cryonase Cold-Active Nuclease (TAKARA Cat: 6232). Incubate for 1 h at 37°C.

2.2 Centrifuge at 9000g for 10min at 4°C, collect the supernatant into a new sterile 50mL centrifuge tube.

2.3 Add 1/10 volume of SD solution, incubate at 37°C for 0.5h; centrifuge at 9000g for 10min at 4°C, collect the supernatant into a new sterile tube.

2.4 Prepare the AAV2 affinity purification column and carefully add the above sample to the column.

2.5 Wash with Washing buffer, at least 10mL.

2.6 The AAV virus was eluted with 3 mL of eluent and collected in a fresh tube, and a total of 3 mL of virus was collected.

2.7 The purified virus was concentrated to 1 mL through a filter.

Example 3: AAV2 animal immunization

The animal immunization antigen adopts the recombinant virus purified in Example 2. C57BL/6 mice were immunized with 10 ¹¹ VG of AAV2 virus by intramuscular immunization. Subsequently, the immunization was repeated every 2 to 3 weeks, so that the experimental mice were boosted 3 times in total. The serum titers of 2 mice (No.: 3839 and 3840) reached more than 10 ⁵ after 3 immunizations (Fig. 1). The spleen of the experimental mouse numbered 3839 was selected 4 days after the last immunization for subsequent antibody discovery.

Example 4: Acquisition of AAV2 Antibody Hybridoma Cell Line and Preparation of Monoclonal Antibody

4.1 Acquisition of hybridoma cells

The mouse spleen in Example 3 was prepared as a single cell suspension, and at the same time a single cell suspension of myeloma cells (SP2/0) was prepared. 5.35×10 ⁷ splenocytes were fused with 2.675×10 ⁷ SP2/0 mouse myeloma cells using electrofusion. Resuspend the fused cells in 150 mL of DMEM + 10% FBS medium containing the hybridoma cell selection agents thymidine, hypoxanthine, and aminopterin, and pipette to 15 cells at a volume of 100 μL/well. in a 96-well plate. The cells in the 96-well plate were incubated in a 37°C, 5% _CO2 incubator. After 7 days of incubation, the presence of antibodies to AAV2 was tested using the ELISA binding described below.

4.2 ELISA binding detection method

Indirect ELISA was used to assess the binding ability of antibodies to AAV2 in the supernatant. ELISA plates were coated with 50 μL/well of 5×10 ⁸ AAV2 in PBS overnight at 4°C. Plates were washed with PBS-T (0.05% Tween) and blocked with 150 [mu]L/well of PBST containing 1% BSA for 1 hour at 37[deg.]C. The blocking solution was then discarded, and 100 μL of hybridoma cell culture supernatant was added to each well, followed by incubation at 37° C. for 1 hour. Plates were washed three times with PBST and incubated with 100 μL/well of horseradish peroxidase-conjugated goat anti-mouse IgG (Fc-specific) secondary antibody (Jackson, 115-035-071 ) for 0.5 hours at 37°C. Plates were washed five times with PBST, then TMB developer solution was added and incubated for 13 minutes at room temperature in the dark. The reaction was terminated by adding 50 μL of 1M HCl stop solution (Sinopharm, 10011018). Read the plate at 450 nm using a microplate reader. Select positive wells with an OD value greater than 1.0 for subsequent experiments.

4.3 Hybridoma subcloning

Subcloning was performed using limiting dilution. Determine the number of cells using a hemocytometer and serial dilutions of cells in DMEM + 10% FBS medium containing the hybridoma cell selection agents thymidine, hypoxanthine, and aminopterin until the cell density reaches 5-15 cells/mL. For each hybridoma, pipette 200 μL of the cell solution into 96 wells at a density of 1-3 cells/well. After culturing the culture in a 37°C, 5% _CO2 incubator for 1 week, select monoclonal cells, and perform the above-mentioned ELISA binding on the supernatant to assess the presence of antibodies against the S protein, picking an OD value greater than 1.0 The monoclonal wells were amplified for subsequent experiments.

Example 5: Production of Hybridoma-Based Monoclonal Antibodies

After the above-mentioned cells were cultured and expanded, they were inoculated in shake flasks at 37° C. for 7 days, and the supernatant was collected for antibody purification. The tubing and Protein A column were depyrogenated with 0.2M NaOH prior to purification. The column was re-equilibrated with buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0). The harvested cell culture supernatant was then diluted 1:1 with 2x the above buffer and filter sterilized. The filtered supernatant was incubated with the protein A column for 2 hours at room temperature, and after washing the column with 1× the above buffer, IgG was eluted with sterile 0.1 M sodium citrate (pH 3.5), and the eluate was collected and washed with one-ninth Neutralize with a volume of sterile 1M Tris-HCl (pH 9). Under sterile conditions, the product was buffer exchanged to PBS (pH 7.4) to remove elution buffer.

The purified antibody 12C10A6 was analyzed by SDS-PAGE using a BioRad electrophoresis system using 10% precast gels (GenScript, M42012C). The gel was stained with Estain 2.0 (GenScript, L00687R) and molecular size and purity were estimated by comparing the stained bands with Protein Ladder (Takara, 3452), as shown in Figure 2, the antibody was 99% pure.

Example 6: Variable Region Sequencing of Monoclonal Antibodies

After the monoclonal antibodies were subtyped using a rapid ELISA mouse antibody subtyping kit (Clonotyping System-HRP, Southern Biotech), TRIzol (Life Technology, 15596-026) was used to identify the monoclonal antibodies from 1×10 ⁶ to 5×10 ⁶ Total RNA was extracted from each hybridoma cell and reverse transcribed into cDNA using antibody subtype-specific primers and universal primers (Prime Script™ 1stStrand cDNA Synthesis Kit, Takara). Murine immunoglobulin heavy and light chain V-region fragments were subsequently amplified by RACE PCR (GenScript) and the resulting PCR fragments were subcloned into the pMD18-T vector system (Takara) and inserted using vector-specific primer pairs Fragments are sequenced. Finally, the unique V-region protein amino acid sequences of 12C10A6 were obtained: 12C10A6 heavy chain variable region amino acid sequence (SEQ ID NO: 1) and 12C10A6 light chain variable region amino acid sequence (SEQ ID NO: 2).

Table 4 Antibody CDR region sequences divided by Kabat rule

Example 7: Binding of recombinant monoclonal antibody supernatants to different serotypes of AAV

ELISA plates were coated with 50 μL/well of 5×10 ⁸ AAV1, AAV5, AAV6, AAV9, AAV2 serotype virus particles in PBS overnight at 4°C. Plates were washed with PBS-T (0.05% Tween) and blocked with 150 [mu]L/well of PBST containing 1% BSA for 1 hour at 37[deg.]C. The blocking solution was then discarded, and 100 μL of hybridoma cell culture supernatant was added to each plate, followed by incubation at 37° C. for 1 hour. Plates were washed three times with PBST and incubated with 100 μL/well of horseradish peroxidase-conjugated goat anti-mouse IgG (Fc-specific) secondary antibody (Jackson, 115-035-071 ) for 0.5 hours at 37°C. Plates were washed five times with PBST, then TMB developer solution was added and incubated for 13 minutes at room temperature in the dark. The reaction was terminated by adding 50 μL of 1M HCl stop solution (Sinopharm, 10011018). Use a microplate reader to read the plate at 450 nm. The higher the absorbance OD value, the more effective antibodies that bind to AAV2 and the higher the binding capacity. The detection results are shown in Table 5 below, where C represents a purchased commercial positive control antibody (CREATIVE DIAGNOSTICS, CABT-B9062), and K represents blank control PBS. It is generally judged that the absorbance is 2.1 times that of the blank control. It can be seen from Table 5 that the binding of the antibody secreted by the 12C10A6 cell line to AAV2 is 16 times that of the blank control, indicating that the antibody secreted by the 12C10A6 cell line can be specific. Sex Recognition AAV2. At the same time, it can be seen from Table 5 that the binding of the antibody secreted by the 12C10A6 cell line to AAV2 is 2.5 times that of the commercial antibody, and the binding capacity is also significantly higher than that of the commercial antibody.

Table 5 Binding detection results of monoclonal antibody recombinant supernatant to different serotypes of AAV

Example 8: WB detection of monoclonal antibodies

8.1 Put 100 μL of AAV2 sample into a 1.5 mL EP tube, add 25 μL of 5x loading buffer (Biyuntian, Cat No: P0015L), and heat the sample in a 95°C metal bath for 10 min. 12000rpm, centrifuge for 10min, take the supernatant to obtain the sample to be tested, load the sample according to the amount of 1.1×10 ¹⁰ , 1.1×10 ⁹ , 1.1×10 ⁸ , 1.1×10 ⁷ and 1.1×10 ⁶ VG AAV2 virus, and detect 12C10A6 for AAV2 Virus detection sensitivity.

8.2 Prepare the lysates of AAV1, AAV5, AAV6, AAV9 and AAV2 according to the virus loading amount of 1.1×10 ⁹ , and load the samples to test the sensitivity of 12C10A6 to different serotype viruses.

8.3 Use GenScript's SurePAGE precast gel for electrophoresis, and transfer the electrophoresed SurePAGE precast gel to an eBlot ^™ L1 fast wet transfer machine.

8.4 After transfer, use 5% milk-PBST to block for 60min at room temperature.

8.5 Pour off the blocking solution, add the primary antibody diluted with 5% milk-PBST, and incubate at 4°C overnight.

8.6 Pour off the antibody, wash three times with PBST, discard the washing solution, add 10 mL of secondary antibody diluted with 5% milk-PBST, and incubate at room temperature for 60 min.

8.7 The antibody was poured out, washed with PBST for three times, and then developed and photographed. The results are shown in Figure 3. It can be seen from the results in Fig. 3 that the lowest dose of 12C10A6 antibody to AAV2 at the concentration of 1 μg/mL is 1.1*10 ⁹ VG.

8.8 Simultaneously load 10 ⁹ VG for each AAV sample, and perform the detection by the above WB method. The results are shown in Figure 4 . From the detection results in Figure 4, it can be seen that 12C10A6 has a positive band that reacts with AAV2 virus samples, but no bands that react with other serotype AAV samples, which can also indicate that 12C10A6 specifically recognizes AAV2.

The anti-AAV2 monoclonal antibody disclosed in this application and its preparation method and application may bring beneficial effects including but not limited to: 1. The anti-AAV2 monoclonal antibody can specifically bind to AAV2. 2. The binding ability of AAV2 monoclonal antibody to AAV2 is obviously better than that of commercial antibodies, which provides possibility and convenience for ELISA and WB detection of AAV2.

Furthermore, unless explicitly stated in the claims, the order of processing elements and sequences described in the present application, the use of numbers and letters, or the use of other names are not intended to limit the order of the procedures and methods of the present application. While the foregoing disclosure discusses by way of various examples some embodiments of the application that are presently believed to be useful, it is to be understood that such details are for purposes of illustration only and that the appended claims are not limited to the disclosed embodiments, but rather The requirements are intended to cover all modifications and equivalent combinations falling within the spirit and scope of the embodiments of the present application.

Similarly, it should be noted that, in order to simplify the expressions disclosed in the present application and thus help the understanding of one or more embodiments of the present application, in the foregoing description of the embodiments of the present application, various features are sometimes combined into one embodiment, in the drawings or descriptions thereof. However, this method of disclosure does not imply that the subject matter of the application requires more features than those mentioned in the claims. Indeed, there are fewer features of an embodiment than all of the features of a single embodiment disclosed above.

Some examples use numbers to describe quantities of components and properties, it should be understood that such numbers used to describe the examples, in some examples, use the modifiers "about", "approximately" or "substantially" to retouch. Unless stated otherwise, "about", "approximately" or "substantially" means that a variation of ±20% is allowed for the stated number. Accordingly, in some embodiments, the numerical parameters set forth in the specification and claims are approximations that can vary depending upon the desired characteristics of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and use a general digit reservation method. Notwithstanding that the numerical fields and parameters used in some embodiments of the present application to confirm the breadth of their ranges are approximations, in particular embodiments such numerical values are set as precisely as practicable.

Each patent, patent application, patent application publication, and other material, such as article, book, specification, publication, document, etc., cited in this application is hereby incorporated by reference in its entirety. Application history documents that are inconsistent with or conflict with the contents of this application are excluded, as are documents (currently or hereafter appended to this application) that limit the broadest scope of the claims of this application. It should be noted that, if there is any inconsistency or conflict between the descriptions, definitions and/or terms used in the attached materials of this application and the content of this application, the descriptions, definitions and/or terms used in this application shall prevail .

Finally, it should be understood that the embodiments described in the present application are only used to illustrate the principles of the embodiments of the present application. Other variations are also possible within the scope of this application. Accordingly, by way of example and not limitation, alternative configurations of embodiments of the present application may be considered consistent with the teachings of the present application. Accordingly, the embodiments of the present application are not limited to the embodiments expressly introduced and described in the present application.

Claims (12)

1. An anti-AAV2 monoclonal antibody or its functional fragment, said antibody or its functional fragment comprising a heavy chain variable region and a light chain variable region, wherein,

   (a) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3,

   The HCDR1 comprises an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 3 or a variant of the amino acid sequence shown comprising up to three amino acid mutations; the HCDR2 comprises an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 4 or a variant shown The amino acid sequence comprises a variant of up to three amino acid mutations; the HCDR3 comprises an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 5 or a variant of the shown amino acid sequence comprising up to three amino acid mutations; and

   (b) the light chain variable region comprises LCDR1, LCDR2 and LCDR3,

   The LCDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 6 or a variant of the amino acid sequence shown comprising up to three amino acid mutations; the LCDR2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 7 or The shown amino acid sequence comprises a variant of up to three amino acid mutations; the LCDR3 sequence comprises a variant selected from the amino acid sequence shown in SEQ ID NO: 8 or a variant of the shown amino acid sequence comprising up to three amino acid mutations.
2. The monoclonal antibody or its functional fragment according to claim 1, wherein,

   The HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO: 3; the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO: 4; the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO: 5 the amino acid sequence of ; and

   The LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO: 6; the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO: 7; the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO: 8 amino acid sequence.
3. The monoclonal antibody or functional fragment thereof according to claim 1 or 2, wherein the heavy chain variable region sequence comprises an amino acid sequence with at least 80% identity to the amino acid sequence shown in SEQ ID NO: 1; and

   The light chain variable region sequence comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 2.
4. The monoclonal antibody or its functional fragment according to claim 3, wherein,

   The heavy chain variable region sequence comprises the amino acid sequence shown in SEQ ID NO:1; and the light chain variable region sequence comprises the amino acid sequence shown in SEQ ID NO:2.
5. An isolated polynucleotide encoding the anti-AAV2 monoclonal antibody or functional fragment thereof of any one of claims 1 to 4.
6. The polynucleotide according to claim 5, wherein the polynucleotide comprises a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody or a functional fragment thereof, and encoding the monoclonal antibody or the nucleotide sequence of the light chain variable region of a functional fragment thereof.
7. An expression vector comprising the polynucleotide of claim 5 or 6.
8. A host cell or cell-free expression system comprising the expression vector according to claim 7.
9. An antibody for ELISA and WB detection, said antibody comprising the monoclonal antibody or its functional fragment according to any one of claims 1 to 4.
10. The application of the monoclonal antibody or its functional fragment according to any one of claims 1 to 4 in detecting AAV2 by ELISA and WB detection methods.
11. The use according to claim 10, wherein the AAV2 is selected from AAV2 recombinantly expressed after purification.
12. The monoclonal antibody or functional fragment thereof according to any one of claims 1 to 4, wherein the antibody is murine, chimeric, humanized or fully human.

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