Affinity polypeptides directed to the full capsid of aav

Abstract

The present disclosure aims to provide an affinity molecule having higher binding to an AAV full capsid than to an empty capsid of AAV. The anti-AAV full capsid molecule having higher binding to an AAV full capsid than to an empty capsid of AAV comprises: a heavy chain variable region comprising CDR1 represented by SEQ ID NO: 6, CDR2 represented by SEQ ID NO: 7, and CDR3 represented by SEQ ID NO: 8 as heavy chain complementarity determining regions; and a light chain variable region comprising CDR1 represented by SEQ ID NO: 9, CDR2 consisting of RAS, and CDR3 represented by SEQ ID NO: 10 as light chain complementarity determining regions.

Description

Technical Field

[0001] This disclosure relates to an affinity polypeptide for the complete capsid of adeno-associated virus (AAV). Background Technology

[0002] In the manufacturing process of adeno-associated virus (AAV), which is used as a gene therapy drug, a mixture of a full capsid containing exogenous DNA (which becomes the active ingredient in the drug) and an empty capsid is obtained. This manufacturing process requires a method for quantifying the full capsid from this mixture.

[0003] As specific methods for quantifying intact capsids, known methods include quantitative PCR (Patent Document 1) and interference scattering mass spectrometry (Patent Document 2).

[0004] Existing technical documents Patent documents Patent Document 1: CN101426935B Patent Document 2: WO2021 / 191079 Summary of the Invention

[0005] The problem that the invention aims to solve Quantitative methods for the complete capsid, such as quantitative PCR or interference scattering mass spectrometry, require expensive specialized equipment and lack simplicity and versatility.

[0006] To quantify the complete capsid using a simple and universal method, affinity molecules are desired. For affinity molecules suitable for such quantification of the complete capsid, their binding affinity to the complete AAV capsid must be higher than their binding affinity to the empty AAV capsid (i.e., selective binding affinity to the complete AAV capsid).

[0007] Therefore, the purpose of this disclosure is to provide a molecule with higher affinity to the complete AAV capsid than the empty capsid of AAV.

[0008] Methods for solving problems The inventors of this application conducted in-depth research and discovered an anti-AAV intact capsid molecule that binds to the intact AAV capsid more strongly than the empty capsid of AAV. This disclosure was completed through further and iterative research based on this insight.

[0009] That is, this disclosure provides the invention in the manner disclosed below.

[0010] Item 1. Anti-AAV intact capsid molecule, which is an antibody that has a higher binding affinity to the intact AAV capsid than the empty AAV capsid.

[0011] Item 2. An intact anti-AAV capsid molecule, comprising: The heavy chain variable region, comprising CDR1 (composed of the amino acid sequence represented by Serial No. 1), CDR2 (composed of the amino acid sequence represented by Serial No. 2), and CDR3 (composed of the amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 3), serves as the heavy chain complementarity-determining region; and The light chain variable region, which includes CDR1 composed of the amino acid sequence represented by sequence number 4, CDR2 composed of RAS, and CDR3 composed of an amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by sequence number 5, serves as the light chain complementarity determination region.

[0012] Item 3. An intact anti-AAV capsid molecule, comprising: The heavy chain variable region, comprising CDR1 (composed of the amino acid sequence represented by sequence number 6), CDR2 (composed of the amino acid sequence represented by sequence number 7), and CDR3 (composed of the amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by sequence number 8) as a heavy chain complementarity-determining region; and The light chain variable region, which includes CDR1 composed of the amino acid sequence represented by sequence number 9, CDR2 composed of RAS, and CDR3 composed of an amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by sequence number 10, serves as the light chain complementarity determination region.

[0013] Item 4. An intact anti-AAV capsid molecule, comprising: The heavy chain variable region, comprising CDR1 (composed of the amino acid sequence represented by Serial No. 11), CDR2 (composed of the amino acid sequence represented by Serial No. 12), and CDR3 (composed of the amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 13) as a heavy chain complementarity-determining region; and The light chain variable region, which includes CDR1 composed of the amino acid sequence represented by Serial No. 14, CDR2 composed of QAS, and CDR3 composed of an amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 15, serves as the light chain complementarity determination region.

[0014] Item 5. An intact anti-AAV capsid molecule, comprising: The heavy chain variable region, comprising CDR1 (composed of the amino acid sequence represented by SEQ ID NO. 16), CDR2 (composed of the amino acid sequence represented by SEQ ID NO. 17), and CDR3 (composed of the amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by SEQ ID NO. 18) as a heavy chain complementarity-determining region; and The light chain variable region, which includes CDR1 composed of the amino acid sequence represented by sequence number 19, CDR2 composed of RAS, and CDR3 composed of the amino acid sequence represented by sequence number 20, serves as the light chain complementarity-determining region.

[0015] Item 6. The anti-AAV intact capsid molecule as described in any one of items 2 to 5, wherein, The aforementioned heavy chain variable region includes: FR1 is composed of amino acid sequences that have more than 70% sequence identity with the amino acid sequences at positions 1-24 of sequence number 21, sequence number 23, sequence number 25, or sequence number 27. FR2 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 33-49 of sequence number 21, positions 34-50 of sequence number 23, positions 34-50 of sequence number 25, or positions 34-50 of sequence number 27. FR3 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 59-95 of sequence number 21, positions 60-96 of sequence number 23, positions 59-95 of sequence number 25, or positions 60-96 of sequence number 27. FR4 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 109-119 of sequence number 21, positions 109-119 of sequence number 23, positions 107-117 of sequence number 25, or positions 113-123 of sequence number 27. The aforementioned light chain variable region includes: FR1 is composed of amino acid sequences that have more than 70% sequence identity with the amino acid sequences at positions 1-26 of sequence number 22, sequence number 24, sequence number 26, or sequence number 28. FR2 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 33-49 of sequence number 22, sequence number 24, sequence number 26, or sequence number 28. FR3 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 53-88 of sequence number 22, sequence number 24, sequence number 26, or sequence number 28. FR4 is composed of amino acid sequences that have more than 70% sequence identity with the amino acid sequences at positions 101-110 of sequence number 22, positions 101-110 of sequence number 24, positions 101-109 of sequence number 26, or positions 98-107 of sequence number 28.

[0016] Item 7. The anti-AAV intact capsid molecule as described in Item 2, wherein the aforementioned heavy chain variable region comprises the amino acid sequence represented by Serial No. 21, and the aforementioned light chain variable region comprises the amino acid sequence represented by Serial No. 22.

[0017] Item 8. The anti-AAV intact capsid molecule as described in Item 3, wherein the aforementioned heavy chain variable region comprises the amino acid sequence represented by Serial No. 23, and the aforementioned light chain variable region comprises the amino acid sequence represented by Serial No. 24.

[0018] Item 9. The anti-AAV intact capsid molecule as described in Item 4, wherein the aforementioned heavy chain variable region comprises the amino acid sequence represented by Serial No. 25, and the aforementioned light chain variable region comprises the amino acid sequence represented by Serial No. 26.

[0019] Item 10. The anti-AAV intact capsid molecule as described in Item 5, wherein the aforementioned heavy chain variable region comprises the amino acid sequence represented by Serial No. 27, and the aforementioned light chain variable region comprises the amino acid sequence represented by Serial No. 28.

[0020] Item 11. The intact anti-AAV capsid molecule as described in any one of items 1 to 10, which is an IgG antibody, a Fab antibody, a Fab' antibody, or an F(ab') antibody. 2 Antibodies, Fv antibodies, scFv antibodies, dsFv antibodies, scFv-Fc antibodies, dsFv-Fc antibodies, Bis-scFv antibodies, minibody antibodies, diabody antibodies, triabody antibodies, or tetrabody antibodies.

[0021] Item 12. An affinity solid phase for binding AAV intact capsids, comprising any one of items 1 to 11, an anti-AAV intact capsid molecule, and a solid phase material for immobilizing the aforementioned anti-AAV intact capsid molecule.

[0022] Item 13. A method for capturing an intact AAV capsid, the method comprising step 1 of capturing the intact AAV capsid by bringing a sample containing an empty AAV capsid and an intact AAV capsid into contact with the intact AAV capsid described in Item 12 by affinity solid-phase contact.

[0023] Item 14. The method of Item 13, further comprising step 2 of quantifying the captured intact AAV capsid by enzyme-linked immunosorbent assay.

[0024] Item 15. Nucleic acid, encoding the anti-AAV intact capsid molecule described in any one of items 1 to 11.

[0025] Item 16. An expression cassette or recombinant vector comprising the nucleic acid described in Item 15.

[0026] Item 17. A transformant obtained by transforming a host using the expression cassette or recombinant vector described in Item 16.

[0027] Item 18. A method for manufacturing an intact anti-AAV capsid molecule, said method comprising the step of culturing the transformant described in Item 17.

[0028] Invention Effects According to this disclosure, an anti-AAV intact capsid molecule can be provided that binds to the intact AAV capsid more strongly than the empty capsid of AAV. Therefore, according to this disclosure, it is possible to quantify the intact capsid using this anti-AAV intact capsid molecule. Attached Figure Description

[0029] [ Figure 1 The diagram illustrates the correlation between the amount of intact AAV9 capsid (anti-AAV-scFv) and the ELISA signal (absorbance) using the intact anti-AAV capsid molecule (anti-AAV-scFv) disclosed herein. Detailed Implementation

[0030] 1. Anti-AAV intact capsid molecules The anti-AAV intact capsid molecule of this disclosure is an antibody that has a higher binding affinity to the AAV intact capsid compared to the empty AAV capsid. Specifically, the anti-AAV intact capsid molecule of this disclosure is a polypeptide with a higher binding affinity to the AAV intact capsid compared to the empty AAV capsid, and has a heavy chain variable region containing a heavy chain complementarity-determining region and a light chain variable region containing a light chain complementarity-determining region, exhibiting higher binding affinity to the AAV intact capsid compared to the empty AAV capsid. Preferably, the anti-AAV intact capsid molecule of this disclosure is a polypeptide with a higher binding affinity to the AAV intact capsid compared to the empty AAV capsid, and has a heavy chain variable region containing a defined heavy chain complementarity-determining region and a light chain variable region containing a defined light chain complementarity-determining region.

[0031] 1-1. Target There is no particular limitation on the serotype of AAV that serves as the target of the intact AAV capsid molecule of this disclosure. Specific serotypes include AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12, and AAV13, with AAV9 being the preferred example.

[0032] AAV empty capsids are empty shells of AAVs that do not contain nucleic acids. A typical example of an AAV empty capsid is a vesicle used to package a target gene (exogenous gene) in the cells to be delivered to a therapeutic recipient.

[0033] The complete AAV capsid is an AAV capsid containing nucleic acids. Examples of such nucleic acids include the viral genome of the AAV, nucleic acid drugs (antisense oligonucleotides, RNAi, aptamers, decoys, etc.), and vectors that introduce genes for gene therapy.

[0034] 1-2. Associativity The anti-AAV intact capsid molecule disclosed herein has a higher binding affinity to the AAV intact capsid compared to the empty AAV capsid.

[0035] The higher binding affinity to the intact AAV capsid compared to the empty AAV capsid can be confirmed by the following method: when the anti-AAV intact capsid molecule of this disclosure is contacted with either the empty or intact AAV capsid under the same conditions, the binding amount of the intact AAV capsid is greater than that of the empty AAV capsid. For example, this can be confirmed by preparing the anti-AAV intact capsid molecule of this disclosure in a form with an affinity tag, contacting it with either the empty or intact AAV capsid under the same conditions, capturing the capsid-bound anti-AAV intact capsid molecule with an antibody (anti-affinity tag antibody) that is specific to the affinity tag and has a signal group (e.g., a fluorescent group), and measuring the signal intensity (e.g., absorbance based on a fluorescent group) of the anti-affinity tag antibody that captured the anti-AAV intact capsid molecule based on the signal group.

[0036] As for the specific degree of binding to the intact AAV capsid, examples can be given: when the anti-AAV intact capsid molecule is contacted with the AAV capsid at pH 7 (25°C) and 25°C for 1 hour, the binding to the intact AAV capsid based on the above absorbance is more than 1.5 times that of the binding to the empty AAV capsid, specifically, it is 1.5 to 5 times, preferably 2 to 5 times, more preferably 2.5 to 5 times, further preferably 3 to 5 times, 3.5 to 5 times, and even more preferably 4 to 5 times.

[0037] 1-3. Complementarity Determining Zone The anti-AAV intact capsid molecule disclosed herein typically comprises three heavy chain complementarity-determining regions (CDR1, CDR2, and CDR3 from the N-terminus) and three light chain complementarity-determining regions (CDR1, CDR2, and CDR3 from the N-terminus).

[0038] In a preferred embodiment, the anti-AAV intact capsid molecule of this disclosure includes the first anti-AAV intact capsid molecule shown in [1], the second anti-AAV intact capsid molecule shown in [2], the third anti-AAV intact capsid molecule shown in [3], and the fourth anti-AAV intact capsid molecule shown in [4] of Table 1.

[0039] [Table 1] The first anti-AAV intact capsid molecule comprises: a heavy chain variable region, which includes CDR1 composed of the amino acid sequence represented by Serial No. 1, CDR2 composed of the amino acid sequence represented by Serial No. 2, and CDR3 composed of an amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 3 as a heavy chain complementarity-determining region; and a light chain variable region, which includes CDR1 composed of the amino acid sequence represented by Serial No. 4, CDR2 composed of RAS, and CDR3 composed of an amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 5 as a light chain complementarity-determining region.

[0040] The second anti-AAV intact capsid molecule comprises: a heavy chain variable region, which includes a CDR1 composed of the amino acid sequence represented by Serial No. 6, a CDR2 composed of the amino acid sequence represented by Serial No. 7, and a CDR3 composed of an amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 8 as a heavy chain complementarity-determining region; and a light chain variable region, which includes a CDR1 composed of the amino acid sequence represented by Serial No. 9, a CDR2 composed of RAS, and a CDR3 composed of an amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 10 as a light chain complementarity-determining region. Among the anti-AAV intact capsid molecules of this disclosure, the second anti-AAV intact capsid molecule is preferred due to its significant binding specificity to the AAV intact capsid.

[0041] The third anti-AAV intact capsid molecule comprises: a heavy chain variable region, which includes CDR1 composed of the amino acid sequence represented by Serial No. 11, CDR2 composed of the amino acid sequence represented by Serial No. 12, and CDR3 composed of an amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 13 as a heavy chain complementarity-determining region; and a light chain variable region, which includes CDR1 composed of the amino acid sequence represented by Serial No. 14, CDR2 composed of QAS, and CDR3 composed of an amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 15 as a light chain complementarity-determining region.

[0042] The fourth anti-AAV intact capsid molecule comprises: a heavy chain variable region, which includes CDR1 composed of the amino acid sequence represented by Serial No. 16, CDR2 composed of the amino acid sequence represented by Serial No. 17, and CDR3 composed of the amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 18 as a heavy chain complementarity-determining region; and a light chain variable region, which includes CDR1 composed of the amino acid sequence represented by Serial No. 19, CDR2 composed of RAS, and CDR3 composed of the amino acid sequence represented by Serial No. 20 as a light chain complementarity-determining region.

[0043] It should be noted that in this invention, "sequence identity" refers to the identity value of the amino acid sequence obtained through the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999) of the BLAST PACKAGE [sgi32bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. The parameters can be set as follows: Gap insertion Cost value: 11, Gap extension Cost value: 1.

[0044] In amino acid sequences with less than 100% sequence identity, where mismatched amino acid residues are replaced by other amino acids from the baseline amino acid sequence, substitution based on similar amino acids (i.e., conserved amino acid substitution) is suitable. Specifically, based on the properties of the amino acid side chains, a classification has been established, preferably replacing the amino acids with those belonging to the same classification.

[0045] Basic amino acids: lysine, arginine, histidine Acidic amino acids: glutamic acid, aspartic acid Neutral amino acids: glycine, alanine, serine, threonine, methionine, cysteine, phenylalanine, tryptophan, tyrosine, leucine, isoleucine, valine, glutamine, asparagine, proline In addition, the aforementioned neutral amino acids can also be classified into amino acids with polar side chains (asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with amide side chains (asparagine, glutamine), amino acids with sulfur side chains (methionine, cysteine), amino acids with aromatic side chains (phenylalanine, tryptophan, tyrosine), amino acids with hydroxyl side chains (serine, threonine, tyrosine), and amino acids with aliphatic side chains (alanine, leucine, isoleucine, valine), etc.

[0046] Regarding methods for replacing specified amino acids in an amino acid sequence with other amino acids, such as the known site-directed mutagenesis (Hashimoto-Gotoh T. et al., Gene, Vol. 152, pp. 271-275 (1995); Zoller MJ. et al., Methods Enzymol. Vol. 100, pp. 468-500 (1983); Kramer W. et al., Nucleic Acids Res. Vol. 12, pp. 9441-9456 (1984); Kramer W. et al., Methods Enzymol. Vol. 154, pp. 350-367 (1987); Kunkel TA., Proc Natl Acad Sci USA., Vol. 82, pp. 488-492 (1985) etc.), this site-directed mutagenesis can be used to replace amino acids in the CDR amino acid sequence. In addition, the library technique described in WO2005 / 080432 can be cited as a method for replacing amino acids with other amino acids.

[0047] 1-4. Frame area The framework regions of the heavy chain variable regions and light chain variable regions of the first to fourth anti-AAV intact capsid molecules are limited to the fact that the anti-AAV intact capsid molecules have a higher binding affinity to the AAV intact capsid compared to the empty capsid of AAV. There are no particular limitations, and the framework regions (FRs) of any animal (human and non-human, preferably rabbit) antibodies can be used.

[0048] For example, the first to fourth anti-AAV intact capsid molecules are preferably the following molecules: The heavy chain variable region contains: FR1 is composed of amino acid sequences that have more than 70% sequence identity with the amino acid sequences at positions 1-24 of sequence number 21, sequence number 23, sequence number 25, or sequence number 27. FR2 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 33-49 of sequence number 21, positions 34-50 of sequence number 23, positions 34-50 of sequence number 25, or positions 34-50 of sequence number 27. FR3 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 59-95 of sequence number 21, positions 60-96 of sequence number 23, positions 59-95 of sequence number 25, or positions 60-96 of sequence number 27. FR4 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 109-119 of sequence number 21, positions 109-119 of sequence number 23, positions 107-117 of sequence number 25, or positions 113-123 of sequence number 27. The aforementioned light chain variable region includes: FR1 is composed of amino acid sequences that have more than 70% sequence identity with the amino acid sequences at positions 1-26 of sequence number 22, sequence number 24, sequence number 26, or sequence number 28. FR2 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 33-49 of sequence number 22, sequence number 24, sequence number 26, or sequence number 28. FR3 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 53-88 of sequence number 22, sequence number 24, sequence number 26, or sequence number 28. FR4 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 101-110 of sequence number 22, positions 101-110 of sequence number 24, positions 101-109 of sequence number 26, or positions 98-107 of sequence number 28. Furthermore, the molecule has a higher binding affinity to the complete AAV capsid compared to the empty capsid of AAV.

[0049] The preferred examples of sequence identity of 70% or more may vary depending on the full length of the amino acid sequence used as a reference. Preferably, it is 80% or more or 85% or more, more preferably 90% or more, even more preferably 92% or more, even more preferably 94% or more or 95% or more, even more preferably 96% or more or 97% or more, and most preferably 100%.

[0050] 1-5. Specific examples of variable regions More preferably, in the first anti-AAV intact capsid molecule, the aforementioned heavy chain variable region contains the amino acid sequence represented by sequence number 21, and the aforementioned light chain variable region contains the amino acid sequence represented by sequence number 22.

[0051] The heavy chain variable region sequence number 21, starting from the N-terminus, consists of {FR1 composed of amino acid sequences from position 1 to 24} - {CDR1 composed of amino acid sequences from position 25 to 32 (equivalent to CDR1 of sequence number 1)} - {FR2 composed of amino acid sequences from position 33 to 49} - {CDR2 composed of amino acid sequences from position 50 to 58 (equivalent to CDR2 of sequence number 2)} - {FR3 composed of amino acid sequences from position 59 to 95} - {CDR3 composed of amino acid sequences from position 96 to 108 (equivalent to CDR3 of sequence number 3)} - {FR4 composed of amino acid sequences from position 109 to 119}. The variable region of the light chain, Serial No. 22, is composed of the following amino acid sequences from the N-terminus: {FR1 composed of amino acid sequences from position 1 to 26} - {CDR1 composed of amino acid sequences from position 27 to 32 (equivalent to CDR1 of Serial No. 4)} - {FR2 composed of amino acid sequences from position 33 to 49} - {CDR2 composed of amino acid sequences from position 50 to 52} - {FR3 composed of amino acid sequences from position 53 to 88 of Serial No. 22} - {CDR3 composed of amino acid sequences from position 89 to 100 (equivalent to CDR3 of Serial No. 5)} - {FR4 composed of amino acid sequences from position 101 to 110 of Serial No. 22}.

[0052] More preferably, in the second anti-AAV intact capsid molecule, the aforementioned heavy chain variable region contains the amino acid sequence represented by sequence number 23, and the aforementioned light chain variable region contains the amino acid sequence represented by sequence number 24.

[0053] The heavy chain variable region sequence number 23, starting from the N-terminus, consists of {FR1 composed of amino acid sequences from position 1 to 24} - {CDR1 composed of amino acid sequences from position 25 to 33 (equivalent to CDR1 in sequence number 6)} - {FR2 composed of amino acid sequences from position 34 to 50} - {CDR2 composed of amino acid sequences from position 51 to 59 (equivalent to CDR2 in sequence number 7)} - {FR3 composed of amino acid sequences from position 60 to 96} - {CDR3 composed of amino acid sequences from position 97 to 108 (equivalent to CDR3 in sequence number 8)} - {FR4 composed of amino acid sequences from position 109 to 119}. The light chain variable region sequence number 24, starting from the N-terminus, consists of {FR1 composed of amino acid sequences from position 1 to 26} - {CDR1 composed of amino acid sequences from position 27 to 32 (equivalent to CDR1 in sequence number 9)} - {FR2 composed of amino acid sequences from position 33 to 49} - {CDR2 composed of amino acid sequences from position 50 to 52} - {FR3 composed of amino acid sequences from position 53 to 88} - {CDR3 composed of amino acid sequences from position 89 to 100 (equivalent to CDR3 in sequence number 10)} - {FR4 composed of amino acid sequences from position 101 to 110}.

[0054] More preferably, in the third anti-AAV intact capsid molecule, the aforementioned heavy chain variable region contains the amino acid sequence represented by sequence number 25, and the aforementioned light chain variable region contains the amino acid sequence represented by sequence number 26.

[0055] The heavy chain variable region sequence number 25, starting from the N-terminus, consists of {FR1 composed of amino acid sequences from position 1 to 24} - {CDR1 composed of amino acid sequences from position 25 to 33 (equivalent to CDR1 in sequence number 11)} - {FR2 composed of amino acid sequences from position 34 to 50} - {CDR2 composed of amino acid sequences from position 51 to 58 (equivalent to CDR2 in sequence number 12)} - {FR3 composed of amino acid sequences from position 59 to 95} - {CDR3 composed of amino acid sequences from position 96 to 106 (equivalent to CDR3 in sequence number 13)} - {FR4 composed of amino acid sequences from position 107 to 117}. The light chain variable region sequence number 26, starting from the N-terminus, consists of {FR1 composed of amino acid sequences from position 1 to 26} - {CDR1 composed of amino acid sequences from position 27 to 32 (equivalent to CDR1 in sequence number 14)} - {FR2 composed of amino acid sequences from position 33 to 49} - {CDR2 composed of amino acid sequences from position 50 to 52} - {FR3 composed of amino acid sequences from position 53 to 88} - {CDR3 composed of amino acid sequences from position 89 to 100 (equivalent to CDR3 in sequence number 15)} - {FR4 composed of amino acid sequences from position 101 to 109}.

[0056] More preferably, in the fourth anti-AAV intact capsid molecule, the aforementioned heavy chain variable region contains the amino acid sequence represented by sequence number 27, and the aforementioned light chain variable region contains the amino acid sequence represented by sequence number 28.

[0057] The heavy chain variable region SEQ ID NO. 27 is composed of the following amino acid sequences from the N-terminus: {FR1 consisting of amino acid sequences from position 1 to 24} - {CDR1 consisting of amino acid sequences from position 25 to 33 (equivalent to CDR1 in SEQ ID NO. 16)} - {FR2 consisting of amino acid sequences from position 34 to 50} - {CDR2 consisting of amino acid sequences from position 51 to 59 (equivalent to CDR2 in SEQ ID NO. 17)} - {FR3 consisting of amino acid sequences from position 60 to 96} - {CDR3 consisting of amino acid sequences from position 97 to 112 (equivalent to CDR3 in SEQ ID NO. 18)} - {FR4 consisting of amino acid sequences from position 113 to 123}. The light chain variable region sequence number 28, starting from the N-terminus, consists of {FR1 composed of amino acid sequences from position 1 to 26} - {CDR1 composed of amino acid sequences from position 27 to 32 (equivalent to CDR1 in sequence number 19)} - {FR2 composed of amino acid sequences from position 33 to 49} - {CDR2 composed of amino acid sequences from position 50 to 52} - {FR3 composed of amino acid sequences from position 53 to 88} - {CDR3 composed of amino acid sequences from position 89 to 97 (equivalent to CDR3 in sequence number 20)} - {FR4 composed of amino acid sequences from position 98 to 107}.

[0058] 1-6. Specific mechanisms of resistance to intact AAV capsid molecules The anti-AAV intact capsid molecule in this disclosure can be a complete antibody or a low molecular weight antibody (an antibody with a molecular weight smaller than that of a complete antibody).

[0059] In the case of a complete antibody-AAV capsid molecule in this disclosure, there are no particular restrictions on its isotype, such as IgG (IgG1, IgG2, IgG3, IgG4), IgA (IgA1, IgA2), IgM, IgD, and IgE.

[0060] In the case of a low molecular weight antibody when the intact anti-AAV capsid molecule in this disclosure is a low molecular weight antibody, specific examples include Fab antibody (approximately 55 kDa), Fab' antibody (approximately 55 kDa), and F(ab'). 2Antibodies (approximately 110 kDa), Fv antibodies (approximately 25 kDa), scFv antibodies (approximately 25 kDa), dsFv antibodies (approximately 25 kDa), scFv-Fc antibodies (approximately 105 kDa), dsFv-Fc antibodies (approximately 105 kDa), Bis-scFv antibodies (approximately 50 kDa), micro antibodies (approximately 80 kDa), bisomic antibodies (approximately 55 kDa), trisomic antibodies (approximately 75 kDa), tetrasomic antibodies (approximately 100 kDa), etc.

[0061] Furthermore, the complete anti-AAV capsid molecule disclosed herein only needs to have a heavy chain variable region containing a defined heavy chain complementarity-determining region and a light chain variable region containing a defined light chain complementarity-determining region, and also includes multivalent specific antibodies (e.g., bispecific antibodies). These fragment antibodies and multispecific antibodies can be prepared according to previously known methods.

[0062] The intact anti-AAV capsid molecule disclosed in this article is typically an isolated monoclonal antibody. There are no particular limitations on the method of preparing the monoclonal antibody; for example, it can be prepared by the hybridoma method disclosed in: "Kohler G, Milstein C., Nature. 1975 Aug 7; 256(5517): 495-497."; the recombinant method described in U.S. Patent No. 4,816,567; the method of isolation from a phage antibody library described in "Clackson et al., Nature. 1991 Aug 15; 352(6336): 624-628." or "Marks et al., J Mol Biol. 1991 Dec 5; 222(3): 581-597."; the method disclosed in "Protein Laboratory Manual, Yotsusha (2003): 92-96."; etc.

[0063] 1-7. Other components The complete anti-AAV capsid molecule disclosed herein only needs to have a higher binding affinity to the complete AAV capsid compared to the empty AAV capsid, and may also include other components besides antibody constituent elements (the aforementioned complementarity-determining region, the aforementioned framework, and the portions outside the complementarity-determining region and framework that constitute the aforementioned “1-6. Specific manner of the anti-AAV complete capsid molecule”).

[0064] Examples of complete anti-AAV capsid molecules in this disclosure, including those containing other components, include conjugated antibodies obtained by incorporating various compounds such as polyethylene glycol, radioactive substances, and toxins as other components; antibodies obtained by incorporating modified sugar chains as other components; and fusion antibodies obtained by fusing other proteins as other components.

[0065] Preferred examples of other components include hinge regions, spacers, purification tags for purifying the intact anti-AAV capsid molecules of this disclosure, and immobilization modification groups for immobilizing the intact anti-AAV capsid molecules of this disclosure onto a solid phase. These components can be used individually or in combination of two or more.

[0066] There is no particular limitation on the number of amino acid residues constituting the hinge region; for example, 1 to 25 amino acids can be mentioned, with 5 to 20 amino acids being preferred. Specific examples of hinge regions include EPKTPKPQ, AHHSEDPS, and EPTPPQPQPQPQPQPNPTTE.

[0067] As a spacer region, a sequence of appropriate length can be selected, such as 1 to 6 amino acids, preferably 2 to 5 amino acids.

[0068] Purification tags bind directly or indirectly to the constituent elements of the antibody, either through a linker group or otherwise. Examples of purification tags include histidine tags (oligohistidines composed of 6 or more histidine residues, preferably 6 to 10 residues). These tags reversibly bind to components with nickel (Ni) on their surface. 2+ ), cobalt (Co) 2+ (e.g., chelate with the solid phase of metal ions that serve as the central metal). Strep-Tag (a tag composed of WSHPQFEK, reversibly binds to a solid phase with streptavidin on the surface). Flag-Tag (a tag composed of DYKDDDDK or DYKDHD-G-DYKDHD-I-DYKDDDDK, reversibly binds to a solid phase with Flag-Tag recognition sites on the surface). Spot-Tag (a tag composed of PDRVRAVSHWSS, reversibly binds to a solid phase with Spot-Tag recognition sites on the surface). C-T Epitope tags such as ag (a tag composed of EPEA, which reversibly binds to a solid phase having a C-Tag recognition site on its surface.) and MyC-Tag (a tag composed of EQKLISEEDL, which reversibly binds to a solid phase having an anti-MyC-Tag antibody on its surface.) and protein tags such as glutathione S-transferase tags (which reversibly bind to a solid phase having a glutathione S-transferase recognition site or glutathione on its surface.) and maltose-binding protein tags (which reversibly bind to a solid phase having a maltose-binding protein recognition site on its surface.) can be used individually or in combination with two or more other purification tags. When using two or more of these purification tags in combination, the purification tags can be bound through suitable spacers.

[0069] Immobilization involves the binding of modifying groups directly or indirectly, via any linker group, to the constituent elements of the antibody. Examples of modification groups for immobilization include amino groups (groups that, together with the carboxyl group of a polypeptide, form a carbamoyl group; immobilized on a solid phase with an active ester group or epoxy group fixed on its surface via amino groups), thiol groups (immobilized on a solid phase modified with a maleimide group via Michael addition reaction), cyclopentadienyl groups (immobilized on a solid phase modified with a quinone group via Diels-Alder reaction), biotin groups (immobilized on a solid phase modified with an avidin group via biotin-avidin interaction), oxyamino groups (immobilized on a solid phase modified with a formyl group via Schiff base), cysteine ​​residues (immobilized on a solid phase modified with a formyl group via the formation of a thiazoline ring, or immobilized on a solid phase modified with a benzyl thioester via transesterification), and modification groups based on the carboxyl group of 2-(2-pyridyldithio)-ethylamine (PDEA) (immobilized on a solid phase modified with a thiol group via disulfide bond), etc.

[0070] The aforementioned purification tags or immobilization modification groups can be introduced into the constituent elements of the antibody by conventional methods, thereby enabling the synthesis of the complete anti-AAV capsid molecule of this disclosure.

[0071] 2. Intact capsid binding of AAV with affinity solid phase The AAV intact capsid binding affinity solid phase disclosed herein comprises the aforementioned "1. anti-AAV intact capsid molecule" and a solid phase material (also referred to as "solid phase" in this specification) that immobilizes the anti-AAV intact capsid molecule.

[0072] There are no particular limitations on the material of the solid phase material. Examples include resins (agarose, agarose gel (Sepharose), dextran, silica gel, polyacrylamide, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, (meth)acrylic polymers, fluororesins, metal complex resins, etc.), glass, metals, and magnetic materials.

[0073] There are no particular limitations on the shape of the solid phase material, and it can be appropriately determined according to the way the AAV is used in conjunction with the affinity solid phase in the complete capsid of this disclosure. Examples of solid phase shapes include plate-like, particle-like, and fibrous forms.

[0074] When the solid material is in the shape of a plate, it can form a chip, or the bottom surface of a container, hole, etc. When the solid material is in the shape of a particle or fiber, it can also be a form that fills a pillar.

[0075] In the affinity solid phase for binding the complete AAV capsid in this disclosure, the immobilization form of the anti-AAV complete capsid molecule is as described in the above description of the immobilization modification groups for "1-7. Other components".

[0076] 3. Methods for capturing complete AAV capsids The method for capturing the complete AAV capsid in this disclosure includes step 1, which involves bringing a sample containing an empty AAV capsid and a complete AAV capsid into contact with the affinity solid phase for binding the complete AAV capsid described in "2. Affinity solid phase for binding the complete AAV capsid" above to capture the aforementioned complete AAV capsid.

[0077] The intact AAV capsid molecules immobilized in the AAV binding affinity solid phase exhibit higher binding affinity to the intact AAV capsid compared to the empty AAV capsid. Therefore, in step 1, by contacting the sample containing both the empty and intact AAV capsids with the affinity solid phase, the intact AAV capsid in the sample is selectively captured. The conditions for contacting the sample with the solid phase are not particularly limited; examples include neutral conditions (pH 6.5-7.7 at 25°C) and, for example, at room temperature (preferably 15-30°C) for, for, for, 1 minute to 3 hours.

[0078] The method for capturing intact AAV capsids in this disclosure can be used, for example, as a method for quantifying intact AAV capsids in the aforementioned sample, or as a method for separating intact AAV capsids in the aforementioned sample.

[0079] When the method for capturing the intact AAV capsid of this disclosure is used as a method for quantifying the intact AAV capsid in the aforementioned sample, a step 2 may be further included to quantify the captured intact AAV capsid by enzyme-linked immunosorbent assay (ELISA). Regarding the ELISA method, any known method can be used; specifically, it can be any of the direct method, indirect method, sandwich method, or competitive method. In step 2, the binding of the affinity solid phase to the intact AAV capsid can be detected based on a signal emitted by a signal group such as a fluorescent label, and therefore, the intact AAV capsid can be quantified based on the intensity of this signal.

[0080] When the method for capturing the complete AAV capsid of this disclosure is used as a method for separating the complete AAV capsid from the above-mentioned sample, a step 3 may be included to separate and recover the captured complete AAV capsid. In this case, the aforementioned solid phase material can be used in the form of a column packed with chromatographic material. In step 3, the complete AAV capsid captured by the affinity solid phase is recovered by elution. Examples of elution methods include pH-based elution (e.g., acid elution, alkali elution) and salt concentration-based elution (e.g., elution based on high-concentration salts). The recovered complete AAV capsid fraction can be further purified using any purification method as needed. The recovered complete AAV capsid fraction or its purified fraction can also be pulverized using methods such as freeze-drying, vacuum drying, or spray drying as needed.

[0081] 4. Nucleic acid The nucleic acid encoding the anti-AAV complete capsid molecule described in "1. Anti-AAV complete capsid molecule" above (hereinafter sometimes referred to as "the nucleic acid of this disclosure") can be appropriately prepared and designed by those skilled in the art based on the amino acid sequence of the anti-AAV complete capsid molecule of this disclosure. The nucleic acid of this disclosure can be DNA or RNA.

[0082] The base sequence of the nucleic acid in this disclosure can be appropriately designed by those skilled in the art based on the amino acid sequence of the complementarity-determining region described in “1-3. Complementarity-determining region” above.

[0083] For example, in the base sequence encoding the complete capsid molecule of the first anti-AAV, the base sequences encoding CDR1 (represented by sequence number 1), CDR2 (represented by sequence number 2), and CDR3 (represented by sequence number 3) can be represented by the base sequences represented by sequence numbers 29, 30, and 31, respectively, as well as the base sequences of DNA that hybridizes with DNA composed of base sequences complementary to these base sequences under stringent conditions. Similarly, the base sequences encoding CDR1 (represented by sequence number 4), CDR2 composed of RAS, and CDR3 (represented by sequence number 5) can be represented by the base sequences represented by sequence numbers 32, 33, and 33, respectively, as well as the base sequences of DNA that hybridizes with DNA composed of base sequences complementary to these base sequences under stringent conditions.

[0084] The base sequences encoding the complete capsid molecule of the second anti-AAV antibody include, for example, the base sequences encoding CDR1 (represented by sequence number 6), CDR2 (represented by sequence number 7), and CDR3 (represented by sequence number 8), as well as the base sequences represented by sequence numbers 34, 35, and 36, and the base sequences of DNA that hybridizes under stringent conditions with DNA composed of base sequences complementary to these base sequences. Similarly, the base sequences encoding CDR1 (represented by sequence number 9), CDR2 composed of RAS, and CDR3 (represented by sequence number 10) include, for example, the base sequences represented by sequence numbers 37, 38, and 38, and the base sequences of DNA that hybridizes under stringent conditions with DNA composed of base sequences complementary to these base sequences.

[0085] The base sequences encoding the complete capsid molecule of the third anti-AAV antibody include, for example, the base sequences encoding CDR1 (represented by sequence number 11), CDR2 (represented by sequence number 12), and CDR3 (represented by sequence number 13), as well as the base sequences represented by sequence numbers 39, 40, and 41, and the base sequences of DNA that hybridizes with DNA composed of base sequences complementary to these base sequences under stringent conditions. Similarly, the base sequences encoding CDR1 (represented by sequence number 14), CDR2 composed of QAS, and CDR3 (represented by sequence number 15) include, for example, the base sequences represented by sequence numbers 42, caggcatcc, and 43, and the base sequences of DNA that hybridizes with DNA composed of base sequences complementary to these base sequences under stringent conditions.

[0086] The base sequences encoding the intact capsid molecule of the fourth anti-AAV antibody include, for example, the base sequences encoding CDR1 (represented by sequence number 16), CDR2 (represented by sequence number 17), and CDR3 (represented by sequence number 18), as well as the base sequences represented by sequence numbers 44, 45, and 46, and the base sequences of DNA that hybridizes under stringent conditions with DNA composed of base sequences complementary to these base sequences. Similarly, the base sequences encoding CDR1 (represented by sequence number 19), CDR2 (composed of RAS), and CDR3 (represented by sequence number 20) include, for example, the base sequences represented by sequence numbers 47, 48, and 48, and the base sequences of DNA that hybridizes under stringent conditions with DNA composed of base sequences complementary to these base sequences.

[0087] The base sequences of the nucleic acids in this disclosure can also be appropriately designed by those skilled in the art based on the amino acid sequences of the frame regions described in "1-4. Frame Regions" above, or the amino acid sequences of the variable regions described in "1-5. Specific Examples of Variable Regions" above. For example, in the base sequence encoding the complete capsid molecule of the first anti-AAV, examples of base sequences encoding the heavy chain variable region and the light chain variable region include the base sequences represented by sequence numbers 49 and 50, which encode the amino acid sequences represented by sequence numbers 21 and 22, respectively, and the base sequences of DNA that hybridize under stringent conditions with DNA composed of base sequences complementary to these base sequences. In the base sequence encoding the complete capsid molecule of the second anti-AAV, examples of base sequences encoding the heavy chain variable region and the light chain variable region include the base sequences represented by sequence numbers 51 and 52, which encode the amino acid sequences represented by sequence numbers 23 and 24, respectively, and the base sequences of DNA that hybridize under stringent conditions with DNA composed of base sequences complementary to these base sequences. In the base sequence encoding the complete capsid molecule of the third anti-AAV, examples of base sequences encoding the heavy chain variable region and the light chain variable region include the base sequences represented by sequence numbers 53 and 54, which encode the amino acid sequences represented by sequence numbers 25 and 26, respectively, and the base sequences of DNA that hybridize with DNA composed of base sequences complementary to these base sequences under stringent conditions. In the base sequence encoding the complete capsid molecule of the fourth anti-AAV, examples of base sequences encoding the heavy chain variable region and the light chain variable region include the base sequences represented by sequence numbers 55 and 56, which encode the amino acid sequences represented by sequence numbers 27 and 28, respectively, and the base sequences of DNA that hybridize with DNA composed of base sequences complementary to these base sequences under stringent conditions.

[0088] It should be noted that "strict conditions" refers to incubation at 50°C to 65°C for 4 hours to overnight in a solution containing 0.5% SDS, 5× Denhardt's (Denhardt's: 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400), and 100 μg / ml salmon sperm DNA in 6×SSC (1×SSC: 0.15M NaCl, 0.015M sodium citrate, pH 7.0). Hybridization under strict conditions is specifically performed as follows: A nylon membrane immobilized with a DNA or cDNA library is prepared and blocked at 65°C in a prehybridization solution containing 6×SSC, 0.5% SDS, 5× Denhardt's, and 100 μg / ml salmon sperm DNA. Then, a labeling agent is added... 32Each probe of P was incubated at 65°C overnight. The nylon membrane was washed in 6×SSC at room temperature for 10 minutes, in 2×SSC containing 0.1% SDS at room temperature for 10 minutes, and in 0.2×SSC containing 0.1% SDS at 45°C for 30 minutes. Then, autoradiography was performed to detect DNA that specifically hybridized with the probe.

[0089] The base sequence of the nucleic acid in this disclosure can also be appropriately designed based on the sequence represented by the amino acid sequence in the other components described in “1-7. Other components” above.

[0090] Furthermore, the nucleic acid of this disclosure can also be obtained by using the nucleic acid encoding the aforementioned anti-AAV complete capsid molecule as a template and obtaining at least the region encoding the anti-AAV complete capsid molecule through PCR or the like. Additionally, the nucleic acid of this disclosure can also be artificially synthesized using gene synthesis methods.

[0091] In addition, the nucleic acid in this disclosure may also include at least one of the base sequences encoding the start codon and the stop codon.

[0092] As a method for obtaining the nucleic acids of this disclosure, hybridization-based methods can be cited.

[0093] The nucleic acids disclosed herein include a variety of degenerate nucleic acids derived from codons. Multiple nucleic acids encoding the same amino acid sequence can be easily artificially produced using known genetic engineering techniques. For example, in the genetic engineering production of peptides, when the codons used on the original gene encoding the target protein are used infrequently in the host, the protein expression level is sometimes low. In this case, by optimizing the codon usage frequency corresponding to the host without changing the encoded amino acid sequence, high expression of the target protein can be achieved.

[0094] As an indicator of codon usage frequency, the sum of the host-optimal codon usage frequencies for each codon is sufficient. The optimal codon is defined as the codon with the highest usage frequency among those corresponding to the same amino acid. Regarding codon usage frequency, there are no particular limitations as long as it corresponds to host optimization. For example, the following codons can be cited as examples of optimal codons in *E. coli*. F: Phenylalanine (ttt), L: Leucine (ctg), I: Isoleucine (att), M: Methionine (atg), V: Valine (gtg), Y: Tyrosine (tat), Stop codon (taa), H: Histidine (cat), Q: Glutamine (cag), N: Asparagine (aat), K: Lysine (aaa), D: Aspartic acid (gat), E: Glutamic acid (gaa), S: Serine (agc), P: Proline (ccg), T: Threonine (acc), A: Alanine (gcg), C: Cysteine ​​(tgc), W: Tryptophan (tggg), R: Arginine (cgc), G: Glycine (ggc).

[0095] 5. Expression cassette or recombinant vector The expression cassette or recombinant vector containing the nucleic acid described in “4. Nucleic Acids” above (hereinafter also referred to as “expression cassette of this disclosure” or “recombinant vector of this disclosure”) contains a nucleic acid encoding a polypeptide of this disclosure.

[0096] The expression cassette or recombinant vector of this disclosure can be obtained by linking a promoter and a terminator to the nucleic acid of this disclosure, or by inserting the expression cassette or the nucleic acid of this disclosure into the expression vector.

[0097] In the expression cassette or recombinant vector of this disclosure, in addition to the promoter and terminator, transcriptional elements such as enhancers, CCAAT boxes, TATA boxes, and SPI sites may also be included as needed, acting as regulatory factors. These regulatory factors only need to be able to effectively link with the DNA of this disclosure. Effective linking means that the various regulatory factors regulating the DNA of this disclosure are linked to the DNA of this disclosure in a state that allows them to function effectively in the host cell.

[0098] In the case where the expression cassette or recombinant vector of this disclosure is designed in such a way that the terminal sequence can be cleaved by a protease after the full-length polypeptide containing the protease recognition sequence is temporarily expressed, the expression cassette or recombinant vector of this disclosure can be configured in a combination of a base sequence encoding the protease recognition sequence and a base sequence encoding the N-terminal sequence and / or the C-terminal sequence.

[0099] As expression vectors, vectors constructed for gene recombination purposes based on bacteriophages, plasmids, or viruses that can autonomously proliferate within the host are suitable. Such expression vectors are known, and those skilled in the art can appropriately select and use suitable combinations with host cells. For example, when using microorganisms as hosts, examples include pBluescript (pBS)II SK(-) (manufactured by Stratagene), pSTV vectors (manufactured by Takara Bio Inc.), pUC vectors (manufactured by Takara Bio Inc.), pET vectors (manufactured by Sigma-Aldrich Japan), pGEX vectors (manufactured by Global Life Sciences Technologies Japan KK (Cytiva)), pCold vectors (manufactured by Takara Bio Inc.), pHY300PLK (manufactured by Takara Bio Inc.), pUB110 (Mckenzie, T. et al., 1986, Plasmid 15(2), p.93-103), pBR322 (manufactured by Takara Bio Inc.), pRS403 (manufactured by Stratagene), and pMW218 / 219 (manufactured by NIPPON GENE CO.,LTD.). When algae or microalgae are used as hosts, examples include pUC19 (manufactured by Takara Bio Inc.), P66 (Chlamydomonas Center), P-322 (Chlamydomonas Center), pPha-T1 (see Yangmin Gong et al., Journal of Basic Microbiology, 2011, vol. 51, pp. 666-672), or pJET1 (manufactured by Thermo Fisher Scientific Inc.). When plant cells are used as hosts, examples include pRI vectors (manufactured by Takara Bio Inc.), pBI vectors (manufactured by Clontech Laboratories, Inc.), and IN3 vectors (manufactured by Inplanta Innovations Inc.).

[0100] 6. Transformation A transformed version (also sometimes referred to as "the transformed version of this disclosure") can be obtained by transforming the host using the expression box or recombination vector of this disclosure.

[0101] As a host for the manufacture of the transformant, there are no particular limitations as long as it can be used to introduce genes, reproduce autonomously, and express the genes described in this disclosure. Suitable examples include bacteria belonging to the genera *Escherichia coli*, *Bacillus*, *Pseudomonas putida*, etc.; actinomycetes; yeasts; filamentous fungi, etc.; and animal cells, insect cells, plant cells, etc. Among the above, *Escherichia coli* is particularly preferred.

[0102] Transformations of this disclosed text can be obtained by introducing the expression cassette or recombinant vector of this disclosed text into a host. The location of the nucleic acid introduced into this disclosed text is not particularly limited, as long as the target gene can be expressed; it can be on a plasmid or on the genome. Specific methods for introducing the expression cassette or recombinant vector of this disclosed text include, for example, the recombinant vector method and the genome editing method.

[0103] The conditions for introducing the expression cassette or recombinant vector of this disclosure into a host can be appropriately set according to the type of host. For example, if the host is a microorganism, methods using calcium-treated competent cells, electroporation, protoplasts, and lithium acetate can be used. If the host is an animal cell, methods such as electroporation, calcium phosphate transfection, and liposome transfection can be used. If the host is an insect cell, methods such as calcium phosphate transfection, liposome transfection, and electroporation can be used. If the host is a plant cell, methods such as electroporation, Agrobacterium-mediated transformation, gene gun transfection, and PEG transfection can be used.

[0104] 7. Method for manufacturing intact AAV capsid molecules This disclosure also provides a method for manufacturing the anti-AAV complete capsid molecule described in "1. Anti-AAV complete capsid molecule". Antibodies with higher binding affinity to the complete AAV capsid than the empty AAV capsid, i.e., anti-AAV complete capsid molecules, can be appropriately manufactured through limited mechanical manipulation.

[0105] 7-1. The anti-AAV intact capsid molecule disclosed herein can be appropriately obtained through the following limited mechanical operations: screening an antibody library by affinity selection targeting the intact AAV capsid, and confirming that the selected antibodies bind to the intact AAV capsid more strongly than the empty AAV capsid. Specifically, the anti-AAV intact capsid molecule can be obtained through the following steps: immunizing an animal with the AAV capsid protein as an antigen, obtaining antibody genes from the lymphocytes of the animal, and constructing an antibody library by introducing the obtained antibody genes containing various VH and VL regions into a phage vector; performing a biopanning process to select phage clones that bind to the intact AAV capsid; and confirming that the antibodies contained in the culture supernatant of the selected phage clones bind to the intact AAV capsid more strongly than the empty AAV capsid.

[0106] 7-2. Regarding the anti-AAV complete capsid molecule of this disclosure, with limited mechanical manipulation, it is possible not only to appropriately manufacture an anti-AAV complete capsid molecule having a CDR composed of an amino acid sequence represented by a specific sequence number, but also to appropriately manufacture an anti-AAV complete capsid molecule having a CDR with a sequence identity of more than 90% and less than 100% with the amino acid sequence represented by the specific sequence number. Specifically, an antibody gene having a highly defined variant sequence (preferably a variant sequence based on a conserved amino acid substitution) with a sequence identity of more than 90% and less than 100% with the amino acid sequence represented by the specific sequence number in the CDR region of the structural domain is introduced into a phage vector to create an antibody phage library. As described in item 7-1 above, phage clones with binding affinity to the complete AAV capsid are selected using biopanning, and it is confirmed that the antibodies produced by the selected phage clones have a higher binding affinity to the complete AAV capsid than to the empty AAV capsid, thereby obtaining the anti-AAV complete capsid molecule of this disclosure.

[0107] 7-3. In a preferred embodiment, the anti-AAV intact capsid molecule of this disclosure can be produced by culturing the aforementioned transformant of this disclosure.

[0108] The culture conditions for the transformants disclosed herein can be appropriately set taking into account the nutritional and physiological characteristics of the host, with liquid culture being a preferred option. Furthermore, in industrial manufacturing cases, aerated and stirred culture is preferred.

[0109] The transformants of this disclosure are cultured, and the culture supernatant or cultured bacterial cells or cells are recovered using methods such as centrifugation. If the polypeptides of this disclosure accumulate in the cultured bacterial cells or cultured cells, the bacterial cells or cells can be treated using mechanical methods such as ultrasound or a French press, or using lysozymes such as lysozyme. If necessary, enzymes such as proteases and surfactants such as sodium dodecyl sulfate (SDS) can be used for solubilization, thereby obtaining a water-soluble fraction containing the intact anti-AAV capsid molecule of this disclosure.

[0110] Alternatively, by selecting a suitable expression vector and host, the expressed anti-AAV intact capsid molecule of this disclosed text can be secreted into the culture medium.

[0111] The culture medium, water-soluble fraction, or protease-treated product containing the complete anti-AAV capsid molecule of this disclosure obtained in the above manner can be directly used for purification, or the complete anti-AAV capsid molecule of this disclosure in the culture medium, water-soluble fraction, or protease-treated product can be concentrated and then used for purification.

[0112] Concentration can be achieved, for example, by vacuum concentration, membrane concentration, salting out, or fractional precipitation based on hydrophilic organic solvents (e.g., methanol, ethanol, and / or acetone).

[0113] The purification of the intact anti-AAV capsid molecules disclosed herein can be performed, for example, by a suitable combination of methods such as gel filtration, hydrophobic chromatography, ion exchange chromatography, and affinity chromatography corresponding to the purification label.

[0114] The intact anti-AAV capsid molecules of this disclosure purified in this manner can also be pulverized as needed using methods such as freeze-drying, vacuum drying, and spray drying.

[0115] Each of the methods disclosed in this specification can be combined with any other feature disclosed in this specification.

[0116] Example The following embodiments illustrate the invention in more detail, but the various components and combinations thereof in each embodiment are merely examples, and appropriate additions, omissions, substitutions, and other modifications can be made to the components without departing from the spirit of the invention. This disclosure is not limited to the embodiments, but only to the scope of the claims.

[0117] [Experimental Example 1] (1) Preparation of antibody library Rabbits were immunized with proteins excised from the AAV capsid protein to induce antibody production. Total RNA was obtained from the spleen, and a cDNA library was constructed using reverse transcription-polymerase chain reaction. Next, using primers, genes in the variable regions (VH domain) of the heavy chain (H chain) and the variable regions (VL domain) of the light chain (L chain) were amplified by PCR. Then, to insert these PCR products into a phage vector, restriction endonuclease-based treatments were performed. Additionally, the phage vector was also treated with specified restriction endonucleases, and the genes treated with the restriction endonucleases were inserted into the restriction endonuclease-treated phage vector. This recombinant phage vector was transformed into *E. coli* TG-1 and inoculated into 10 ml of 2×YT medium (containing 1% glucose and 50 mg / L ampicillin). The culture was incubated overnight at 37°C and 200 rpm in a 200 ml Erlenmeyer flask (pre-culture).

[0118] The pre-culture medium was inoculated into 50 ml of 2×YT medium (containing 1% glucose and 50 mg / L ampicillin) with an OD600 of 0.1 and cultured at 30°C with shaking at 200 rpm. After incubation to approximately OD 1.0, helper phage VCSM13 was added to the culture medium with a multiplicity of infection (MOI) of 20 and incubated at 37°C for 30 minutes. The mixture was then centrifuged at 3000g for 10 minutes at 37°C, the supernatant was removed, and the phage was gently resuspended in 50 ml of 2×YT medium (containing 50 mg / L ampicillin and 35 mg / L kanamycin). The mixture was shaken at 30°C for at least 12 hours, producing phages that presented single-chain antibodies in the culture supernatant. The supernatant was recovered by centrifugation, concentrated by PEG precipitation, and a library of phages presenting single-chain antibodies was obtained in 1 ml of PBS.

[0119] (2) Biological panning Intact AAV9 capsid samples were physically adsorbed onto immunotubes in a buffer solution (137 mmol / L NaCl, 8.1 mmol / L Na₂HPO₄, 2.68 mmol / L KCl, 1.47 mmol / L KH₂PO₄, pH 7.4). The tubes were then blocked with a solution prepared by dissolving and dispersing BSA at a concentration of 2% in the buffer solution (137 mmol / L NaCl, 8.1 mmol / L Na₂HPO₄, 2.68 mmol / L KCl, 1.47 mmol / L KH₂PO₄, pH 7.4). After incubation for a period of time, the tubes were washed three times with a buffer solution containing 0.1% Tween 20 (137 mmol / L NaCl, 8.1 mmol / L Na₂HPO₄, 2.68 mmol / L KCl, 1.47 mmol / L KH₂PO₄, pH 7.4).

[0120] Add the phage library (approximately 1.0 × 10⁻⁶) prepared in (1) above to the tube. 11 Dissolve and disperse the PFU in a buffer containing 2% BSA (137 mmol / L NaCl, 8.1 mmol / L Na₂HPO₄, 2.68 mmol / L KCl, 1.47 mmol / L KH₂PO₄, pH 7.4) and incubate at 25°C for 1 hour. After incubation, remove the solution and wash the tube 5 times with a buffer containing 0.1% Tween 20 (137 mmol / L NaCl, 8.1 mmol / L Na₂HPO₄, 2.68 mmol / L KCl, 1.47 mmol / L KH₂PO₄, pH 7.4). Add 0.1 M glycine hydrochloride (Glycine-HCl) (pH 2.2) and incubate at 25°C for 10 minutes. Then, recover and neutralize the solution in the tube, and infect E. coli with TG-1. E. coli TG-1 infected with phages that present single-chain antibodies was plated on LB agar medium (with ampicillin added to a final concentration of 50 mg / L) and incubated overnight at 37°C to form colonies. Ninety-six clones were then randomly selected.

[0121] (3) Experimental steps 1. Add a solution (PBS) containing 0.1 μg of AAV to a Maxisorp 96-well plate and incubate at room temperature for 1 hour. For AAV samples, for AAV9, use either a sample containing an intact capsid or an empty capsid, i.e., “AAV9 intact capsid sample” and “AAV9 empty capsid sample”.

[0122] 2. Wash the wells twice with PBS.

[0123] 3. Add 2% BSA blocking solution to the culture plate and shake and stir at 25°C for 1 hour.

[0124] 4. Wash the wells three times with PBST (0.1% Tween).

[0125] 5. The 96 clones selected in (2) above were cultured at 37°C for 24 hours using 2YT culture. The anti-AAV-scFv contained in the culture supernatant was purified and quantified using His MultiTrap FF according to the protocol. Among the 96 clones, the first anti-AAV-scFv[1], the second anti-AAV-scFv[2], the third anti-AAV-scFv[3], and the fourth anti-AAV-scFv[4] were composed of the amino acid sequence of the variable region shown in Table 2A, the amino acid sequence of the linker and the amino acid sequence of the tag shown in Table 2B. Any anti-AAV-scFv is composed of the heavy chain variable region, the linker, the light chain variable region and the tag in sequence from the N-terminus. The aqueous solutions of these anti-AAV-scFv[1]~[4] were added to the culture plate and shaken at 25°C for 1 hour (pH is about 7).

[0126] [Table 2A] [Table 2B] 6. Wash the wells three times with PBST (0.1% Tween).

[0127] 7. Add the HRP-modified His-Tag antibody to the culture plate and vortex at 25°C for 1 hour.

[0128] 8. Wash the wells three times with PBST (0.1% Tween).

[0129] 9. Add the substrate for HRP enzyme (1-Step Ultra TMB-substrate solution).

[0130] 10. After incubating at room temperature until the color develops, add sulfuric acid and hydrochloric acid to stop the reaction.

[0131] 11. Measure the absorbance at 450 nm and 650 nm for each well. Subtract the absorbance at 650 nm from the absorbance at 450 nm as the absorbance value of the ELISA signal.

[0132] 12. Derive the ratio (IF / IE) of the absorbance of the intact capsid sample to the absorbance (IE) of the empty capsid sample, and the ratio (IE / IB) of the absorbance of the empty capsid sample to the absorbance (IB) of the blank control (BSA).

[0133] result The ratios IF / IE and IE / IB obtained for the first anti-AAV-scFv[1], the second anti-AAV-scFv[2], the third anti-AAV-scFv[3], and the fourth anti-AAV-scFv[4] are shown in Table 3 below.

[0134] [Table 3] As shown in Table 3, the anti-AAV-scFv of [1] to [4] all showed specific binding to the intact capsid of AAV.

[0135] [Experimental Example 2] Using the second antibody AAV-scFv[2], the amount of AAV added to the culture plate in step “1” of Experimental Example 1 was changed. Otherwise, the same procedure as in Experimental Example 1 was performed, and the absorbance at 450 nm and 650 nm was measured for each well. The value obtained by subtracting the absorbance at 650 nm from the absorbance at 450 nm was taken as the absorbance value of the ELISA signal. Figure 1 The graph shows absorbance on the vertical axis and the amount of added AAV on the horizontal axis.

[0136] like Figure 1 As shown, there was no substantial difference in absorbance for the blank control (BSA) and the empty capsid (empty), and no concentration dependence was observed. In contrast, for the intact capsid (intact), the absorbance increased in a concentration-dependent manner. That is, it was observed that the intact AAV capsid could be selectively quantified using anti-AAV-scFv.

Claims

1. Anti-AAV intact capsid molecule, which is an antibody that has a higher binding affinity to the intact AAV capsid than the empty AAV capsid.

2. Anti-AAV intact capsid molecule, which includes: The heavy chain variable region, comprising CDR1 (composed of the amino acid sequence represented by Serial No. 1), CDR2 (composed of the amino acid sequence represented by Serial No. 2), and CDR3 (composed of the amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 3), serves as the heavy chain complementarity-determining region; and The light chain variable region, which includes CDR1 composed of the amino acid sequence represented by sequence number 4, CDR2 composed of RAS, and CDR3 composed of an amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by sequence number 5, serves as the light chain complementarity determination region.

3. Anti-AAV intact capsid molecules, which include: The heavy chain variable region, comprising CDR1 (composed of the amino acid sequence represented by sequence number 6), CDR2 (composed of the amino acid sequence represented by sequence number 7), and CDR3 (composed of the amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by sequence number 8) as a heavy chain complementarity-determining region; and The light chain variable region, which includes CDR1 composed of the amino acid sequence represented by sequence number 9, CDR2 composed of RAS, and CDR3 composed of an amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by sequence number 10, serves as the light chain complementarity determination region.

4. Anti-AAV intact capsid molecules, which include: The heavy chain variable region, comprising CDR1 (composed of the amino acid sequence represented by Serial No. 11), CDR2 (composed of the amino acid sequence represented by Serial No. 12), and CDR3 (composed of the amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 13) as a heavy chain complementarity-determining region; and The light chain variable region, which includes CDR1 composed of the amino acid sequence represented by Serial No. 14, CDR2 composed of QAS, and CDR3 composed of an amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by Serial No. 15, serves as the light chain complementarity determination region.

5. Anti-AAV intact capsid molecules, which include: The heavy chain variable region, comprising CDR1 (composed of the amino acid sequence represented by SEQ ID NO. 16), CDR2 (composed of the amino acid sequence represented by SEQ ID NO. 17), and CDR3 (composed of the amino acid sequence having more than 90% sequence identity with the amino acid sequence represented by SEQ ID NO. 18) as a heavy chain complementarity-determining region; and The light chain variable region, which includes CDR1 composed of the amino acid sequence represented by sequence number 19, CDR2 composed of RAS, and CDR3 composed of the amino acid sequence represented by sequence number 20, serves as the light chain complementarity-determining region.

6. The anti-AAV intact capsid molecule as described in any one of claims 2 to 5, wherein, The heavy chain variable region includes: FR1 is composed of amino acid sequences that have more than 70% sequence identity with the amino acid sequences at positions 1-24 of sequence number 21, sequence number 23, sequence number 25, or sequence number 27. FR2 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 33-49 of sequence number 21, positions 34-50 of sequence number 23, positions 34-50 of sequence number 25, or positions 34-50 of sequence number 27. FR3 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 59-95 of sequence number 21, positions 60-96 of sequence number 23, positions 59-95 of sequence number 25, or positions 60-96 of sequence number 27. FR4 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 109-119 of sequence number 21, positions 109-119 of sequence number 23, positions 107-117 of sequence number 25, or positions 113-123 of sequence number 27. The light chain variable region includes: FR1 is composed of amino acid sequences that have more than 70% sequence identity with the amino acid sequences at positions 1-26 of sequence number 22, sequence number 24, sequence number 26, or sequence number 28. FR2 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 33-49 of sequence number 22, sequence number 24, sequence number 26, or sequence number 28. FR3 is composed of amino acid sequences that share more than 70% sequence identity with the amino acid sequences at positions 53-88 of sequence number 22, sequence number 24, sequence number 26, or sequence number 28. FR4 is composed of amino acid sequences that have more than 70% sequence identity with the amino acid sequences at positions 101-110 of sequence number 22, positions 101-110 of sequence number 24, positions 101-109 of sequence number 26, or positions 98-107 of sequence number 28.

7. The anti-AAV intact capsid molecule as described in claim 2, wherein, The heavy chain variable region contains the amino acid sequence represented by sequence number 21, and the light chain variable region contains the amino acid sequence represented by sequence number 22.

8. The anti-AAV intact capsid molecule as described in claim 3, wherein, The heavy chain variable region contains the amino acid sequence represented by sequence number 23, and the light chain variable region contains the amino acid sequence represented by sequence number 24.

9. The anti-AAV intact capsid molecule as described in claim 4, wherein, The heavy chain variable region contains the amino acid sequence represented by sequence number 25, and the light chain variable region contains the amino acid sequence represented by sequence number 26.

10. The anti-AAV intact capsid molecule as described in claim 5, wherein, The heavy chain variable region contains the amino acid sequence represented by sequence number 27, and the light chain variable region contains the amino acid sequence represented by sequence number 28.

11. The anti-AAV intact capsid molecule as described in any one of claims 1 to 5, wherein it is an IgG antibody, a Fab antibody, a Fab' antibody, or an F(ab') antibody. 2 Antibodies, Fv antibodies, scFv antibodies, dsFv antibodies, scFv-Fc antibodies, dsFv-Fc antibodies, Bis-scFv antibodies, micro antibodies, disomic antibodies, trisomic antibodies, or tetrasomic antibodies.

12. An affinity solid phase for binding AAV intact capsids, comprising an anti-AAV intact capsid molecule as described in any one of claims 1 to 5, and a solid phase material for immobilizing the anti-AAV intact capsid molecule.

13. A method for capturing an intact AAV capsid, the method comprising step 1 of capturing the intact AAV capsid by bringing a sample containing an empty AAV capsid and an intact AAV capsid into contact with an affinity solid phase of the intact AAV capsid of claim 12.

14. The method of claim 13, further comprising step 2 of quantifying the captured intact AAV capsid by enzyme-linked immunosorbent assay (ELISA).

15. A nucleic acid encoding an anti-AAV intact capsid molecule as described in any one of claims 1 to 5.

16. An expression cassette or recombinant vector comprising the nucleic acid of claim 15.

17. A transformant obtained by transforming a host using the expression cassette or recombinant vector of claim 16.

18. A method for manufacturing an intact anti-AAV capsid molecule, the method comprising the step of culturing the transformant of claim 17.

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