Affinity polypeptide for full capsids of aav

Abstract

The purpose of the present disclosure is to provide an affinity molecule that exhibits a higher affinity to AAV full capsids than capsids empty of AAV. An anti-AAV full capsid molecule of the present invention includes, as a heavy chain complementarity-determining region, a heavy chain variable region including CDR1 represented by SEQ ID NO: 6, CDR2 represented by SEQ ID NO: 7, and CDR3 represented by SEQ ID NO: 8, and includes, as a light chain complementarity-determining region, a light chain variable region including CDR1 represented by SEQ ID NO: 9, CDR2 formed of RAS, and CDR3 represented by SEQ ID NO: 10. The anti-AAV full capsid molecule exhibits a higher affinity to AVV full capsids than capsids empty of AAV.

Description

Affinity polypeptide for AAV full capsid

[0001] The present disclosure relates to affinity polypeptides for the full capsid of adeno-associated virus (AAV).

[0002] In the manufacturing process of adeno-associated virus (AAV) used as a gene therapy drug, a mixture of full capsids, which contain foreign DNA and serve as an active pharmaceutical ingredient, and empty capsids is obtained. In this manufacturing process, a method for quantifying the amount of full capsids from this mixture is required.

[0003] Specific methods for quantifying full capsids include quantitative PCR (Patent Document 1) and coherent scattering mass spectrometry (Patent Document 2).

[0004] CN101426935BWO2021 / 191079

[0005] Methods for quantifying full capsids using quantitative PCR or interference scattering mass spectrometry require expensive dedicated equipment and lack simplicity and versatility.

[0006] To quantify full capsids simply and generally, it is desirable to use an affinity molecule. Such an affinity molecule suitable for quantifying full capsids must have higher binding affinity for AAV full capsids than for AAV empty capsids (i.e., selective binding affinity for AAV full capsids).

[0007] Therefore, an object of the present disclosure is to provide an affinity molecule that has higher binding affinity to AAV full capsids than to AAV empty capsids.

[0008] As a result of extensive research, the present inventors have discovered an anti-AAV full capsid molecule that has stronger binding affinity to AAV full capsids than to AAV empty capsids. The present disclosure was completed through further research based on this finding.

[0009] That is, the present disclosure provides the following aspects of the invention: Item 1. An anti-AAV full capsid molecule, which is an antibody having higher binding affinity for AAV full capsids than for AAV empty capsids. Item 2. An anti-AAV full capsid molecule comprising: a heavy chain variable region comprising, as heavy chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable region comprising, as light chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, CDR2 consisting of RAS, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5. Item 3. Item 3. An anti-AAV full capsid molecule comprising: a heavy chain variable region comprising, as heavy chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:6, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:7, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:8; and a light chain variable region comprising, as light chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:9, CDR2 consisting of RAS, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:10. An anti-AAV full capsid molecule comprising: a heavy chain variable region comprising, as heavy chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 13; and a light chain variable region comprising, as light chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, CDR2 consisting of QAS, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 15.Item 5. An anti-AAV full capsid molecule comprising: a heavy chain variable region comprising, as heavy chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 16, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 17, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 18; and a light chain variable region comprising, as light chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 19, CDR2 consisting of RAS, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 20.Item 6. The heavy chain variable region comprises: FR1 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 1 to 24 of SEQ ID NO:21, positions 1 to 24 of SEQ ID NO:23, positions 1 to 24 of SEQ ID NO:25, or positions 1 to 24 of SEQ ID NO:27; FR2 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 33 to 49 of SEQ ID NO:21, positions 34 to 50 of SEQ ID NO:23, positions 34 to 50 of SEQ ID NO:25, or positions 34 to 50 of SEQ ID NO:27; FR3 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 59 to 95 of SEQ ID NO:21, positions 60 to 96 of SEQ ID NO:23, positions 59 to 95 of SEQ ID NO:25, or positions 60 to 96 of SEQ ID NO:27; and the light chain variable region comprises an FR4 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 109 to 119 of SEQ ID NO:21, positions 109 to 119 of SEQ ID NO:23, positions 107 to 117 of SEQ ID NO:25, or positions 113 to 123 of SEQ ID NO:27, wherein the light chain variable region comprises: an FR1 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 1 to 26 of SEQ ID NO:22, positions 1 to 26 of SEQ ID NO:24, positions 1 to 26 of SEQ ID NO:26, or positions 1 to 26 of SEQ ID NO:28; an FR2 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 33 to 49 of SEQ ID NO:22, positions 33 to 49 of SEQ ID NO:24, positions 33 to 49 of SEQ ID NO:26, or positions 33 to 49 of SEQ ID NO:28; Item 7. The anti-AAV full capsid molecule of any of Items 2 to 5, comprising an FR3 consisting of an amino acid sequence having 70% or more sequence identity to the amino acid sequence of positions 53 to 88 of SEQ ID NO: 22, positions 53 to 88 of SEQ ID NO: 24, positions 53 to 88 of SEQ ID NO: 26, or positions 53 to 88 of SEQ ID NO: 28, and an FR4 consisting of an amino acid sequence having 70% or more sequence identity to the amino acid sequence of positions 101 to 110 of SEQ ID NO: 22, positions 101 to 110 of SEQ ID NO: 24, positions 101 to 109 of SEQ ID NO: 26, or positions 98 to 107 of SEQ ID NO: 28. Item 7. The anti-AAV full capsid molecule of Items 2, wherein the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 21 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 22.Item 8. The anti-AAV full capsid molecule of Item 3, wherein the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 23, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 24. Item 9. The anti-AAV full capsid molecule of Item 4, wherein the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 26. Item 10. The anti-AAV full capsid molecule of Item 5, wherein the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 28. Item 11. An IgG antibody, a Fab antibody, a Fab' antibody, or an F(ab'). 2 Item 12. An affinity solid phase for binding to AAV full capsids, comprising the anti-AAV full capsid molecule of any of Items 1 to 11 and a solid phase material to which the anti-AAV full capsid molecule is immobilized. Item 13. A method for capturing AAV full capsids, comprising Step 1 of contacting a sample containing AAV empty capsids and AAV full capsids with the affinity solid phase for binding to AAV full capsids of Item 12, and capturing the AAV full capsids. Item 14. Item 13. The method according to Item 13, further comprising a step 2 of quantifying the captured AAV full capsid by enzyme-linked immunosorbent assay. Item 15. A nucleic acid encoding the anti-AAV full capsid molecule of any one of Items 1 to 11. Item 16. An expression cassette or recombinant vector comprising the nucleic acid of Item 15. Item 17. A transformant obtained by transforming a host with the expression cassette or recombinant vector of Item 16. Item 18. A method for producing an anti-AAV full capsid molecule, comprising a step of culturing the transformant of Item 17.

[0010] According to the present disclosure, there is provided an anti-AAV full capsid molecule that has a higher binding affinity to AAV full capsids than to AAV empty capsids, thereby enabling the quantification of full capsids using the anti-AAV full capsid molecule.

[0011] 1 shows the relationship between the amount of AAV9 full capsid and the ELISA signal (absorbance) using the anti-AAV full capsid molecule (anti-AAV-scFv) of the present disclosure.

[0012] 1. Anti-AAV full capsid molecule The anti-AAV full capsid molecule of the present disclosure is an antibody that has a higher binding affinity to AAV full capsids than to AAV empty capsids. That is, the anti-AAV full capsid molecule of the present disclosure is a polypeptide that has a higher binding affinity to AAV full capsids than to AAV empty capsids, and has a heavy chain variable region including a heavy chain complementarity-determining region and a light chain variable region including a light chain complementarity-determining region that show a higher binding affinity to AAV full capsids than to AAV empty capsids. Preferably, the anti-AAV full capsid molecule of the present disclosure is a polypeptide that has a higher binding affinity to AAV full capsids than to AAV empty capsids, and has a heavy chain variable region including a predetermined heavy chain complementarity-determining region and a light chain variable region including a predetermined light chain complementarity-determining region.

[0013] 1-1. Target The AAV serotype targeted by the anti-AAV full capsid molecule of the present disclosure is not particularly limited. Specific serotypes include AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12, and AAV13, preferably AAV9.

[0014] An AAV empty capsid is an empty outer shell of an AAV that does not contain any nucleic acid inside. A typical example of an AAV empty capsid is a vesicle used to package a target gene (foreign gene) to be delivered to cells of a therapeutic target.

[0015] An AAV full capsid is an AAV capsid containing a nucleic acid therein. Examples of the nucleic acid include the AAV viral genome, nucleic acid medicines (antisense oligonucleotides, RNAi, aptamers, decoys, etc.), and vectors into which genes for gene therapy have been introduced.

[0016] 1-2. Binding Property The anti-AAV full capsid molecule of the present disclosure has higher binding property to AAV full capsid than to AAV empty capsid.

[0017] The fact that the anti-AAV full capsid molecule of the present disclosure has a higher binding affinity to AAV full capsids than to AAV empty capsids can be confirmed by the fact that, when the anti-AAV full capsid molecule of the present disclosure is contacted with an AAV empty capsid or an AAV full capsid under the same conditions, the amount of AAV full capsid bound is greater than the amount of AAV empty capsid bound. For example, this can be confirmed by preparing the anti-AAV full capsid molecule of the present disclosure in a form having an affinity tag attached, contacting it with an AAV empty capsid or an AAV full capsid under the same conditions, capturing the anti-AAV full capsid molecule bound to the capsid with an antibody (anti-affinity tag antibody) specific to the affinity tag and having a signal group (e.g., a fluorescent group), and measuring the signal intensity (e.g., absorbance based on the fluorescent group) of the anti-affinity tag antibody that captured the anti-AAV full capsid molecule.

[0018] A specific level of binding affinity to AAV full capsids is, when the anti-AAV full capsid molecule is contacted with an AAV capsid at pH 7 (25°C) for 1 hour at 25°C, the binding affinity to AAV full capsids based on the above absorbance is 1.5 times or more, specifically 1.5 to 5 times, preferably 2 to 5 times, more preferably 2.5 to 5 times, even more preferably 3 to 5 times, 3.5 to 5 times, and even more preferably 4 to 5 times, the binding affinity to AAV empty capsids.

[0019] 1-3. Complementarity-Determining Regions The anti-AAV full capsid molecule of the present disclosure 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).

[0020] In a preferred embodiment, the anti-AAV full capsid molecule of the present disclosure comprises a first anti-AAV full capsid molecule shown in [1] of Table 1, a second anti-AAV full capsid molecule shown in [2], a third anti-AAV full capsid molecule shown in [3], and a fourth anti-AAV full capsid molecule shown in [4].

[0021]

[0022] The first anti-AAV full capsid molecule comprises a heavy chain variable region comprising, as heavy chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable region comprising, as light chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, CDR2 consisting of RAS, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5.

[0023] The second anti-AAV full capsid molecule comprises a heavy chain variable region comprising, as heavy chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 6, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 7, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, and a light chain variable region comprising, as light chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 9, CDR2 consisting of RAS, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. Among the anti-AAV full capsid molecules of the present disclosure, the second anti-AAV full capsid molecule is preferred in that it has remarkable binding specificity for AAV full capsids.

[0024] The third anti-AAV full capsid molecule comprises a heavy chain variable region comprising, as heavy chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 13; and a light chain variable region comprising, as light chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, CDR2 consisting of QAS, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0025] The fourth anti-AAV full capsid molecule comprises a heavy chain variable region comprising, as heavy chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 16, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 17, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 18; and a light chain variable region comprising, as light chain complementarity determining regions, CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 19, CDR2 consisting of RAS, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 20.

[0026] In the present invention, the term "sequence identity" refers to the amino acid sequence identity value obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) of BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters are set as follows: Gap insertion cost value: 11, Gap extension cost value: 1.

[0027] In the amino acid sequences having a sequence identity of less than 100%, when the mismatched amino acid residue is substituted with another amino acid from the reference amino acid sequence, the substitution is preferably a substitution with a similar amino acid (i.e., a conservative amino acid substitution). Specifically, the following classifications have been established based on the properties of the amino acid side chain, and substitution with an amino acid belonging to the same classification is preferred. 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 Furthermore, the neutral amino acids can be classified into those with polar side chains (asparagine, glutamine, serine, threonine, tyrosine, cysteine), those with nonpolar side chains (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), those with amide-containing side chains (asparagine, glutamine), those with sulfur-containing side chains (methionine, cysteine), those with aromatic side chains (phenylalanine, tryptophan, tyrosine), those with hydroxyl-containing side chains (serine, threonine, tyrosine), and those with aliphatic side chains (alanine, leucine, isoleucine, valine).

[0028] Methods for substituting a predetermined amino acid in an amino acid sequence with another amino acid include, for example, 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., are known, and amino acid substitutions can be made in the amino acid sequence of a CDR using this site-directed mutagenesis method. Methods for substituting with other amino acids also include the library technology described in WO2005 / 080432.

[0029] 1-4. Framework Regions The framework regions of the heavy chain variable region and light chain variable region of the first to fourth anti-AAV full capsid molecules are not particularly limited, as long as the anti-AAV full capsid molecules have higher binding affinity to AAV full capsids than to AAV empty capsids, and framework regions (FRs) of antibodies from any animal (human and non-human, preferably rabbit) can be used.

[0030] For example, the first to fourth anti-AAV full capsid molecules preferably comprise a heavy chain variable region comprising: FR1 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 1 to 24 of SEQ ID NO:21, positions 1 to 24 of SEQ ID NO:23, positions 1 to 24 of SEQ ID NO:25, or positions 1 to 24 of SEQ ID NO:27; FR2 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 33 to 49 of SEQ ID NO:21, positions 34 to 50 of SEQ ID NO:23, positions 34 to 50 of SEQ ID NO:25, or positions 34 to 50 of SEQ ID NO:27; FR3 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 59 to 95 of SEQ ID NO:21, positions 60 to 96 of SEQ ID NO:23, positions 59 to 95 of SEQ ID NO:25, or positions 60 to 96 of SEQ ID NO:27; and the light chain variable region comprises an FR4 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 109 to 119 of SEQ ID NO:21, positions 109 to 119 of SEQ ID NO:23, positions 107 to 117 of SEQ ID NO:25, or positions 113 to 123 of SEQ ID NO:27, wherein the light chain variable region comprises: an FR1 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 1 to 26 of SEQ ID NO:22, positions 1 to 26 of SEQ ID NO:24, positions 1 to 26 of SEQ ID NO:26, or positions 1 to 26 of SEQ ID NO:28; an FR2 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 33 to 49 of SEQ ID NO:22, positions 33 to 49 of SEQ ID NO:24, positions 33 to 49 of SEQ ID NO:26, or positions 33 to 49 of SEQ ID NO:28; The molecule comprises an FR3 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 53 to 88 of SEQ ID NO:22, positions 53 to 88 of SEQ ID NO:24, positions 53 to 88 of SEQ ID NO:26, or positions 53 to 88 of SEQ ID NO:28, and an FR4 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 101 to 110 of SEQ ID NO:22, positions 101 to 110 of SEQ ID NO:24, positions 101 to 109 of SEQ ID NO:26, or positions 98 to 107 of SEQ ID NO:28, and has higher binding affinity to AAV full capsids than to AAV empty capsids.

[0031] Preferred examples of the above-mentioned sequence identity of 70% or more may vary depending on the full length of the reference amino acid sequence, but include preferably 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, still more preferably 96% or more or 97% or more, and most preferably 100%.

[0032] 1-5. Specific Examples of Variable Regions More preferably, the first anti-AAV full capsid molecule has a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:22.

[0033] The heavy chain variable region of SEQ ID NO: 21 is composed of, from the N-terminus, {FR1 consisting of the amino acid sequence of positions 1 to 24} - {CDR1 consisting of the amino acid sequence of positions 25 to 32 (corresponding to CDR1 of SEQ ID NO: 1)} - {FR2 consisting of the amino acid sequence of positions 33 to 49} - {CDR2 consisting of the amino acid sequence of positions 50 to 58 (corresponding to CDR2 of SEQ ID NO: 2)} - {FR3 consisting of the amino acid sequence of positions 59 to 95} - {CDR3 consisting of the amino acid sequence of positions 96 to 108 (corresponding to CDR3 of SEQ ID NO: 3)} - {FR4 consisting of the amino acid sequence of positions 109 to 119}. The light chain variable region of SEQ ID NO:22 is composed of, from the N-terminus, {FR1 consisting of the sequence of amino acids 1 to 26} - {CDR1 consisting of the sequence of amino acids 27 to 32 (corresponding to CDR1 of SEQ ID NO:4)} - {FR2 consisting of the sequence of amino acids 33 to 49} - {CDR2 consisting of the sequence of amino acids 50 to 52} - {FR3 consisting of the sequence of amino acids 53 to 88 of SEQ ID NO:22} - {CDR3 consisting of the sequence of amino acids 89 to 100 (corresponding to CDR3 of SEQ ID NO:5)} - {FR4 consisting of the sequence of amino acids 101 to 110 of SEQ ID NO:22}.

[0034] More preferably, the second anti-AAV full capsid molecule has a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:24.

[0035] The heavy chain variable region of SEQ ID NO: 23 is composed of, from the N-terminus, {FR1 consisting of the amino acid sequence of positions 1 to 24} - {CDR1 consisting of the amino acid sequence of positions 25 to 33 (corresponding to CDR1 of SEQ ID NO: 6)} - {FR2 consisting of the amino acid sequence of positions 34 to 50} - {CDR2 consisting of the amino acid sequence of positions 51 to 59 (corresponding to CDR2 of SEQ ID NO: 7)} - {FR3 consisting of the amino acid sequence of positions 60 to 96} - {CDR3 consisting of the amino acid sequence of positions 97 to 108 (corresponding to CDR3 of SEQ ID NO: 8)} - {FR4 consisting of the amino acid sequence of positions 109 to 119}. The light chain variable region of SEQ ID NO: 24 is composed of, from the N-terminus, {FR1 consisting of the amino acid sequence of positions 1 to 26} - {CDR1 consisting of the amino acid sequence of positions 27 to 32 (corresponding to CDR1 of SEQ ID NO: 9)} - {FR2 consisting of the amino acid sequence of positions 33 to 49} - {CDR2 consisting of the amino acid sequence of positions 50 to 52} - {FR3 consisting of the amino acid sequence of positions 53 to 88} - {CDR3 consisting of the amino acid sequence of positions 89 to 100 (corresponding to CDR3 of SEQ ID NO: 10)} - {FR4 consisting of the amino acid sequence of positions 101 to 110}.

[0036] More preferably, the third anti-AAV full capsid molecule has a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:25 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:26.

[0037] The heavy chain variable region of SEQ ID NO: 25 is composed of, from the N-terminus, {FR1 consisting of the amino acid sequence of positions 1 to 24} - {CDR1 consisting of the amino acid sequence of positions 25 to 33 (corresponding to CDR1 of SEQ ID NO: 11)} - {FR2 consisting of the amino acid sequence of positions 34 to 50} - {CDR2 consisting of the amino acid sequence of positions 51 to 58 (corresponding to CDR2 of SEQ ID NO: 12)} - {FR3 consisting of the amino acid sequence of positions 59 to 95} - {CDR3 consisting of the amino acid sequence of positions 96 to 106 (corresponding to CDR3 of SEQ ID NO: 13)} - {FR4 consisting of the amino acid sequence of positions 107 to 117}. The light chain variable region of SEQ ID NO: 26 is composed of, from the N-terminus, {FR1 consisting of the amino acid sequence of positions 1 to 26} - {CDR1 consisting of the amino acid sequence of positions 27 to 32 (corresponding to CDR1 of SEQ ID NO: 14)} - {FR2 consisting of the amino acid sequence of positions 33 to 49} - {CDR2 consisting of the amino acid sequence of positions 50 to 52} - {FR3 consisting of the amino acid sequence of positions 53 to 88} - {CDR3 consisting of the amino acid sequence of positions 89 to 100 (corresponding to CDR3 of SEQ ID NO: 15)} - {FR4 consisting of the amino acid sequence of positions 101 to 109}.

[0038] More preferably, the fourth anti-AAV full capsid molecule has a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:27 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:28.

[0039] The heavy chain variable region of SEQ ID NO:27 is composed of, from the N-terminus, {FR1 consisting of the amino acid sequence of positions 1 to 24} - {CDR1 consisting of the amino acid sequence of positions 25 to 33 (corresponding to CDR1 of SEQ ID NO:16)} - {FR2 consisting of the amino acid sequence of positions 34 to 50 of SEQ ID NO:27} - {CDR2 consisting of the amino acid sequence of positions 51 to 59 (corresponding to CDR2 of SEQ ID NO:17)} - {FR3 consisting of the amino acid sequence of positions 60 to 96 of SEQ ID NO:27} - {CDR3 consisting of the amino acid sequence of positions 97 to 112 (corresponding to CDR3 of SEQ ID NO:18)} - {FR4 consisting of the amino acid sequence of positions 113 to 123 of SEQ ID NO:27}. The light chain variable region of SEQ ID NO: 28 is composed of, from the N-terminus, {FR1 consisting of the amino acid sequence of positions 1 to 26} - {CDR1 consisting of the amino acid sequence of positions 27 to 32 (corresponding to CDR1 of SEQ ID NO: 19)} - {FR2 consisting of the amino acid sequence of positions 33 to 49} - {CDR2 consisting of the amino acid sequence of positions 50 to 52} - {FR3 consisting of the amino acid sequence of positions 53 to 88} - {CDR3 consisting of the amino acid sequence of positions 89 to 97 (corresponding to CDR3 of SEQ ID NO: 20)} - {FR4 consisting of the amino acid sequence of positions 98 to 107}.

[0040] 1-6. Specific Forms of Anti-AAV Full Capsid Molecule The anti-AAV full capsid molecule of the present disclosure may be a complete antibody or a small antibody (an antibody having a smaller molecular weight than a complete antibody).

[0041] When the anti-AAV full capsid molecule of the present disclosure is a complete antibody, its isotype is not particularly limited, and examples include IgG (IgG1, IgG2, IgG3, IgG4), IgA (IgA1, IgA2), IgM, IgD, and IgE.

[0042] When the anti-AAV full capsid molecule of the present disclosure is a low molecular weight antibody, specific examples thereof include a Fab antibody (about 55 kDa), a Fab' antibody (about 55 kDa), and a F(ab') antibody. 2Examples of such antibodies include antibodies (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), minibodies (approximately 80 kDa), diabodies (approximately 55 kDa), triabodies (approximately 75 kDa), and tetrabodies (approximately 100 kDa).

[0043] Furthermore, the anti-AAV full capsid molecule of the present disclosure also encompasses multispecific antibodies (e.g., bispecific antibodies) as long as they have a heavy chain variable region containing a predetermined heavy chain complementarity-determining region and a light chain variable region containing a predetermined light chain complementarity-determining region. These antibody fragments and multispecific antibodies can be prepared according to conventionally known methods.

[0044] The anti-AAV full capsid molecules of the present disclosure are typically isolated monoclonal antibodies. The method for producing monoclonal antibodies is not particularly limited, and can be, for example, the hybridoma method described in "Kohler G, Milstein C., Nature. 1975 Aug. 7; 256(5517): 495-497."; the recombinant method described in U.S. Pat. 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."; or the method described in "Protein Experiment Handbook, Yodosha (2003): 92-96."

[0045] 1-7. Other Components The anti-AAV full capsid molecule of the present disclosure may contain components other than the antibody components (the above-described complementarity-determining regions, the above-described framework, and portions other than the complementarity-determining regions and framework that constitute the above-described "1-6. Specific Form of the Anti-AAV Full Capsid Molecule"), so long as it has higher binding affinity to AAV full capsids than to AAV empty capsids.

[0046] Examples of the anti-AAV full capsid molecule of the present disclosure that contains other components include a conjugated antibody to which various compounds such as polyethylene glycol, radioactive substances, and toxins are bound as other components, an antibody to which a modified sugar chain is bound as other components, and a fusion antibody to which another protein is fused as other components.

[0047] Preferred examples of other components include a hinge region, a spacer, a purification tag that enables purification of the anti-AAV full capsid molecule of the present disclosure, an immobilization modification group that enables immobilization of the anti-AAV full capsid molecule of the present disclosure to a solid phase, etc. These components may be used alone or in combination of two or more.

[0048] The number of amino acid residues constituting the hinge region is not particularly limited, but may be, for example, 1 to 25 aa, preferably 5 to 20 aa. Specific examples of the hinge region include EPKTPKPQ, AHHSEDPS, EPTPPQPQPQPQPQPNPTTE, etc.

[0049] As the spacer, a sequence of an appropriate length may be selected, for example, 1 to 6 amino acids, preferably 2 to 5 amino acids.

[0050] The purification tag is bound to the antibody component directly or indirectly via an optional linker group. Examples of the purification tag include a histidine tag (an oligohistidine consisting of 6 or more, preferably 6 to 10, histidine residues), a nickel (Ni 2+ ), cobalt (Co 2+) or the like, which reversibly chelates to a solid phase having on its surface a metal ion serving as a central metal; Strep tag (a tag consisting of WSHPQFEK, which reversibly binds to a solid phase having streptavidin on its surface); Flag tag (a tag consisting of DYKDDDDK or DYKDHD-G-DYKDHD-I-DYKDDDDK, which reversibly binds to a solid phase having a Flag tag recognition site on its surface); Spot tag (a tag consisting of PDRVRAVSHWSS, which reversibly binds to a solid phase having a Spot tag recognition site on its surface); C tag (a tag consisting of EPEA, which reversibly binds to a solid phase having a C tag recognition site on its surface); Examples of suitable purification tags include epitope tags such as Myc tag (a tag consisting 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 tag (which reversibly binds to a solid phase having a glutathione-S-transferase recognition site or glutathione on its surface); and maltose-binding protein tag (which reversibly binds to a solid phase having a maltose-binding protein recognition site on its surface). In other configurations, these purification tags may be used alone or in combination of two or more. When two or more types of these purification tags are used in combination, the purification tags can be attached via an appropriate spacer.

[0051] The modifying group for immobilization is bound to the component of the antibody directly or indirectly via an optional linker group. The modifying group for immobilization includes an amino group (a group that forms a carbamoyl group together with a carboxyl group of a polypeptide; it is immobilized via the amino group to a solid phase having an active ester group or an epoxy group immobilized on its surface), a thiol group (immobilized by a Michael addition reaction to a solid phase whose surface is modified with a maleimide group), a cyclopentadienyl group (immobilized by a Diels-Alder reaction to a solid phase whose surface is modified with a quinone group), a biotinyl group (immobilized by a biotin-avidin interaction to a solid phase whose surface is modified with an avidin group), and the like. by thiol groups), oxyamino groups (immobilized by a Schiff base on a solid phase surface-modified with a formyl group), cysteine ​​residues (immobilized by thiazoline ring formation on a solid phase surface-modified with a formyl group, or immobilized by transesterification on a solid phase surface-modified with a benzylthioester), and carboxyl group modified groups with 2-(2-pyridinyldithio)-ethanamine (PDEA) (immobilized by a disulfide bond on a solid phase surface-modified with a thiol group).

[0052] The above-mentioned purification tags or immobilization modifying groups can be introduced into antibody components by conventional methods, thereby synthesizing the anti-AAV full capsid molecules of the present disclosure.

[0053] 2. AAV full capsid-binding affinity solid phase The AAV full capsid-binding affinity solid phase of the present disclosure comprises the above-mentioned "1. Anti-AAV full capsid molecule" and a solid phase material to which the anti-AAV full capsid molecule is immobilized (also referred to herein simply as "solid phase").

[0054] The material of the solid phase material is not particularly limited, and examples thereof include resins (agarose, sepharose, dextran, silica gel, polyacrylamide, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, (meth)acrylic acid-based polymers, fluororesins, metal complex resins, etc.), glass, metals, magnetic materials, etc.

[0055] The shape of the solid phase material is not particularly limited and can be appropriately determined depending on the mode of use of the affinity solid phase for binding to AAV full capsids of the present disclosure. Examples of the shape of the solid phase include flat plates, particles, fibers, etc.

[0056] When the solid phase material has a flat plate shape, the flat solid phase may form a chip or the bottom surface of a container, well, etc. When the solid phase material has a particulate or fibrous shape, the particulate or fibrous solid phase may be packed in a column.

[0057] In the AAV full capsid-binding affinity solid phase of the present disclosure, the mode of immobilization of anti-AAV full capsid molecules is as described above in the explanation of the immobilization modifying group in "1-7. Other Configurations."

[0058] 3. Method for capturing AAV full capsids The method for capturing AAV full capsids of the present disclosure includes Step 1 of contacting a sample containing AAV empty capsids and AAV full capsids with the affinity solid phase for binding AAV full capsids described above in "2. Affinity solid phase for binding AAV full capsids," and capturing the AAV full capsids.

[0059] Because the anti-AAV full capsid molecule immobilized on the AAV full capsid-binding affinity solid phase exhibits higher binding affinity for AAV full capsids than for AAV empty capsids, AAV full capsids in the sample are selectively captured by contacting a sample containing AAV empty capsids and AAV full capsids with the affinity solid phase in step 1. The conditions for contacting the sample with the solid phase are not particularly limited, and include, for example, neutral conditions (e.g., pH 6.5 to 7.7 at 25°C) and at room temperature (preferably 15 to 30°C) for 1 minute to 3 hours.

[0060] The method for capturing AAV full capsids of the present disclosure can be used, for example, in a method for quantifying AAV full capsids in the above-mentioned sample or a method for separating AAV full capsids in the above-mentioned sample.

[0061] When the AAV full capsid capture method of the present disclosure is used in the method for quantifying AAV full capsids in the sample, the method can further include step 2, in which the captured AAV full capsid is quantified by enzyme-linked immunosorbent assay. The enzyme-linked immunosorbent assay can be performed using any known method, specifically, any of a direct method, an indirect method, a sandwich method, and a competitive method. In step 2, the binding between the affinity solid phase and the AAV full capsid can be detected based on a signal emitted by a signal group such as a fluorescent label, and therefore the AAV full capsid can be quantified based on the intensity of the signal.

[0062] When the AAV full capsid capture method of the present disclosure is used in a method for separating AAV full capsids in a sample, the method can further include step 3 of separating and recovering the captured AAV full capsids. In this case, the solid phase material can be used in a form packed in a chromatography column. In step 3, the AAV full capsids captured on the affinity solid phase are recovered by elution. Examples of elution methods include elution by adjusting the pH (e.g., acid elution, alkaline elution), elution by adjusting the salt concentration (e.g., elution with a high salt concentration), etc. The recovered AAV full capsid fraction can be further purified by any purification method, if necessary. The recovered AAV full capsid fraction or a purified fraction thereof may be powdered by lyophilization, vacuum drying, spray drying, etc., if necessary.

[0063] 4. Nucleic Acids Nucleic acids encoding the anti-AAV full capsid molecules described above in "1. Anti-AAV full capsid molecules" (hereinafter also referred to as "nucleic acids of the present disclosure") can be appropriately prepared and designed by those skilled in the art according to the amino acid sequences of the anti-AAV full capsid molecules of the present disclosure. The nucleic acids of the present disclosure may be DNA or RNA.

[0064] The base sequence of the nucleic acid of the present disclosure can be appropriately designed by those skilled in the art according to the amino acid sequence of the complementarity determining region described above in "1-3. Complementarity determining region."

[0065] For example, in the nucleotide sequence encoding the first anti-AAV full capsid molecule, the nucleotide sequences encoding CDR1 represented by SEQ ID NO: 1, CDR2 represented by SEQ ID NO: 2, and CDR3 represented by SEQ ID NO: 3 include the nucleotide sequences represented by SEQ ID NOs: 29, 30, and 31, respectively, and DNA nucleotide sequences that hybridize under stringent conditions with DNA consisting of nucleotide sequences complementary to these nucleotide sequences. The nucleotide sequences encoding CDR1 represented by SEQ ID NO: 4, CDR2 consisting of RAS, and CDR3 represented by SEQ ID NO: 5 include the nucleotide sequences represented by SEQ ID NO: 32, agggcatcc, and SEQ ID NO: 33, respectively, and DNA nucleotide sequences that hybridize under stringent conditions with DNA consisting of nucleotide sequences complementary to these nucleotide sequences.

[0066] In the nucleotide sequence encoding the second anti-AAV full capsid molecule, the nucleotide sequences encoding CDR1 represented by SEQ ID NO:6, CDR2 represented by SEQ ID NO:7, and CDR3 represented by SEQ ID NO:8 include the nucleotide sequences represented by SEQ ID NOs:34, 35, and 36, respectively, and DNA nucleotide sequences that hybridize under stringent conditions with DNA consisting of nucleotide sequences complementary to these nucleotide sequences. The nucleotide sequences encoding CDR1 represented by SEQ ID NO:9, CDR2 consisting of RAS, and CDR3 represented by SEQ ID NO:10 include the nucleotide sequences represented by SEQ ID NO:37, agggcatcc, and SEQ ID NO:38, respectively, and DNA nucleotide sequences that hybridize under stringent conditions with DNA consisting of nucleotide sequences complementary to these nucleotide sequences.

[0067] In the nucleotide sequence encoding the third anti-AAV full capsid molecule, the nucleotide sequences encoding CDR1 represented by SEQ ID NO:11, CDR2 represented by SEQ ID NO:12, and CDR3 represented by SEQ ID NO:13 include the nucleotide sequences represented by SEQ ID NOs:39, 40, and 41, respectively, and DNA nucleotide sequences that hybridize under stringent conditions with DNA consisting of nucleotide sequences complementary to these nucleotide sequences. The nucleotide sequences encoding CDR1 represented by SEQ ID NO:14, CDR2 consisting of QAS, and CDR3 represented by SEQ ID NO:15 include the nucleotide sequences represented by SEQ ID NO:42, caggcatcc, and SEQ ID NO:43, respectively, and DNA nucleotide sequences that hybridize under stringent conditions with DNA consisting of nucleotide sequences complementary to these nucleotide sequences.

[0068] In the nucleotide sequence encoding the fourth anti-AAV full capsid molecule, the nucleotide sequences encoding CDR1 represented by SEQ ID NO:16, CDR2 represented by SEQ ID NO:17, and CDR3 represented by SEQ ID NO:18 include the nucleotide sequences represented by SEQ ID NOs:44, 45, and 46, respectively, and DNA nucleotide sequences that hybridize under stringent conditions with DNA consisting of nucleotide sequences complementary to these nucleotide sequences. The nucleotide sequences encoding CDR1 represented by SEQ ID NO:19, CDR2 consisting of RAS, and CDR3 represented by SEQ ID NO:20 include the nucleotide sequences represented by SEQ ID NO:47, agggcatcc, and SEQ ID NO:48, respectively, and DNA nucleotide sequences that hybridize under stringent conditions with DNA consisting of nucleotide sequences complementary to these nucleotide sequences.

[0069] The nucleotide sequences of the nucleic acids of the present disclosure can be further designed appropriately by those skilled in the art based on the amino acid sequences of the framework regions described above in "1-4. Framework Regions" or the amino acid sequences of the variable regions described above in "1-5. Specific Examples of Variable Regions." For example, in the nucleotide sequence encoding the first anti-AAV full capsid molecule, examples of the nucleotide sequences encoding the heavy chain variable region and the light chain variable region include the nucleotide sequences represented by SEQ ID NOs: 49 and 50, which encode the amino acid sequences represented by SEQ ID NOs: 21 and 22, respectively, and DNA sequences that hybridize under stringent conditions with DNAs comprising nucleotide sequences complementary to these nucleotide sequences. In the nucleotide sequence encoding the second anti-AAV full capsid molecule, examples of the nucleotide sequences encoding the heavy chain variable region and the light chain variable region include the nucleotide sequences represented by SEQ ID NOs: 51 and 52, which encode the amino acid sequences represented by SEQ ID NOs: 23 and 24, respectively, and DNA sequences that hybridize under stringent conditions with DNAs comprising nucleotide sequences complementary to these nucleotide sequences. In the nucleotide sequence encoding the third anti-AAV full capsid molecule, the nucleotide sequences encoding the heavy chain variable region and the light chain variable region include the nucleotide sequences represented by SEQ ID NOs: 53 and 54, which encode the amino acid sequences represented by SEQ ID NOs: 25 and 26, respectively, and the nucleotide sequences of DNA that hybridize under stringent conditions with DNA consisting of nucleotide sequences complementary to these nucleotide sequences. In the nucleotide sequence encoding the fourth anti-AAV full capsid molecule, the nucleotide sequences encoding the heavy chain variable region and the light chain variable region include the nucleotide sequences represented by SEQ ID NOs: 55 and 56, which encode the amino acid sequences represented by SEQ ID NOs: 27 and 28, respectively, and the nucleotide sequences of DNA that hybridize under stringent conditions with DNA consisting of nucleotide sequences complementary to these nucleotide sequences.

[0070] The term "stringent conditions" refers to conditions in which the mixture is incubated at 50°C to 65°C for 4 hours to overnight in 6xSSC (1xSSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5x Denhartz's (0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400), and 100µg / ml salmon sperm DNA. Hybridization under stringent conditions is specifically carried out by the following method. That is, a nylon membrane on which a DNA library or cDNA library is immobilized is prepared, and the nylon membrane is blocked at 65°C in a prehybridization solution containing 6xSSC, 0.5% SDS, 5x Denhartz's, and 100µg / ml salmon sperm DNA. Thereafter, 32 Each P-labeled probe is added and incubated overnight at 65° C. The nylon membrane is 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, and then autoradiography is performed to detect DNA that has specifically hybridized with the probe.

[0071] The base sequence of the nucleic acid of the present disclosure can also be appropriately designed according to the other structures described above in "1-7. Other Structures" that are represented by amino acid sequences.

[0072] The nucleic acid of the present disclosure can also be obtained by using the above-mentioned nucleic acid encoding the anti-AAV full capsid molecule as a template to obtain at least a region encoding the anti-AAV full capsid molecule by PCR, etc. The nucleic acid of the present disclosure can also be artificially synthesized by gene synthesis methods.

[0073] Furthermore, the nucleic acid of the present disclosure may further include at least one of a base sequence encoding an initiation codon and a termination codon.

[0074] Methods for obtaining the nucleic acids of the present disclosure include hybridization-based methods.

[0075] The nucleic acids of the present disclosure include various nucleic acids resulting from codon degeneracy. Various nucleic acids encoding the same amino acid sequence can be artificially produced easily using known genetic engineering techniques. For example, in the production of a polypeptide by genetic engineering, if the codons used in the original gene encoding the target protein are infrequently used in the host, the expression level of the protein may be low. In such cases, high expression of the target protein can be achieved by optimizing the codon usage frequency for the host without changing the encoded amino acid sequence.

[0076] The sum of the host-optimal codon usage frequencies for each codon can be used as an index of codon usage frequency. An optimal codon is defined as the codon with the highest usage frequency among codons corresponding to the same amino acid. The codon usage frequency is not particularly limited as long as it is optimized for the host. For example, the following is an example of an optimal codon for 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 (tgg), R: arginine (cgc), G: glycine (ggc).

[0077] 5. Expression Cassette or Recombinant Vector An expression cassette or recombinant vector containing the nucleic acid described above in "4. Nucleic Acid" (hereinafter also referred to as "expression cassette of the present disclosure" or "recombinant vector of the present disclosure") contains a nucleic acid encoding a polypeptide of the present disclosure.

[0078] An expression cassette or recombinant vector of the present disclosure can be obtained by linking a promoter and a terminator to a nucleic acid of the present disclosure, or by inserting an expression cassette or a nucleic acid of the present disclosure into an expression vector.

[0079] The expression cassette or recombinant vector of the present disclosure may contain, as regulatory elements, a promoter and a terminator, as well as transcription elements such as an enhancer, a CCAAT box, a TATA box, and an SPI site, as necessary. These regulatory elements may be operably linked to the DNA of the present disclosure. "Operably linked" means that the DNA of the present disclosure is linked to various regulatory elements that regulate the DNA of the present disclosure in a state that allows it to operate in a host cell.

[0080] When the expression cassette or recombinant vector of the present disclosure is designed so that a terminal sequence can be cleaved with a protease after a full-length polypeptide containing a protease recognition sequence has been expressed, the expression cassette or recombinant vector of the present disclosure can be configured to include a combination of a base sequence encoding the protease recognition sequence and a base sequence encoding an N-terminal sequence and / or a C-terminal sequence.

[0081] The expression vector is preferably one constructed for genetic recombination from a phage, plasmid, or virus that can autonomously replicate in a host. Such expression vectors are known, and those skilled in the art can select and use an appropriate combination with a host cell. For example, when a microorganism is used as a host, examples of suitable vectors include pBluescript (pBS) II SK(-) (Stratagene), pSTV-based vectors (Takara Bio), pUC-based vectors (Takara Bio), pET-based vectors (Sigma-Aldrich Japan LLC), pGEX-based vectors (Global Life Science Technologies Japan (Cytiva)), pCold-based vectors (Takara Bio), pHY300PLK (Takara Bio), pUB110 (Mckenzie, T. et al., 1986, Plasmid 15(2), pp. 93-103), pBR322 (Takara Bio), pRS403 (Stratagene), and pMW218 / 219 (Nippon Gene). When algae or microalgae are used as hosts, examples of vectors 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). When plant cells are used as hosts, examples of vectors include pRI-based vectors (manufactured by Takara Bio Inc.), pBI-based vectors (manufactured by Clontech), and IN3-based vectors (manufactured by Inplanta Innovations).

[0082] 6. Transformant A transformant (hereinafter sometimes referred to as "the transformant of the present disclosure") can be obtained by transforming a host with an expression cassette or recombinant vector of the present disclosure.

[0083] The host used to produce the transformant is not particularly limited as long as it can be introduced with a gene, is capable of autonomous proliferation, and is capable of expressing the genetic traits of the present disclosure, and suitable examples include microorganisms such as bacteria belonging to the genus Escherichia, such as Escherichia coli, the genus Bacillus, such as Bacillus subtilis, and the genus Pseudomonas, such as Pseudomonas putida; actinomycetes; yeast; and filamentous fungi, but may also be animal cells, insect cells, plant cells, etc. Among these, Escherichia coli is particularly preferred.

[0084] The transformant of the present disclosure can be obtained by introducing an expression cassette or recombinant vector of the present disclosure into a host. The location where the nucleic acid of the present disclosure is introduced is not particularly limited as long as the gene of interest can be expressed, and may be on a plasmid or on the genome. Specific methods for introducing the expression cassette or recombinant vector of the present disclosure include, for example, recombinant vector methods and genome editing methods.

[0085] Conditions for introducing the expression cassette or recombinant vector of the present disclosure into a host may be appropriately set depending on the type of host, etc. When the host is a microorganism, examples of such methods include a method using competent cells treated with calcium ions, electroporation, the spheroplast method, and the lithium acetate method. When the host is an animal cell, examples of such methods include electroporation, the calcium phosphate method, and the lipofection method. When the host is an insect cell, examples of such methods include the calcium phosphate method, the lipofection method, and the electroporation method. When the host is a plant cell, examples of such methods include the electroporation method, the Agrobacterium method, the particle gun method, and the PEG method.

[0086] 7. Method for Producing Anti-AAV Full Capsid Molecules The present disclosure also provides methods for producing the anti-AAV full capsid molecules described above in "1. Anti-AAV full capsid molecules." Anti-AAV full capsid molecules, which are antibodies that have higher binding affinity for AAV full capsids than for AAV empty capsids, can be produced appropriately using limited mechanical work.

[0087] 7-1. The anti-AAV full capsid molecules of the present disclosure can be suitably obtained by a limited, mechanical process of selecting antibodies from an antibody library by affinity selection for AAV full capsids and confirming that the selected antibodies have higher binding affinity to AAV full capsids than to AAV empty capsids. Specifically, anti-AAV full capsid molecules can be obtained by the following steps: immunizing an animal with an AAV capsid protein as an antigen, obtaining antibody genes from the animal's lymphocytes, and introducing the obtained antibody genes, which combine various VH and VL regions, into a phagemid vector to construct an antibody library; performing biopanning to select phage clones that have binding affinity to AAV full capsids; and confirming that the binding affinity of antibodies contained in the culture supernatant of the selected phage clones to AAV full capsids is higher than to AAV empty capsids.

[0088] 7-2. The anti-AAV full capsid molecules of the present disclosure can be appropriately produced by limited mechanical operations, not only those having CDRs consisting of amino acid sequences represented by specific SEQ ID NOs, but also those having CDRs with 90% to less than 100% sequence identity to the amino acid sequence represented by a specific SEQ ID NO. Specifically, an antibody phage library is produced by introducing into a phagemid vector antibodies having extremely limited sequence variations (preferably sequences with conservative amino acid substitutions) with 90% to less than 100% sequence identity to the amino acid sequence represented by a specific SEQ ID NO in the CDR region of the structural domain. As described in Section 7-1 above, phage clones having binding ability to AAV full capsids are selected by biopanning, and the binding ability of the antibodies produced by the selected phage clones to AAV full capsids is confirmed to be higher than that to AAV empty capsids, thereby obtaining the anti-AAV full capsid molecules of the present disclosure.

[0089] 7-3. In a preferred embodiment, the anti-AAV full capsid molecule of the present disclosure can be produced by culturing the transformant of the present disclosure.

[0090] The culture conditions for the transformant of the present disclosure may be appropriately determined taking into consideration the nutritional and physiological properties of the host, and are preferably liquid culture. For industrial production, aeration and agitation culture is preferred.

[0091] The transformant of the present disclosure is cultured, and the culture supernatant or cultured bacterial cells or cells are recovered by centrifugation, etc. If the polypeptide of the present disclosure has accumulated in the cultured bacterial cells or cells, the bacterial cells or cells can be treated with a mechanical method such as ultrasound or a French press, or with a lytic enzyme such as lysozyme, and then solubilized, as necessary, using an enzyme such as protease or a surfactant such as sodium dodecyl sulfate (SDS), to obtain a water-soluble fraction containing the anti-AAV full capsid molecule of the present disclosure.

[0092] Furthermore, by selecting an appropriate expression vector and host, the expressed anti-AAV full capsid molecule of the present disclosure may be secreted into the culture medium.

[0093] The culture medium, water-soluble fraction, or protease-treated product containing the anti-AAV full capsid molecule of the present disclosure obtained as described above may be subjected to a purification treatment as is, or the anti-AAV full capsid molecule of the present disclosure in the culture medium, water-soluble fraction, or protease-treated product may be concentrated and then subjected to a purification treatment.

[0094] The concentration can be carried out by, for example, vacuum concentration, membrane concentration, salting out treatment, fractional precipitation using a hydrophilic organic solvent (for example, methanol, ethanol and / or acetone), or the like.

[0095] The anti-AAV full capsid molecule of the present disclosure can be purified by appropriately combining methods such as gel filtration, hydrophobic chromatography, ion exchange chromatography, and affinity chromatography corresponding to a purification tag.

[0096] The anti-AAV full capsid molecule of the present disclosure purified in this manner may be powdered by freeze-drying, vacuum drying, spray drying, or the like, if necessary.

[0097] Each feature disclosed herein may be combined with any other feature disclosed herein.

[0098] The present invention will be described in more detail below with reference to examples, but the configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations may be made as appropriate within the scope of the present invention. The present disclosure is not limited by the embodiments, but is limited only by the scope of the claims.

[0099] [Test Example 1] (1) Preparation of Antibody Library Rabbits were immunized with proteins excised from AAV capsid proteins as antigens to produce antibodies. Total RNA was obtained from the spleen, and a cDNA library was constructed using reverse transcription polymerase chain reaction (RTC). Next, primers were used to amplify the heavy chain (H chain) variable region (VH domain) gene and the light chain (L chain) variable region (VL domain) gene by PCR. These PCR products were then treated with restriction enzymes to insert them into a phagemid vector. The phagemid vector was also treated with the appropriate restriction enzymes, and each restriction enzyme-treated gene was inserted into the restriction enzyme-treated phagemid vector. Escherichia coli TG-1 was transformed with this recombinant phagemid vector and inoculated into 10 ml of 2xYT medium (containing 1% glucose and 50 mg / L ampicillin). The cells were cultured overnight in a 200 ml Erlenmeyer flask at 37°C and 200 rpm (preculture).

[0100] The preculture was inoculated into 50 ml of 2xYT medium (containing 1% glucose and 50 mg / L ampicillin) to an OD600 of 0.1 and cultured at 30°C with shaking at 200 rpm. After culturing to approximately OD=1.0, helper phage VCSM13 was added to the culture to a multiplicity of infection (MOI) of 20 and incubated at 37°C for 30 minutes. The culture was centrifuged at 3000 g for 10 minutes at 37°C, the supernatant was removed, and the culture was gently suspended in 50 ml of 2xYT medium (containing 50 mg / L ampicillin and 35 mg / L kanamycin). After shaking at 30°C and 200 rpm for 12 hours or more, phages displaying single-chain antibodies were produced in the culture supernatant. The supernatant was collected by centrifugation, concentrated by PEG precipitation, and a library of phage displaying single-chain antibodies was isolated in 1 ml of PBS.

[0101] (2) Biopanning: AAV9 full capsid samples were physically adsorbed onto immunotubes in a buffer solution (137 mmol / L NaCl, 8.1 mmol / L NaHPO, 2.68 mmol / L KCl, 1.47 mmol / L KHPO, pH 7.4). Each tube was then blocked with a solution of 2% BSA dissolved and dispersed in the buffer solution (137 mmol / L NaCl, 8.1 mmol / L NaHPO, 2.68 mmol / L KCl, 1.47 mmol / L KHPO, pH 7.4). After incubation for a while, the plate was 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).

[0102] The phage library prepared in (1) above (approximately 1.0 × 10 11The IgG (pfu) was dissolved and dispersed in a buffer solution (137 mmol / L NaCl, 8.1 mmol / L NaHPO, 2.68 mmol / L KCl, 1.47 mmol / L KHPO, pH 7.4) containing 2% BSA, and the solution was added to the tube and incubated for 1 hour at 25°C. After incubation, the solution was removed, and the tube was washed five times with a buffer solution (137 mmol / L NaCl, 8.1 mmol / L NaHPO, 2.68 mmol / L KCl, 1.47 mmol / L KHPO, pH 7.4) containing 0.1% Tween 20. 0.1 M glycine-HCl (pH 2.2) was added, and the tube was incubated for 10 minutes at 25°C. The solution was then recovered, neutralized, and used to infect E. coli TG-1. E. coli TG-1 infected with phages displaying single-chain antibodies was plated on LB agar medium (ampicillin added to a final concentration of 50 mg / L) and incubated overnight at 37°C to form colonies, from which 96 clones were randomly selected.

[0103] (3) Experimental Procedure: 1. A solution (PBS) containing 0.1 μg of AAV was applied to a Maxisorp 96-well plate and allowed to stand at room temperature for 1 hour. Two types of AAV samples were used: AAV9 full capsid and empty capsid samples (i.e., "AAV9 Full Capsid Sample" and "AAV9 Empty Capsid Sample"). 2. The wells were washed twice with PBS. 3. 2% BSA blocking solution was applied to the plate and shaken at 25°C for 1 hour. 4. The wells were washed three times with PBST (0.1% Tween). 5. Separately, the 96 clones selected in (2) above were cultured in 2YT medium at 37°C for 24 hours. The anti-AAV-scFv contained in the culture supernatant was purified and quantified using His MultiTrap FF according to the protocol. The 96 clones included 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], each consisting of the amino acid sequences of the variable regions shown in Table 2A and the amino acid sequences of the linker and tag shown in Table 2B. Each anti-AAV-scFv consisted of a heavy chain variable region, a linker, a light chain variable region, and a tag, in this order from the N-terminus. Aqueous solutions of these anti-AAV-scFvs [1] to [4] were applied to a plate and shaken at 25°C for 1 hour (pH approximately 7).

[0104]

[0105]

[0106] 6. The wells were washed three times with PBST (0.1% Tween). 7. The HRP-conjugated His-Tag antibody was applied to the plate and shaken for 1 hour at 25°C. 8. The wells were washed three times with PBST (0.1% Tween). 9. The HRP enzyme substrate (1-Step Ultra TMB-Substrate Solution) was added. 10. After incubation at room temperature for color development, sulfuric acid or hydrochloric acid was added to stop the reaction. 11. The absorbance at 450 nm and 650 nm was measured for each well, and the absorbance at 650 nm was subtracted from the absorbance at 450 nm to determine the absorbance of the ELISA signal. 12. The ratio of the absorbance in the full capsid sample (IF) to the absorbance in the empty capsid sample (IE) (IF / IE), and the ratio of the absorbance in the empty capsid sample (IE) to the absorbance in the blank (BSA) (IE / IB) were calculated.

[0107] Results 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.

[0108]

[0109] As shown in Table 3, all of the anti-AAV-scFvs [1] to [4] were found to have specific binding to the AAV full capsid.

[0110] Test Example 2 The same procedures as in Test Example 1 were performed using the second anti-AAV-scFv [2], except that the amount of AAV applied to the plate in step "1" of the experimental procedure in Test Example 1 was varied. The absorbance at 450 nm and 650 nm was measured for each well, and the value obtained by subtracting the absorbance at 650 nm from the absorbance at 450 nm was used as the absorbance value of the ELISA signal. A graph with absorbance on the vertical axis and the amount of AAV applied on the horizontal axis is shown in Figure 1.

[0111] As shown in Figure 1, there was virtually no difference in absorbance between the blank (BSA) and empty capsids (Empty), and no concentration dependency was observed, whereas the absorbance of the full capsid (Full) increased in a concentration-dependent manner, demonstrating that AAV full capsids can be selectively quantified using anti-AAV-scFv.

Claims

1. An anti-AAV full capsid molecule, which is an antibody having higher binding affinity to AAV full capsid than to AAV empty capsid.

2. An anti-AAV full capsid molecule comprising a heavy chain variable region comprising, as heavy chain complementarity determining regions, CDR1 consisting of the amino acid sequence represented by SEQ ID NO:1, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:2, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:3, and a light chain variable region comprising, as light chain complementarity determining regions, CDR1 consisting of the amino acid sequence represented by SEQ ID NO:4, CDR2 consisting of RAS, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

5.

3. An anti-AAV full capsid molecule comprising a heavy chain variable region comprising, as heavy chain complementarity determining regions, CDR1 consisting of the amino acid sequence represented by SEQ ID NO:6, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:7, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:8, and a light chain variable region comprising, as light chain complementarity determining regions, CDR1 consisting of the amino acid sequence represented by SEQ ID NO:9, CDR2 consisting of RAS, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

10.

4. An anti-AAV full capsid molecule comprising a heavy chain variable region comprising, as heavy chain complementarity determining regions, CDR1 consisting of the amino acid sequence represented by SEQ ID NO:11, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:12, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:13, and a light chain variable region comprising, as light chain complementarity determining regions, CDR1 consisting of the amino acid sequence represented by SEQ ID NO:14, CDR2 consisting of QAS, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

15.

5. An anti-AAV full capsid molecule comprising a heavy chain variable region comprising, as heavy chain complementarity determining regions, CDR1 consisting of the amino acid sequence represented by SEQ ID NO:16, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:17, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:18, and a light chain variable region comprising, as light chain complementarity determining regions, CDR1 consisting of the amino acid sequence represented by SEQ ID NO:19, CDR2 consisting of RAS, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO:

20.

6. The heavy chain variable region comprises: FR1 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 1 to 24 of SEQ ID NO:21, positions 1 to 24 of SEQ ID NO:23, positions 1 to 24 of SEQ ID NO:25, or positions 1 to 24 of SEQ ID NO:27; FR2 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 33 to 49 of SEQ ID NO:21, positions 34 to 50 of SEQ ID NO:23, positions 34 to 50 of SEQ ID NO:25, or positions 34 to 50 of SEQ ID NO:27; FR3 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 59 to 95 of SEQ ID NO:21, positions 60 to 96 of SEQ ID NO:23, positions 59 to 95 of SEQ ID NO:25, or positions 60 to 96 of SEQ ID NO:27; and the light chain variable region comprises an FR4 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 109 to 119 of SEQ ID NO:21, positions 109 to 119 of SEQ ID NO:23, positions 107 to 117 of SEQ ID NO:25, or positions 113 to 123 of SEQ ID NO:27, wherein the light chain variable region comprises an FR1 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 1 to 26 of SEQ ID NO:22, positions 1 to 26 of SEQ ID NO:24, positions 1 to 26 of SEQ ID NO:26, or positions 1 to 26 of SEQ ID NO:28, an FR2 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 33 to 49 of SEQ ID NO:22, positions 33 to 49 of SEQ ID NO:24, positions 33 to 49 of SEQ ID NO:26, or positions 33 to 49 of SEQ ID NO:28, The anti-AAV full capsid molecule according to any one of claims 2 to 5, comprising: an FR3 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 53 to 88 of SEQ ID NO:22, positions 53 to 88 of SEQ ID NO:24, positions 53 to 88 of SEQ ID NO:26, or positions 53 to 88 of SEQ ID NO:28; and an FR4 consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of positions 101 to 110 of SEQ ID NO:22, positions 101 to 110 of SEQ ID NO:24, positions 101 to 109 of SEQ ID NO:26, or positions 98 to 107 of SEQ ID NO:

28.

7. The anti-AAV full capsid molecule of claim 2, wherein the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:21 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:

22.

8. The anti-AAV full capsid molecule of claim 3, wherein the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:23 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:

24.

9. The anti-AAV full capsid molecule of claim 4, wherein the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:25 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:

26.

10. The anti-AAV full capsid molecule of claim 5, wherein the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:27 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:

28.

11. IgG antibody, Fab antibody, Fab' antibody, F(ab') 2 The anti-AAV full capsid molecule of any one of claims 1 to 5, which is an antibody, an Fv antibody, an scFv antibody, a dsFv antibody, an scFv-Fc antibody, a dsFv-Fc antibody, a Bis-scFv antibody, a minibody, a diabody, a triabody, or a tetrabody.

12. An affinity solid phase for binding to an AAV full capsid, comprising an anti-AAV full capsid molecule according to any one of claims 1 to 5 and a solid phase material on which the anti-AAV full capsid molecule is immobilized.

13. A method for capturing an AAV full capsid, comprising step 1 of contacting a sample containing an AAV empty capsid and an AAV full capsid with the AAV full capsid-binding affinity solid phase described in claim 12, and capturing the AAV full capsid.

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

15. A nucleic acid encoding an anti-AAV full capsid molecule according to 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 with the expression cassette or recombinant vector described in claim 16.

18. A method for producing an anti-AAV full capsid molecule, comprising the step of culturing the transformant according to claim 17.