CN116917314A

CN116917314A - anti-AAV 2 monoclonal antibody, preparation method and application thereof

Info

Publication number: CN116917314A
Application number: CN202280013675.2A
Authority: CN
Inventors: 丁莉丹; 邹江波; 史敏龙; 陈晖�; 武波
Original assignee: Nanjing Jinsirui Science and Technology Biology Corp
Current assignee: Nanjing Jinsirui Science and Technology Biology Corp
Priority date: 2021-02-07
Filing date: 2022-02-07
Publication date: 2023-10-20
Also published as: WO2022166949A1

Abstract

The anti-AAV 2 monoclonal antibody can be specifically combined with AAV2, the combination capacity is obviously better than that of commercial antibodies, and possibility and convenience are provided for ELISA and WB detection of AAV 2.

Description

anti-AAV 2 monoclonal antibody, preparation method and application thereof

Priority statement

The present application claims priority from chinese patent application No. 202110174898.4 filed 2/7 at 2021, which is incorporated herein by reference in its entirety.

Technical Field

The application belongs to the field of downstream detection of monoclonal antibodies, and relates to an anti-AAV 2ELISA and Western Blot (WB) applied monoclonal antibody. The application also relates to a preparation method and application of the AAV2 monoclonal antibody.

Background

AAV (Adeno-associated virus), an Adeno-associated virus, belongs to the family of parvoviruses. The diameter is about 20-25nM, the genome is single-stranded DNA, the length is about 4.7kb, and the genome is composed of two ITR (inverted terminal repeat) ends. The core elements of AAV are mainly composed of four nonstructural proteins (Rep 78, rep68, rep52, and Rep 40) and three structural proteins (VP 1, VP2, and VP 3). VP1, VP2 and VP3 interact according to approximately 1:1:10 to form a structure approximating an icosahedron.

Currently, AAV has been widely used in gene therapy, and a number of AAV drugs have been marketed. There have been various ways to support large-scale production of AAV as vectors for gene delivery, including early process development and GMP-level production. However, due to the specificity of AAV, about 40% -70% of individuals are infected with AAV, which results in the inclusion of a certain amount of AAV antibodies in humans. These antibodies present have a significant impact on the sustained efficacy of AAV.

To better understand the relationship between AAV and antibodies and identify potential antigens, we have to have antibodies that recognize AAV for early AAV-related studies. Meanwhile, as research of gene therapy is in progress, more scientists are put into the field of gene therapy, and demands for high-quality AAV are increased, so that the significance of developing detection antibodies is very prominent. AAV has multiple serotypes, and at least 10 AAV of different serotypes are now found, including AAV 1-9 and AAV-DJ. The main difference between AAV of different serotypes is their different capsid proteins, which also results in different AAV's ability to infect different tissues and cell types. AAV2 has very low immunogenicity, is not easily subject to immune rejection by the body, and has the ability to infect dividing and non-dividing cells. It has been reported to have a longer expression of heterologous genes, and therefore AAV2 has been the most widely studied. Currently, the engineering of AAV vectors is focused mainly on coat proteins, and in engineering, both wild-type and novel AAV vectors need to be detected and characterized, which requires AAV antibodies with very good specificity and affinity.

Disclosure of Invention

In one aspect, the application provides a monoclonal antibody against AAV2, or a functional fragment thereof, 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 comprising a sequence selected from the group consisting of SEQ ID NOs: 3 or a variant of the indicated amino acid sequence comprising up to three (e.g., one, two, or three) amino acid mutations; the HCDR2 comprises an amino acid sequence selected from SEQ ID NOs: 4 or a variant of the indicated amino acid sequence comprising at most three amino acid mutations; the HCDR3 comprises an amino acid sequence selected from SEQ ID NOs: 5 or a variant of the indicated amino acid sequence comprising at most three amino acid mutations; and (b) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the LCDR1 sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 6 or a variant of the indicated amino acid sequence comprising at most three amino acid mutations; the LCDR2 sequence comprises a sequence selected from SEQ ID NOs: 7 or a variant of the indicated amino acid sequence comprising at most three amino acid mutations; the LCDR3 sequence comprises a sequence selected from SEQ ID NOs: 8 or a variant of the indicated amino acid sequence comprising at most three amino acid mutations.

In some embodiments, the HCDR1 sequence comprises a sequence selected from SEQ ID NOs: 3, and the HCDR2 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, and the HCDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, and a polypeptide sequence shown in the figure; the LCDR1 sequence comprises a sequence selected from SEQ ID NOs: 6, said LCDR2 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, and the LCDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, and a polypeptide having the amino acid sequence shown in FIG. 8.

In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the group consisting of: the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 3. 4 and 5, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively as shown in SEQ ID NO: 6. 7 and 8.

In some embodiments, the heavy chain variable region sequence comprises a sequence identical to SEQ ID NO:1, the amino acid sequence having at least 80% identity to the amino acid sequence set forth in seq id no; the light chain variable region sequence comprises a sequence identical to SEQ ID NO:2 has an amino acid sequence having at least 80% identity. In some embodiments, the heavy chain variable region sequence comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 1; the light chain variable region sequence comprises a sequence identical to SEQ ID NO:2 has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

In some embodiments, the heavy chain variable region sequence comprises SEQ ID NO:1, and a polypeptide sequence shown in 1; the light chain variable region sequence comprises SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in2.

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, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

In another aspect, the application provides isolated polynucleotides encoding the above-described anti-AAV 2 monoclonal antibodies or functional fragments thereof.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding the heavy chain variable region of the anti-AAV 2 monoclonal antibody or functional fragment thereof described above, and a nucleotide sequence encoding the light chain variable region of the anti-AAV 2 monoclonal antibody or functional fragment thereof.

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

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

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

On the other hand, the application provides the application of the anti-AAV 2 monoclonal antibody or the functional fragment thereof in the detection of AAV2 by ELISA and WB detection methods.

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

In another aspect, the application provides a method of making an anti-AAV 2 monoclonal antibody, or functional fragment thereof, comprising: (1) Immunizing an animal with the purified AAV2, generating an immune response against AAV2 in the animal; (2) Taking hybridoma cells obtained by fusing spleen cells and myeloma cells of the animals, and screening to obtain positive clones specifically recognizing AAV 2; (3) Subcloning the positive parent clone to obtain a stable hybridoma cell strain; (4) Carrying out gene sequencing on the hybridoma cell strain to obtain variable region coding sequences of heavy chains and light chains of AAV 2; (5) And (3) carrying out antibody production by using the stable hybridoma cell strain or carrying out recombinant antibody production by using variable region coding sequences to obtain the monoclonal antibody of the functional AAV2.

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

Drawings

The application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. The embodiments are not limiting, in which like numerals represent like structures, wherein:

FIG. 1 is a graph showing the results of murine serum titer assays after immunization according to some embodiments of the application;

FIG. 2 is a diagram showing SDS-PAGE identification after purification of monoclonal antibodies according to some embodiments of the application;

FIG. 3 is a graph showing the WB assay of monoclonal antibodies with different titers of AAV2 according to some embodiments of the application;

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

Detailed Description

In order to more clearly illustrate the technical solution of the embodiments of the present application, the drawings that are required to be used in the description of the embodiments will be briefly described below. It is apparent that the drawings in the following description are only some examples or embodiments of the present application, and it is apparent to those of ordinary skill in the art that the present application may be applied to other similar situations according to the drawings without inventive effort. Unless otherwise apparent from the context of the language or otherwise specified, like reference numerals in the figures refer to like structures or operations.

As used in the specification and in the claims, the terms "a," "an," "the," and/or "the" are not specific to a singular, but may include a plurality, unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that the steps and elements are explicitly identified, and they do not constitute an exclusive list, as other steps or elements may be included in a method or apparatus.

Embodiments of the present application will be described in detail below with reference to examples. Unless defined otherwise, 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)", i.e. Adeno-associated virus, belongs to the family picoviridae. The diameter is about 20-25nM, the genome is single-stranded DNA, the length is about 4.7kb, and the genome is composed of two ITR (inverted terminal repeat) ends.

The term "antibody" is intended to mean an immunoglobulin molecule consisting of four polypeptide chains, wherein two heavy chains (H) and two light chains (L) are interconnected by disulfide bonds (i.e., a "complete antibody molecule"), as well as multimers thereof (e.g., 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 domains CH1, CH2 and CH 3). Each light chain consists of a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). VH and VL regions can be further subdivided into regions of hypervariability termed Complementarity Determining Regions (CDRs) with regions of greater conservation interposed therebetween termed Framework Regions (FR). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to hydroxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the application, the FR of the antibody (or antigen binding fragment thereof) may be identical to the human germline sequence or may be modified naturally or artificially.

The term "monoclonal antibody" refers to a homogeneous antibody directed against only one specific epitope. In contrast to typical polyclonal antibody preparations, which include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope on the antigen. The modifier "monoclonal" refers to a homogeneous characteristic of the antibody and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies of the application are preferably produced by recombinant DNA methods or obtained by screening methods described elsewhere in the application.

The term "mutation" refers to a polypeptide of a monoclonal antibody or functional fragment thereof comprising alterations, i.e., substitutions, insertions and/or deletions, of one or more amino acid residue(s) at one or more position(s). Substitution refers to the replacement of an amino acid occupying a position with a different amino acid; deletions refer to the removal of an amino acid occupying a position; whereas insertion refers to the addition of 1-3 amino acids next to an amino acid occupying a position and after that.

The term "isolated polynucleotide" refers to a polynucleotide that is not naturally occurring in nature, including polynucleotides isolated from nature (including in vivo) by biological techniques, and also includes synthetic polynucleotides. The isolated polynucleotide may be genomic DNA, cDNA, mRNA or other RNA synthesized, or a combination thereof. It is noted that one skilled in the art can design nucleotide sequences that are not exactly identical but all encode identical amino acid sequences from the amino acid sequences of the heavy chain variable region and the light chain variable region provided herein, based on codon degeneracy. Such modified nucleotide sequences are also included within the scope of the present application.

When referring to a polynucleotide, the term "vector" refers to any molecule (e.g., nucleic acid, plasmid, virus, etc.) used to transfer nucleotide coding information 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, and generally includes portions of the gene of interest, promoters, terminators, marker genes, and the like.

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

The term "antibody functional fragment" means antigen binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen binding or variable regions (e.g., one or more CDRs) of the parent antibody (parental antibody). The 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, including but not limited to sdabs (single domain antibodies), fabs (e.g., antibodies obtained by papain digestion), F (ab') 2 (e.g., obtained by pepsin digestion), fv, or scFv (e.g., obtained by molecular biology techniques).

The term "amino acid substitution" refers to the replacement of an existing amino acid residue with a different amino acid residue in a predetermined (initial) amino acid sequence. In general, it is well recognized by those skilled in The art that single amino acid substitutions in The nonessential region of a polypeptide do not substantially alter biological activity (see, e.g., watson et al, molecular Biology of The Gene (molecular biology of The gene), the Benjamin/Cummings Pub.Co., page 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 substitutions
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 an antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed in a variety of ways within the skill in the art to determine percent amino acid sequence identity, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment for the full length of sequences compared.

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

Example 1: recombinant AAV2 packaging

1.1 cell preparation: the day before packaging, about 2.8X10 are inoculated ⁷ Each Lenti-X293T (TKARA, cat: 632180) cell was placed in a T225 flask, and DMEM (Gibco, cat: 10569-010) +10% FBS (Gibco, cat: 10099-141C) medium was added to the flask to a final volume of 50mL/T225. Culturing for about 24 hours to a cell confluence of about 80% and then transfection.

1.2 transfection: transfection with PEI MAX was performed and prepared according to 5×T225.

a. The plasmid (TAKARA Cat: 6230) was mixed with Opti-MEM (Gibco, cat: 31985-070) in the following manner as shown in Table 2:

TABLE 2 plasmid and Opti-MEM Mixed System

Reagent(s)	Concentration of	Volume of
pAAV–MCS	1μg/μL	293μL
pAAV-RC	1μg/μL	293μL
pHelper Vector	1μg/μL	293μL
Opti-MEM	Not labeled	8.25mL

b. PEI-MAX (Polyplus, cat: 24765-2) was mixed with Opti-MEM in the following Table 3:

TABLE 3 PEI-MAX and Opti-MEM Mixed System

Reagent(s)	Concentration of	Volume of
PEI-MAX	2.5mg/mL	1.76mL
Opti-MEM	Not labeled	8.2mL

c. Mixing a and b, and standing at room temperature for 10min;

d. 3.5mL of the c-mixture was added dropwise to a 1 xT 225 flask, and the cells were then placed at 37℃with 5% CO ₂ Culturing in incubator.

1.3 liquid exchange: at least 6h post-transfection, the medium was changed from dmem+10% fbs to fresh dmem+5% fbs.

1.4 virus harvesting: after 72h of incubation, virus harvest was performed, 1/80 (V/V) of 0.5M EDTA (pH 8) was added to the culture supernatant and mixed. The mixture was left at room temperature for 10min. The digested cells were collected in 50mL centrifuge tubes, 1750g, and centrifuged at 4℃for 10min. The centrifuged supernatant was collected into a new centrifuge tube. The cell-containing centrifuge tube was centrifuged again at 1750g at 4℃for about 1 to 2 minutes, 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 subjected to virus purification according to the kit instructions (TAKARA Cat: 6232) as follows:

2.1 transfer the supernatant from example 1 to a fresh 50mL centrifuge tube and add 1/100 volume of Crronase Cold-Active Nuclear (TAKARA Cat: 6232). Incubate at 37℃for 1h.

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

2.3 adding 1/10 volume of SD solution, and incubating for 0.5h at 37 ℃; centrifuge at 4℃for 10min at 9000g and collect the supernatant into a new sterile tube.

2.4 AAV2 affinity purification columns were prepared and the samples were carefully added to the columns.

2.5 Washing with Wash buffer, at least 10mL.

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

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

Example 3: AAV2 animal immunization

Animal immunization antigens the recombinant viruses purified in example 2 were used. Will 10 ¹¹ AAV2 virus of VG immunized C57BL/6 mice by muscle immunization. Subsequently, the immunization was repeated every 2 to 3 weeks, thereby boosting the experimental mice 3 times. Serum titers of 2 mice (number: 3839 and 3840) reached 10 after 3 immunizations ⁵ The above (fig. 1). The mice numbered 3839 were selected for subsequent antibody discovery by spleen isolation 4 days after the last immunization.

Example 4: acquisition of AAV2 antibody hybridoma cell line and preparation of monoclonal antibody

4.1 obtaining of hybridoma cells

The spleen of the mice in example 3 was subjected to single cell suspension preparation while myeloma cell (SP 2/0) single cell suspension was prepared. 5.35×10 using electrofusion ⁷ Spleen cells and 2.675×10 ⁷ The SP2/0 mouse myeloma cells were fused. The fused cells were resuspended in 150mL of DMEM+10% FBS medium containing the hybridoma cell selection agents thymine nucleoside pyrimidine, hypoxanthine and aminopterin, and pipetted into 15 96-well plates at a volume of 100. Mu.L/well using a pipette. Cells in 96-well plates were incubated at 37℃with 5% CO ₂ Incubation in incubator. After 7 days of incubation, the presence of antibodies to AAV2 was initially tested using ELISA binding as described below.

4.2ELISA binding detection method

Indirect ELISA was used to assess the binding capacity of antibodies in the supernatant to AAV2. ELISA plates were prepared with 5X 10 in 50. Mu.L/well PBS ⁸ Is coated overnight at 4 ℃. Plates were washed with PBS-T (0.05% Tween) and blocked with 150. Mu.L/well of 1% BSA in PBST for 1 hour at 37 ℃. The blocking solution was then discarded, 100. Mu.L of hybridoma cell culture supernatant was added to each well, and then incubated at 37℃for 1 hour. Plates were washed three times with PBST and incubated with 100. Mu.L/well horseradish peroxidase conjugated goat anti-mouse IgG (Fc-specific) secondary antibody (Jackson, 115-035-071) for 0.5 hours at 37 ℃. Plates were washed five times with PBST, then TMB color development was added and incubated in the dark for 13 minutes at room temperature. The reaction was quenched by the addition of 50. Mu.L of 1M HCl stop solution (Guo Yao, 10011018). Plates were read at 450nm using an microplate reader. Positive wells with OD values greater than 1.0 were selected for subsequent experiments.

4.3 subcloning of hybridomas

Subcloning was performed using limiting dilution. Using a hemocytometer and in containing hybridoma cellsThe number of cells was determined by serial dilutions of the selection agents thymine pyrimidine, hypoxanthine and aminopterin in dmem+10% fbs medium until the cell density reached 5-15 cells/mL. For each hybridoma, 200 μl of the cell solution was transferred to 96 wells with a pipette at a density of 1-3 cells/well. Cultures were incubated at 37℃with 5% CO ₂ After 1 week of incubation in the incubator, monoclonal cells were selected and the supernatant was subjected to the ELISA binding described above to evaluate the presence of antibodies against S protein, and subsequent experiments were performed by selecting the expansion of monoclonal wells with OD values greater than 1.0.

Example 5: production of hybridoma cell-based monoclonal antibodies

After the cell culture was expanded, the cells were inoculated into a shake flask and cultured at 37℃for 7 days, and the supernatant was collected for antibody purification. Prior to purification, the tubing and protein a column were depyrogenated with 0.2M NaOH. The column was re-equilibrated with buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0). The harvested cell culture supernatant is then diluted 1:1 with 2 Xthe above buffer and sterilized by filtration. The filtered supernatant and protein a column were incubated for 2 hours at room temperature and after washing the column with 1 x the above buffer, igG was eluted using sterile 0.1M sodium citrate (pH 3.5), and the eluate was collected and neutralized with one-ninth volume of sterile 1M Tris-HCl (pH 9). Under sterile conditions, the product buffer was exchanged to PBS (pH 7.4) to remove the elution buffer.

Purified antibody 12C10A6 was analyzed by SDS-PAGE using 10% pre-gel (GenScript, M42012C) by BioRad electrophoresis system. The gel was stained with Estain2.0 (GenScript, L00687R) and molecular size and purity were estimated by comparing the stained band with Protein Ladder (Takara, 3452), as shown in FIG. 2, with 99% purity.

Example 6: variable region sequencing of monoclonal antibodies

After subtype identification of monoclonal antibodies using a rapid ELISA mouse antibody subtype identification kit (Clonotyping System-HRP, southern Biotech), TRIzol (Life Technology, 15596-026) was used from 1X 10 ⁶ ～5×10 ⁶ Total RNA was extracted from hybridoma cells and antibody subzone was usedThe type-specific primer and the universal primer (Prime Script 1stStrand cDNA Synthesis Kit,Takara) were reverse transcribed into cDNA. The murine immunoglobulin heavy and light chain V-region fragments were then amplified by RACE PCR (GenScript) and the resulting PCR fragments subcloned into the pMD18-T vector system (Takara) and the insert fragments sequenced using vector-specific primers. Finally, the unique V-region protein amino acid sequence of 12C10A6 is 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 regularly divided by Kabat

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

ELISA plates were prepared with 50. Mu.L/well of 5X 10 ⁸ PBS of AAV1, AAV5, AAV6, AAV9, AAV2 serotype viral particles was coated overnight at 4 ℃. Plates were washed with PBS-T (0.05% Tween) and blocked with 150. Mu.L/well of 1% BSA in PBST for 1 hour at 37 ℃. The blocking solution was then discarded, 100. Mu.L of hybridoma cell culture supernatant was added to each plate, and then incubated at 37℃for 1 hour. Plates were washed three times with PBST and incubated with 100. Mu.L/well horseradish peroxidase conjugated goat anti-mouse IgG (Fc-specific) secondary antibody (Jackson, 115-035-071) for 0.5 hours at 37 ℃. Plates were washed five times with PBST, then TMB color development was added and incubated in the dark at room temperature for 13 minutes. The reaction was quenched by the addition of 50. Mu.L of 1M HCl stop solution (Guo Yao, 10011018). Reading the plate at 450nm using a microplate reader, the higher the absorbance OD value, the more effective antibodies that bind AAV2, the higher the binding capacity. The results are shown in Table 5 below, wherein C represents a commercially available positive control antibody (CREATIVE DIAGNOSTICS, CABT-B9062) and K represents blank PBS. As a result of judging that the absorbance was 2.1 times that of the blank, positive data was obtained, it was found from Table 5 that the binding of the antibody secreted by the 12C10A6 cell line obtained by us to AAV2 was the blankIs 16 times larger than the standard, indicating that antibodies secreted by the 12C10A6 cell line can specifically recognize AAV2. Meanwhile, it can be seen from Table 5 that the binding of the antibody secreted by the 12C10A6 cell line to AAV2 was 2.5 times that of the commercial antibody, and the binding capacity was also significantly higher than that of the commercial antibody.

TABLE 5 detection of binding of monoclonal antibody recombinant supernatants to AAV of different serotypes

Example 8: WB detection of monoclonal antibodies

8.1 AAV2 samples were placed in 1.5mL EP tubes at 100. Mu.L, 25. Mu.L of 5xloading buffer (Biyun day, cat No: P0015L) was added, and the samples were heated in a metal bath at 95℃for 10min. Centrifuging at 12000rpm for 10min, collecting supernatant to obtain sample to be tested, and measuring 1.1X10 ¹⁰ 、1.1×10 ⁹ 、1.1×10 ⁸ 、1.1×10 ⁷ And 1.1X10 ⁶ The VG AAV2 viral load was loaded and the sensitivity of 12C10A6 to AAV2 virus detection was measured.

8.2 according to 1.1X10 ⁹ The lysate of AAV1, AAV5, AAV6, AAV9 and AAV2 is prepared, and the sample is loaded, so as to detect the sensitivity of 12C10A6 to the detection of different serotypes of virus.

8.3 electrophoresis was performed using a gold SurePAGE preform using eBlot ^TM And (3) carrying out film transfer by an L1 rapid wet transfer instrument.

8.4 after the end of the transfer, the membrane was blocked with 5% mill-PBST for 60min at room temperature.

8.5 pouring off the blocking solution, adding the primary antibody diluted with 5% mill-PBST, and incubating overnight at 4 ℃.

8.6 pouring out antibody, PBST washing three times, pouring out washing solution, adding 10mL of secondary antibody diluted with 5% milk-PBST, and incubating at room temperature for 60min.

8.7 antibody was decanted, washed three times with PBST, and developed and photographed, and the results are shown in FIG. 3. As can be seen from the results of FIG. 3, the 12C10A6 antibody was found to be at 1. Mu.g/mAt L concentration, the minimum recognition dose for AAV2 was 1.1×10 ⁹ VG。

8.8 simultaneous adaptation to each AAV sample 10 ⁹ VG was loaded and the WB method was tested as described above, and the results are shown in FIG. 4. From the results of the assay in FIG. 4, it can be seen that 12C10A6 has a positive band reactive with AAV2 virus samples, whereas it has no band reactive with AAV samples of other serotypes, and it can also be demonstrated that 12C10A6 specifically recognizes AAV2.

The AAV 2-resistant monoclonal antibodies disclosed by the application and the preparation method and application thereof have the beneficial effects that may be brought about, but are not limited to: 1. monoclonal antibodies against AAV2 are capable of specifically binding to AAV2. 2. The binding capacity of the monoclonal antibody of AAV2 and AAV2 is obviously better than that of commercial antibodies, and the monoclonal antibody provides possibility and convenience for ELISA and WB detection of AAV2.

Furthermore, the order in which the elements and sequences are presented, the use of numerical letters, or other designations are used in the application is not intended to limit the sequence of the processes and methods unless specifically recited in the claims. While certain presently useful application embodiments have been discussed in the foregoing disclosure by way of various examples, it is to be understood that such details are for the purpose of illustration only and that the appended claims are not to be limited to the disclosed embodiments, but rather are intended to cover all modifications and equivalent combinations that fall within the spirit and scope of the embodiments of the present application.

Similarly, it should be appreciated that in order to simplify the present disclosure and thereby facilitate an understanding of one or more embodiments of the application, various features are sometimes grouped together in a single embodiment, figure, or description thereof. This method of disclosure, however, is not intended to imply that more features than are required by the subject application. Indeed, less than all of the features of a single embodiment disclosed above.

In some embodiments, numbers describing the components, number of attributes are used, it being understood that such numbers being used in the description of embodiments are modified in some examples by the modifier "about," approximately, "or" substantially. Unless otherwise indicated, "about," "approximately," or "substantially" indicate that the number allows for a 20% variation. Accordingly, in some embodiments, numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and employ a method for preserving the general number of digits. Although the numerical ranges and parameters set forth herein are approximations in some embodiments for use in determining the breadth of the range, in particular embodiments, the numerical values set forth herein are as precisely as possible.

Each patent, patent application publication, and other material, such as articles, books, specifications, publications, documents, etc., cited herein is hereby incorporated by reference in its entirety. Except for the application history file that is inconsistent or conflicting with this disclosure, the file (currently or later attached to this disclosure) that limits the broadest scope of the claims of this disclosure is also excluded. It is noted that the description, definition, and/or use of the term in the appended claims controls the description, definition, and/or use of the term in this application if there is a discrepancy or conflict between the description, definition, and/or use of the term in the appended claims.

Finally, it should be understood that the embodiments described herein are merely illustrative of the principles of the embodiments of the present application. Other variations are also possible within the scope of the application. Thus, by way of example, and not limitation, alternative configurations of embodiments of the application may be considered in keeping with the teachings of the application. Accordingly, the embodiments of the present application are not limited to the embodiments explicitly described and depicted herein.

Claims

An anti-AAV 2 monoclonal antibody or functional fragment thereof 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 SEQ ID NOs: 3 or a variant of the indicated amino acid sequence comprising at most three amino acid mutations; the HCDR2 comprises an amino acid sequence selected from SEQ ID NOs: 4 or a variant of the indicated amino acid sequence comprising at most three amino acid mutations; the HCDR3 comprises an amino acid sequence selected from SEQ ID NOs: 5 or a variant of the indicated amino acid sequence comprising at most three amino acid mutations; and

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

the LCDR1 sequence comprises a sequence selected from SEQ ID NOs: 6 or a variant of the indicated amino acid sequence comprising at most three amino acid mutations; the LCDR2 sequence comprises a sequence selected from SEQ ID NOs: 7 or a variant of the indicated amino acid sequence comprising at most three amino acid mutations; the LCDR3 sequence comprises a sequence selected from SEQ ID NOs: 8 or a variant of the indicated amino acid sequence comprising at most three amino acid mutations.
The monoclonal antibody or a functional fragment thereof according to claim 1, wherein,

the HCDR1 sequence comprises a sequence selected from SEQ ID NOs: 3, an amino acid sequence shown in 3; the HCDR2 sequence comprises a sequence selected from SEQ ID NOs: 4, and a polypeptide sequence shown in the figure; the HCDR3 sequence comprises a sequence selected from SEQ ID NOs: 5, and a polypeptide sequence shown in the figure; and

the LCDR1 sequence comprises a sequence selected from SEQ ID NOs: 6, an amino acid sequence shown in figure 6; the LCDR2 sequence comprises a sequence selected from SEQ ID NOs: 7; the LCDR3 sequence comprises a sequence selected from SEQ ID NOs: 8, and a polypeptide having the amino acid sequence shown in FIG. 8.
The monoclonal antibody or functional fragment thereof according to claim 1 or 2, wherein the heavy chain variable region sequence comprises a sequence identical to SEQ ID NO:1, the amino acid sequence having at least 80% identity to the amino acid sequence set forth in seq id no; and

the light chain variable region sequence comprises a sequence identical to SEQ ID NO:2 has an amino acid sequence having at least 80% identity.
A monoclonal antibody or a functional fragment thereof according to claim 3, wherein,

the heavy chain variable region sequence comprises SEQ ID NO:1, and a polypeptide sequence shown in the specification; the light chain variable region sequence comprises SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in2.
An isolated polynucleotide encoding the anti-AAV 2 monoclonal antibody or functional fragment thereof of any one of claims 1-4.
The polynucleotide of claim 5, wherein the polynucleotide comprises a nucleotide sequence encoding a heavy chain variable region of the monoclonal antibody or functional fragment thereof, and a nucleotide sequence encoding a light chain variable region of the monoclonal antibody or functional fragment thereof.
An expression vector comprising the polynucleotide of claim 5 or 6.
A host cell or cell-free expression system comprising the expression vector of claim 7.
An antibody for ELISA and WB detection comprising the monoclonal antibody or functional fragment thereof according to any of claims 1-4.
Use of the monoclonal antibody or functional fragment thereof according to any one of claims 1-4 for detecting AAV2 by ELISA and WB detection methods.
The use of claim 10, wherein the AAV2 is selected from the group consisting of AAV2 recombinantly expressed after purification.
The monoclonal antibody or functional fragment thereof according to any one of claims 1-4, wherein the antibody is murine, chimeric, humanized or fully human.