An anti-AAV8 antibody and its application in AAV8 titer determination

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to an anti-AAV8 antibody and an application thereof in AAV8 titer determination; the anti-AAV8 antibody 22H8 disclosed in the present invention only recognizes AAV8 particle conformational epitopes, and does not recognize disaggregated VP1, 2, and 3 subunits; it does not bind to virus particles of AAV2, 5, 6, and 9 serotypes, and has strong specificity; at the same time, the linear range of the AAV8 titer determination Elisa kit is 1.80E+09 capsids / ml-2.81E+07 capsids / ml, the sensitivity is 2E+07 capsids / ml, and the precision CV% is less than 5%; its AAV8 linear range is wider than that of competing products currently on the market, and it is more convenient to use.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an anti-AAV8 antibody and an application thereof in AAV8 titer determination. Background Art

[0002] Adeno-associated virus (AAV) has high safety and clinical value as a gene therapy delivery vector and has become one of the main platforms for in vivo gene introduction. AAV virus particles / vectors are composed of 60 VP subunits, which are assembled to form icosahedral protein capsid particles with a diameter of about 26nm. The ratio of the three structural proteins VP1:VP2:VP3 that assemble the capsid is 1:1:10. VP1, 2, and 3 only differ in the N-terminal sequence of the protein. VP1 and 2 both contain the amino acid sequence of VP3, and there are additional other amino acid sequences at the N-terminus. The AAV icosahedron has five-fold, three-fold, and two-fold symmetry axes. Each of the 60 VP subunits is formed by multiple β-fold chains to form a barrel-shaped core, connecting the loop rings of the secondary structure of the β-fold chain, and forming a protruding spike (spine area) around the three-fold symmetry axis on the capsid surface, which is the recognition area of ​​specific receptors and antibodies.

[0003] The sequence and structure of AAV determine the differences in the binding effects of different serotypes of AAV with host receptors, resulting in different infection efficiencies of different serotypes of AAV for different tissues and cells, and tissue tropism. There are currently more than a dozen serotypes of AAV vectors in clinical trials, most of which are concentrated in the four organ tissues of the eye, liver, muscle and central nervous system, among which the most frequently used is AAV2. AAV8 and 9 have high tissue specificity for skeletal muscle and myocardium, and are used in a variety of muscle diseases, including gene delivery for Duchenne muscular dystrophy (DMD). Since 2015, the number of clinical trials of AAV8 and 9 as gene delivery vectors for the central nervous system has continued to increase.

[0004] For therapeutic AAV8 vectors, accurate titer determination is an important part of drug quality control. The ELISA method can usually be used to determine the AAV8 vector capsid titer, with antibodies that specifically bind to the AAV8 vector as capture and detection antibodies. The ELISA method has the advantages of reliability and good reproducibility. However, AAV8 has low immunogenicity as a gene therapy delivery vector, which brings certain difficulties to mouse immunization and antibody development. For AAV vectors of different serotypes used in gene therapy, especially AAV vectors that have been optimized and modified in sequence structure, there is a need to develop AAV-specific antibodies and quantitative methods. The preparation of this patented anti-AAV8 antibody immune screening and quantitative kit is of reference value. Summary of the invention

[0005] The present invention develops a specific antibody 22H8 for AAV8 vector particles through mouse immunization and antibody screening, which can specifically recognize and bind to AAV8 virus particles / AAV8 vector particles.

[0006] The first aspect of the present invention discloses an anti-AAV8 antibody.

[0007] a), the antibody comprises a light chain variable region and a heavy chain variable region; according to the IMGT numbering system,

[0008] The light chain variable region includes CDR-L1 of the amino acid sequence shown in SEQ ID NO: 3;

[0009] CDR-L1: QSLLESDGKTY (SEQ ID NO: 3);

[0010] CDR-L2 with the amino acid sequence shown in LVS;

[0011] and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 4;

[0012] CDR-L3: WQGMHFPPT (SEQ ID NO: 4);

[0013] The heavy chain variable region includes CDR-H1 of the amino acid sequence shown in SEQ ID NO: 5;

[0014] CDR-H1: GTFFSSYS (SEQ ID NO: 5);

[0015] CDR-H2 of the amino acid sequence shown in SEQ ID NO: 6;

[0016] CDR-H2: ITSGGDYT (SEQ ID NO: 6)

[0017] and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 7;

[0018] CDR-H3: IGDYYGSTYWYFAV (SEQ ID NO: 7)

[0019] b) A protein derived from a) in which the amino acid sequence in a) is increased, decreased or replaced and which has the ability to recognize and bind to AAV8 like the antibody in a).

[0020] The anti-AAV8 antibody comprises an antibody light chain variable region having an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 90% sequence similarity thereto;

[0021] DVVMTQNPFSLSVTIGQPASISCKSSQSLLESDGKTYLNWLLQRPGQSPQRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGMHFPPPTFGVGTKLELK (SEQ ID NO: 1);

[0022] The anti-AAV8 antibody comprises an antibody heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 90% sequence similarity thereto;

[0023] DVKLVESGGGLVKPGGSLKLPCAASGFTFSSYSMSWVRETPEKRLEWVATITSGGDYTYYPDSVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCIGDYYGSTYWYFAVWGAGTTVTVSS (SEQ ID NO: 2).

[0024] The second aspect of the present invention discloses a recombinant antibody, wherein the recombinant antibody has:

[0025] a) the sequence of an anti-AAV8 antibody as described above; and

[0026] b) Optional signal peptides for promoting secretory expression of the antibody and / or tag sequences for purification and detection.

[0027] The amino acid sequence of the signal peptide for the recombinant antibody light chain is shown in SEQ ID NO: 8;

[0028] MSPAQFLFLLVLWIRETNG (SEQ ID NO: 8);

[0029] The amino acid sequence of the signal peptide for the recombinant antibody heavy chain is shown in SEQ ID NO: 9;

[0030] MNFGLRLIFLVLTLNGVQC (SEQ ID NO: 9).

[0031] Wherein, the amino acid sequence of the recombinant antibody light chain is shown in SEQ ID NO: 10;

[0032] MSPAQFLFLLVLWIRETNGDVVMTQNPFSLSVTIGQPASISCKSSQSLLESDGKTYLNWLLQRPGQSPQRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCWQGMHFPPT FGVGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 10);

[0033] The amino acid sequence of the recombinant antibody heavy chain is shown in SEQ ID NO: 11;

[0034] MNFGLRLIFLVLTLNGVQCDVKLVESGGGLVKPGGSLKLPCAASGFTFSSYSMSWVRETPEKRLEWVATITSGGDYTYYPDSVTISRDNARNTLYLQMSSLKSEDTAMYYCIGD YYGSTYWYFAVWGAGTTVTVSSATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRI EPRIPKPSTPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPI ERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPGK (SEQ ID NO: 11).

[0035] Isolated nucleic acid molecules encoding the above-mentioned recombinant antibody light and heavy chains; nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating from cells and tissues containing the gene using standard techniques.

[0036] The nucleotide sequence encoding the recombinant antibody light chain is shown in SEQ ID NO: 12;

[0037] ATGTCACCAGCACAATTTCTCTTTCTTTTGGTGTTGTGGATTAGGGAAACAAACGGCGACGTTGTCATGACCCAGAACCCTTTTTCCCTGTCCGTGACCATCGGCCAGCCAGCATCTATTAGCTGCAAGAGTAGCCAGAGTTTGCTGGAGTCTGACGGCAAGACTTATCTGAATTGGCTGCTGCAGAGGCCTGGACAGTCCCCTCAACGCCTCATTTATCTCGTGTCCAAACTGGACTCAGGGGTGCCTGATCGCTTCACTGGGAGTGGATCAGGCACCGACTTTACTCTGAAGATTAGCCGCGTTGAAGCCGAAGATGTGGGTGTCTACTATTGCTGGCAAGGCATGCACTTCCCACCCACTTTTGGGGTGGGGACAAAACTGGAACTGAAACGCGCCGATGCCGCTCCTACAGTGAGCATCTTTCCTCCTTCCTCCGAGCAGCTGACAAGCGGCGGCGCCAGCGTGGTGTGTTTCCTGAACAACTTCTATCCTAAGGACATCAATGTGAAGTGGAAGATCGACGGCAGCGAGAGACAGAACGGCGTGCTGAACTCCTGGACCGACCAGGATTCCAAGGACTCCACCTACTCCATGTCCTCCACACTGACCCTGACCAAGGATGAGTACGAGAGGCACAACAGCTACACATGCGAGGCCACACACAAGACCTCCACCAGCCCTATCGTGAAGAGCTTCAATAGAAACGAGTGC (SEQ ID NO: 12);

[0038] The nucleotide sequence encoding the recombinant antibody heavy chain is shown in SEQ ID NO: 13;

[0039]

[0040] The third aspect of the present invention discloses the use of the above-mentioned anti-AAV8 antibodies and recombinant antibodies in the preparation of products for detecting AAV8 virus particles / AAV8 vectors. Preferably, the products include ELISA test kits or colloidal gold test strips.

[0041] The term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0042] It should be understood that the amino acid names herein are identified by internationally accepted single-letter English letters, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V).

[0043] The present invention achieves the following beneficial technical effects:

[0044] The anti-AAV8 antibody 22H8 disclosed in the present invention only recognizes the conformational epitope of AAV8 particles, and does not recognize the disaggregated VP1, 2, and 3 subunits; it does not bind to virus particles of AAV2, 5, 6, and 9 serotypes, and has strong specificity; at the same time, the linear range of the AAV8 titer determination ELISA kit is 1.80E+09 capsids / ml-2.81E+07 capsids / ml, the sensitivity is 2E+07 capsids / ml, and the precision CV% is less than 5%; its AAV8 quantitative linear range is wider than the linear range of competing products currently on the market, and it is more convenient to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of the binding between different serotypes of AAV and 22H8 antibody in Example 2.

[0046] Figure 2 Schematic diagram of the binding of 22H8 antibody to AAV8 particles and denatured AAV8 VP subunits in Example 3.

[0047] Figure 3 This is a standard curve diagram of the AAV8 Elisa quantitative kit based on the 22H8 antibody in Example 4.

[0048] Figure 4 This is the standard curve of the competitor AAV8 Elisa quantitative kit in Example 4. DETAILED DESCRIPTION

[0049] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The raw materials and equipment used in the examples are well known to those skilled in the art, and can be purchased or easily obtained or prepared on the market. Among them, the experimental materials are:

[0050] 1. AAV8 / 2 / 5 / 6 / 9-GFP was purchased from Weigen Biotechnology;

[0051] 2. Microplate reader: Varioskan™ Lux purchased from Thermo Fisher Scientific;

[0052] 3. ELISA plate; plate washer; 37°C constant temperature shaking incubator purchased from Thermo Fisher Scientific;

[0053] 4. Immunized mice were purchased from Shanghai Shengchang Biotechnology Co., Ltd.

[0054] Example 1. AAV8 antigen immunization and antibody screening

[0055] 1. Mouse immunization:

[0056] Take 1ul of AAV8-GFP (1E+13 capsids / ml), dilute it 100 times with PBS and mix it with an equal volume of aluminum hydroxide adjuvant. After mixing, 200μl / mouse is used for subcutaneous multi-point immunization of 6-8 week old female Balb / C mice. Two weeks after each immunization, the same dose and method are used for boosting immunization. After four immunizations, the serum titer is tested and the titer reaches 1:800000. Take 100μl of diluted AAV8-GFP (without adjuvant) for intraperitoneal boosting immunization once, and perform myeloma cell SP2 / 0 fusion three days later.

[0057] 2. Screening of hybridoma cell lines:

[0058] Three days after the booster immunization of mice, spleen cells from mice were fused with myeloma cells SP2 / 0 in the logarithmic growth phase by PEG1500 to prepare hybridoma cells. Ten days later, indirect ELISA detection was performed by coating AAV8-GFP and AAV2 / 5 / 6 / 9-GFP to screen out cell lines that only reacted with AAV8-GFP but not with other serotypes. After limiting dilution to a monoclonal state, they were expanded and frozen. The supernatant of the expanded cultured cells was used by indirect ELISA to obtain the cell line 22H8 with high activity and specific anti-AAV8 antibodies.

[0059] 3. Hybridoma monoclonal antibody sequencing and recombinant expression purification:

[0060] The selected 22H8 hybridoma cell line was expanded and cultured to a density of 1E+07 cells / ml, and sent for hybridoma monoclonal antibody sequencing (Baiying Biotechnology Co., Ltd.). The variable region sequences of the 22H8 heavy chain and light chain antibodies were fused with the antibody constant region sequences (mouse IgG3 / mouse Kappa), and the mammalian codon optimized gene synthesis was performed, and they were constructed into mammalian cell expression vectors. The expression vectors can be any commercial mammalian cell expression vectors such as pTT5 and pCDNA3.1. The plasmid was transfected into Expi293 mammalian cells to secrete and express 22H8 antibodies, and the cell expression supernatant was purified by protein A affinity to obtain the AAV8-specific recombinant antibody 22H8. It is used for antibody characterization, antibody biotin labeling and kit development.

[0061] Example 2, 22H8 Antibody Specificity

[0062] AAV2 / 5 / 6 / 8 / 9-GFP was coated at a concentration of 1E+09 capsids / ml in a microplate. The recombinant monoclonal 22H8 antibody was diluted 3 times with an initial concentration of 10ug / ml. 100μl was added to each well. Goat anti-mouse Fc-HRP antibody 0.5ug / ml was used as the secondary antibody. Figure 1 The results showed that the 22H8 mouse monoclonal antibody only reacted with AAV8, but not with AAV2 / 5 / 6 / 9, which are AAV serotypes commonly used for gene delivery, indicating that the recombinant monoclonal 22H8 antibody has good specificity.

[0063] Example 3: 22H8 antibody binds to AAV8 particles but not to denatured VP subunits

[0064] Sandwich ELISA, 22H8 antibody was coated on the ELISA plate at a concentration of 2ug / ml, AAV8-GFP (1E+13capsids / ml) was diluted 1000 times with PBST, and a portion of the diluted sample was heated at 95℃ for 5 minutes for depolymerization and denaturation, and the diluted undenatured AAV8-GFP particles and the denatured AAV8 (VP1 / VP2 / VP3) samples were added to the ELISA plate coated with 22H8 antibody for binding, and then detected with biotin-labeled 22H8 antibody 2ug / ml, as shown in Figure 2. Figure 2 It was shown that the 22H8 antibody only binds to the undepolymerized AAV8-GFP capsid, but not to the denatured and depolymerized AAV8 VP1, 2, and 3 subunits.

[0065] Example 4 Comparison of the linear range of the AAV8 Elisa quantitative kit based on the 22H8 antibody and the competitive kit

[0066] 1. Preparation of AAV8 Elisa quantitative kit reagents:

[0067] (1) Prepare 1× washing solution: 1X PBS, 0.05% Tween 20;

[0068] (2) Prepare 1× assay buffer: 1X PBS, 0.05% Tween 20, 3% BSA;

[0069] (3) Prepare the detection antibody: Use activated biotin (NHS-biotin) and mix it with the 22H8 antibody at a mass ratio of 1:10, label it at 37°C for half an hour, and dialyze it in 1XPBS overnight. Dilute the dialyzed detection antibody to 0.5ug / ml using 1X analytical buffer for later use;

[0070] (4) Prepare stop solution: dilute 98% concentrated sulfuric acid with ultrapure water to 1 M / L;

[0071] (5) Standard: Dilute AAV8 (1E+13 capsids / ml) to 1.80E+09 capsids / ml with 1X assay buffer;

[0072] (6) Prepare SA-HRP working solution: dilute 1 mg / ml SA-HRP 10,000 times with 1X assay buffer.

[0073] 2. AAV8 Elisa Quantification Kit Operation Steps:

[0074] (1) Antibody coating: Dilute 22H8 antibody to 5ug / ml using 1XPBS, add 100ul to each well of the ELISA plate, and incubate at 4°C overnight.

[0075] (2) Plate strip blocking: Wash the coated ELISA plate once, add 1X assay buffer, 200ul per well, incubate at 37℃ for 2 hours, and pat dry the ELISA plate for later use.

[0076] (3) Dilution of standard: Take 200 μl of standard 1.80E+09 capsids / ml; dilute in 2-fold ratio to 9E+08 capsids / ml, 4.5E+08 capsids / ml, 2.25E+08 capsids / ml, 1.13E+08 capsids / ml, 5.63E+07 capsids / ml, 2.81E+07 capsids / ml, a total of 7 titers, use 1× assay buffer as the zero point, and set up duplicate wells for each titer standard;

[0077] (4) Sample addition: Add the standard or sample to a 96-well reaction plate, 100 μl per well, and incubate at 37°C for 60 min with shaking.

[0078] (5) Add detection antibody: Wash the 96-well plate after the previous step 4 times, 300 μl / well, pat dry, add the diluted detection antibody, 100 μl per well, and react at 37°C with constant temperature shaking for 60 min;

[0079] (6) Adding SA-HRP: Wash the 96-well plate after the previous step 4 times, 300 μl / well, pat dry, add SA-HRP, 100 μl per well, and react at 37°C with constant temperature shaking for 60 min;

[0080] (7) Colorimetric reading: Wash the 96-well plate in step (6) 4 times, 300 μl / well, pat dry, add 100 μl / well of colorimetric solution, and develop at 37°C in the dark for 10 min. Add 50 μl / well of stop solution to terminate the reaction. Immediately read the OD value at a wavelength of 450 nm using a Varioskan™ Lux microplate reader.

[0081] The linear range of the kit is determined by the nature of the antibody itself. Figure 3 As shown, the linear range of AAV8 Elisa kit is 1.80E+09 capsids / ml-2.81E+07 capsids / ml, and the sensitivity is 2E+07 capsids / ml ( Figure 3 ); A competitive kit on the market has a linear range of 4.80E+08 capsids / ml-7.5E+06 capsids / ml ( Figure 4 ). The AAV8 Elisa quantitative kit of the present invention has a wider linear range than competing products, and can reduce the sample dilution multiple during use, which is more convenient.

[0082] Example 5. Precision of AAV8 Elisa Quantification Kit

[0083] The AAV8 capsid particles were diluted with 1X dilution buffer to high, medium and low titers of 1.92E+09 capsids / ml, 4.8E+08 capsids / ml and 1.2E+08 capsids / ml. The AAV8 Elisa quantitative kit was used to continuously detect the high, medium and low titers of AAV8 particles diluted within the linear range for 10 days, once a day. The results in Table 1 show that the CV% are within the allowable error range (CV% < 5%). The kit has high precision and good repeatability.

[0084] Table 1: Precision of AAV8 Elisa Quantification Kit

[0085]

[0086] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An anti-AAV8 antibody, It is characterized in that a), the antibody comprises a light chain variable region and a heavy chain variable region; The amino acid sequence of the light chain variable region CDR-L1 is shown in SEQ ID NO: 3; The amino acid sequence of CDR-L2 is LVS; The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 4; The amino acid sequence of the heavy chain variable region CDR-H1 is as shown in SEQ ID NO: 5; The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 6; The amino acid sequence of CDR-H3 is shown in SEQ ID NO:

7.

2. The anti-AAV8 antibody according to claim 1, It is characterized in that The antibody comprises an antibody light chain variable region of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90% sequence similarity thereto.

3. The anti-AAV8 antibody according to claim 1, It is characterized in that The antibody comprises an antibody heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 90% sequence similarity thereto.

4. A recombinant antibody, It is characterized in that The recombinant antibody has: a) the sequence of the anti-AAV8 antibody according to claim 1; and b) Optional signal peptides for promoting secretory expression of the antibody and / or tag sequences for purification and detection.

5. The recombinant antibody according to claim 4, It is characterized in that The signal peptide amino acid sequence for the recombinant antibody light chain is shown in SEQ ID NO: 8; the signal peptide amino acid sequence for the recombinant antibody heavy chain is shown in SEQ ID NO:

9.

6. The recombinant antibody according to claim 5, It is characterized in that The amino acid sequence of the recombinant antibody light chain is shown in SEQ ID NO: 10; the amino acid sequence of the recombinant antibody heavy chain is shown in SEQ ID NO:

11.

7. An isolated nucleic acid molecule encoding the light chain and heavy chain of the recombinant antibody according to claim 6; The nucleotide sequence encoding the recombinant antibody light chain is shown in SEQ ID NO: 12; The nucleotide sequence encoding the recombinant antibody heavy chain is shown in SEQ ID NO:

13.

8. Use of the anti-AAV8 antibody according to any one of claims 1 to 3, or the recombinant antibody according to any one of claims 4 to 6, in the preparation of a product for detecting AAV8 viral particles / AAV8 vectors.

9. The use according to claim 8, It is characterized in that The product includes an ELISA detection kit or a colloidal gold test strip.

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