Antibody binding to AAV capsid

Abstract

The purpose of the present disclosure is to provide a molecule that shows different binding properties between a full capsid and an empty capsid of AAV. An antibody is provided that has a higher binding ability to an AAV empty capsid than to an AAV full capsid, under a condition (I) of pH 7.4 or a condition (II) of pH 3.0.

Description

Antibodies that bind to AAV capsids

[0001] The present disclosure relates to antibodies having binding ability to adeno-associated virus (AAV) capsids, and in particular to antibodies having higher binding ability to AAV empty capsids than to AAV full capsids.

[0002] In the field of recombinant adeno-associated viruses (AAV), such as the development or production of AAVs for gene therapy applications, various methodologies have been reported for purifying AAV capsids (also called AAV particles) from producer cells or culture media. For example, affinity chromatography using monoclonal antibodies that recognize AAV coat proteins can remove free viral proteins and other impurities from assembled capsids.

[0003] On the other hand, affinity chromatography cannot selectively separate full capsids (also called complete particles) that contain nucleic acids from empty capsids (also called empty particles) that do not contain any nucleic acids, and therefore must be used in combination with other purification methods (Non-Patent Document 1).

[0004] Methods for purifying full capsids from a mixture of full and empty capsids include, for example, the production and purification of recombinant AAV from the cell lysate fraction of transfected human embryonic kidney-derived 293 (HEK293) cells, which are primarily based on cesium chloride or iodixanol density ultracentrifugation.

[0005] Since density ultracentrifugation-based methods are not suitable for mass production, chromatography-based methods are desirable from the viewpoint of purification scale. From this viewpoint, a method for producing and purifying recombinant AAV1 from serum-free culture supernatant using ion exchange chromatography and gel filtration chromatography has been proposed (Non-Patent Document 1). This document explains that since it is difficult to separate recombinant AAV from empty capsids using affinity chromatography, ion exchange chromatography is used, taking advantage of the fact that the isoelectric point of the capsid protein differs depending on whether or not the vector genome is present (Non-Patent Document 1).

[0006] Production, Processing, and Characterization of Synthetic AAV Gene Therapy Vectors. Biotechnol J (2021) 16, e2000025. 10.1002 / biot.202000025.Molecular Therapy - Methods & Clinical Development (2016) 3, 15058; doi:10.1038 / mtm.2015.58

[0007] Affinity chromatography using an antibody that binds to the AAV capsid is used to purify the AAV capsid by removing free viral proteins and other impurities, but there is still room for improvement depending on the nature of the mixture to be purified and / or the target of affinity capture, etc. Therefore, there is a need for more options for antibodies that can be used to purify the AAV capsid.

[0008] Therefore, an object (first object) of the present disclosure is to provide an antibody that binds to an AAV capsid.

[0009] Furthermore, among methods for purifying full capsids from a mixture of full capsids and empty capsids, the method described in Non-Patent Document 2 is based on chromatography and therefore contributes to improving the purification scale. However, this method requires the use of two types of chromatography and is only capable of purifying AAV1 full capsids. For this reason, new options for chromatography-based methods that enable AAV full capsid purification are needed. As Non-Patent Documents 1 and 2 describe the difficulty of separating full capsids and empty capsids using affinity chromatography, the existence of molecules that exhibit different binding properties for AAV full capsids and empty capsids is unknown. If such molecules, for example, antibodies, can be found, it is expected that full capsids can be purified using affinity chromatography.

[0010] Therefore, another object (second object) of the present disclosure is to provide an antibody that preferably exhibits different binding affinity to AAV full capsids and empty capsids.

[0011] As a result of intensive research, the present inventors have found an antibody that binds to AAV capsids under pH 7.4 conditions. They have also found an antibody that binds to AAV capsids under pH 7.4 conditions and has a higher binding ability to AAV empty capsids than to AAV full capsids under pH 3.0 conditions. They have also found an antibody that binds to AAV capsids under pH 7.4 conditions and has a higher binding ability to AAV empty capsids than to AAV full capsids under pH 7.4 conditions. The present disclosure has been completed through further research based on these findings.

[0012] That is, the present disclosure provides the following aspects of the invention. Item 1. An antibody that has binding ability to AAV capsids under condition (I) at pH 7.4 and has higher binding ability to AAV empty capsids than to AAV full capsids under condition (II) at pH 3.0. A preferred example of the antibody of Item 1 (also referred to as the "antibody of the first embodiment") is the antibody of [1] shown in Item 4 or Item 5. Item 2. The antibody of Item 1, wherein, when a mixture of equal amounts of the AAV full capsid and the AAV empty capsid is subjected to condition (II), the ratio of the amount of binding of the empty capsid to the amount of binding of the AAV full capsid is set to 1, is 3 or more.

[0013] Item 3. An antibody having a higher binding affinity to AAV empty capsid than to AAV full capsid under the condition (I) of pH 7.4. Preferred examples of the antibody of Item 3 (also referred to as the "antibody of the second embodiment") include the antibodies [2] to

[10] ,

[12] ,

[14] , and

[16] shown in Item 4 or Item 5.

[0014] Item 4. An antibody having one or more structural domains comprising CDR1 to CDR3 of the following [1] to

[16] , and having binding ability to an AAV capsid under condition (I) at pH 7.4: [1] 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; [2] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6; [3] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 7, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 8, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 9; [4] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12; [5] [6] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 13, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 14, 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; [6] 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; [7] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 19, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 20, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 21; [8] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 22, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 23, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24;[9] CDR1 consisting of the amino acid sequence represented by SEQ ID NO:25, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:26, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:27;

[10] CDR1 consisting of the amino acid sequence represented by SEQ ID NO:28, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:29, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:30;

[11] CDR1 consisting of the amino acid sequence represented by SEQ ID NO:31, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:32, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:33;

[12] CDR1 consisting of the amino acid sequence represented by SEQ ID NO:34, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:35, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:36;

[13] CDR1 consisting of the amino acid sequence represented by SEQ ID NO:37, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:38, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO:39;

[14] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 40, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 41, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 42;

[15] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 43, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 44, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 45;

[16] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 46, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 47, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 48.

[0015] Item 5. An antibody having one or more structural domains containing CDRs of the following [1] to

[16] , and having the ability to bind to an AAV capsid under condition (I) at pH 7.4: [1] A CDR consisting of an amino acid sequence that has 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO: 49, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 113 to 123; [2] A CDR consisting of an amino acid sequence that has 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO: 50, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 115 to 125; [3] [4] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 51, excluding framework regions consisting of positions 1 to 28, 37 to 53, 62 to 99, and 120 to 130; [4] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 52, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 118 to 128; [5] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 53, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 114 to 124; [6] [6] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 54, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 118 to 128; [7] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 55, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 116 to 126;[8] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 56, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 112 to 122; [9] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 57, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 108 to 117;

[10] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 58, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 113 to 123;

[11] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 59, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 111 to 121;

[12] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 60, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 112 to 122;

[13] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 61, excluding framework regions consisting of positions 1 to 22, 31 to 47, 56 to 93, and 100 to 110;

[14] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a structural domain consisting of the amino acid sequence represented by SEQ ID NO: 62, excluding framework regions consisting of positions 1 to 25, positions 34 to 50, positions 58 to 95, and positions 111 to 121;

[15] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 63, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 105 to 115;

[16] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 64, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 108 to 118;

[0016] Item 6. The antibody according to Item 4 or 5, which has a higher binding affinity to AAV empty capsids than to AAV full capsids under condition (II) at pH 3.0. (Included in the antibody of the first embodiment.) Item 7. The antibody according to Item 4 or 5, which has a higher binding affinity to AAV empty capsids than to AAV full capsids under condition (I) at pH 7.4. (Included in the antibody of the second embodiment.) Item 8. The antibody according to any of Item 1 to 7, which is a heavy chain antibody, a VHH antibody, or a VHH antibody multimer.

[0017] Item 9. An affinity solid phase comprising the antibody according to any one of Items 1 to 8 and a solid phase material to which the antibody is immobilized. Item 10. An affinity solid phase comprising the antibody according to any one of Items 1 to 3 and 6 to 8 and a solid phase material to which the antibody is immobilized, and used for purifying AAV full capsids from a sample containing AAV empty capsids and AAV full capsids.

[0018] Item 11. A method for producing a sample from which AAV has been purified, comprising: Step 1: contacting a sample containing AAV and impurities with the affinity solid phase according to Item 9; and Step 2: eluting the AAV. (A preferred embodiment of the method for producing a sample from which AAV has been purified according to Item 11 is included in the method for producing a sample from which AAV full capsid has been purified according to Item 14.) Item 12. In Step 1, 10 10Item 13. The production method according to Item 11, wherein the step 1 is carried out at a pH of 6.5 to 8.5 and at 2 to 37°C, and the step 2 is carried out at a pH of 1 to 5 and at 2 to 37°C.

[0019] Item 14. A method for producing a sample from which AAV full capsids have been purified, comprising: Step 1' of contacting a sample containing AAV empty capsids and AAV full capsids with the affinity solid phase of Item 10; and Step 2' of eluting the AAV full capsids. Item 15. The production method of Item 14, in which Step 1' is carried out at pH 6.5 to 8.5 and 2 to 37°C, and Step 2' is carried out at pH 1 to 5 and 2 to 37°C. Item 16. The production method of Item 14, in which Step 1' and Step 2' are carried out at pH 1 to 5 and 2 to 37°C. (This includes the case where the antibody of the first embodiment is used.) Item 17. The production method of Item 14, in which Step 1' and Step 2' are carried out at pH 6.5 to 8.5 and 2 to 37°C. (This includes the case where the antibody of the second embodiment is used.) Item 18. Item 18. The method according to any one of Items 14 to 17, wherein the elution solution in step 2' contains 0.13 to 2 M of a water-soluble salt.

[0020] Item 19. A nucleic acid encoding the antibody according to any one of Items 4 to 8. Item 20. An expression cassette or recombinant vector comprising the nucleic acid according to Item 19. Item 21. A transformant obtained by transforming a host with the expression cassette or recombinant vector according to Item 20. Item 22. A method for producing an antibody, comprising the step of culturing the transformant according to Item 21.

[0021] According to the present disclosure, an antibody that binds to an AAV capsid is provided, and preferably an antibody that exhibits different binding properties to an AAV full capsid and an empty capsid.

[0022] 1 shows a schematic conceptual diagram of AAV full capsids and AAV empty capsids, and is the result of subjecting an AAV culture supernatant to affinity column chromatography using an affinity solid phase on which an antibody of the present disclosure is immobilized, and shows the AAV capsid contents in the culture supernatant applied to the affinity solid phase (Apply), in the fraction (PT) that leaked from the column during application, in the wash fraction (wash) with 1×PBS buffer (pH 7.4) at 25°C, and in the elution fraction (Elution) obtained at 25°C using 20 mM citrate buffer (pH 2.0) as an eluent (Test Example 3).

[0033] Figure 1 shows the results of affinity column chromatography of an AAV culture supernatant using an affinity solid phase on which an antibody of the present disclosure is immobilized, showing the HCP (host cell protein) content in Apply, PT, wash, and Elution (Test Example 3).

[0034] Figure 1 shows the results of affinity column chromatography of an AAV culture supernatant using an affinity solid phase on which an antibody of the present disclosure is immobilized, showing the DNA content in Apply, PT, wash, and Elution (Test Example 3).

[0035] Figure 1 shows the results of affinity column chromatography of an equal amount of AAV full capsid or AAV empty capsid using an affinity solid phase on which an antibody of the present disclosure is immobilized, showing the elution amount of each capsid at 25°C using 20 mM citrate buffer (pH 2.5 or 3.0) as the eluent (Test Example 5).

[0033] Figure 1 shows the results of affinity column chromatography using an affinity solid phase on which an antibody of the present disclosure has been immobilized, in which the same amount of AAV full capsid or AAV empty capsid was subjected to the same column chromatography using an affinity solid phase on which an antibody of the present disclosure has been immobilized, and shows the elution amount of each capsid when 20 mM citrate buffer (pH 3.0, 25°C) containing 0.5 to 2.0 mM NaCl was used as the eluent at 25°C (Test Example 6). Figure 1 shows the results of affinity column chromatography using an affinity solid phase on which an antibody of the present disclosure has been immobilized, in which the same amount of AAV full capsid or AAV empty capsid was subjected to the same column chromatography using an affinity solid phase on which an antibody of the present disclosure has been immobilized, and shows the elution amount of each capsid when 20 mM citrate buffer (pH 3.5) containing 250 mM NaCl was used as the eluent at 4°C (Test Example 6).The same amount of AAV full capsid or AAV empty capsid was subjected to affinity column chromatography using an affinity solid phase on which an antibody of the present disclosure was immobilized. The results show the amount of each capsid eluted at 25°C using a 20 mM citrate buffer solution (pH 3.5) containing 250 mM NaCl as the eluent (Test Example 6).

[0023] 1. Antibodies The antibodies of the present disclosure are antibodies having binding ability to adeno-associated virus capsids (AAV capsids). Preferred antibodies of the present disclosure are antibodies having higher binding ability to AAV empty capsids than to AAV full capsids.

[0024] 1-1. AAV In the present disclosure, the AAV serotype 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 AAV8 and AAV9, more preferably AAV8.

[0025] The term "AAV capsid" (also referred to as "AAV particle") encompasses both an AAV empty capsid and an AAV full capsid. The AAV capsid may be at least one of an AAV empty capsid and an AAV full capsid, but is preferably both an AAV empty capsid and an AAV full capsid.

[0026] An AAV empty capsid (also called an empty particle) 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.

[0027] An AAV full capsid (also referred to as a complete particle) is an AAV capsid containing nucleic acids therein, such as the AAV viral genome, nucleic acid medicines (antisense oligonucleotides, RNAi, aptamers, decoys, etc.), and vectors carrying genes for gene therapy.

[0028] 1-2. Binding Property The antibodies of the present disclosure have the ability to bind to AAV capsids under a condition of pH 7.4 (hereinafter also referred to as "condition (I)"). That is, the antibodies of the present disclosure have the ability to bind to at least either an AAV empty capsid or an AAV full capsid, preferably at least the AAV empty capsid of the AAV empty capsid and the AAV full capsid, under condition (I).

[0029] The temperature under the condition (I) that confers the ability to bind to AAV capsids may be at least any temperature selected from 2 to 37° C. Alternatively, the condition (I) that confers the ability to bind to AAV capsids may be established by adjusting the pH to 7.4 at 25° C. using phosphate buffered saline containing 0.13 to 4 M NaCl, and then adjusting the temperature to any temperature between 2 and 37° C. as necessary.

[0030] Without being bound by theory, it is presumed that the antibody of the present disclosure has the property of being concentrated by panning using a solution containing a relatively high concentration of salt and not containing a surfactant, and therefore, the antibody has the property of binding by utilizing hydrophobic interactions.

[0031] 1-3. Cross-reactivity 1-3-1. First embodiment The antibody of the first embodiment of the present disclosure has a higher binding affinity to AAV empty capsids than to AAV full capsids under a pH 3.0 condition (hereinafter also referred to as "condition (II)").

[0032] The fact that the antibody of the first embodiment of the present disclosure has a higher binding ability to AAV empty capsids than to AAV full capsids under condition (II) can be evaluated by determining the binding ability of the antibody of the present disclosure to each of AAV full capsids and AAV empty capsids under condition (II) using any method that can evaluate the level of binding ability between an antigen and an antibody, and comparing the levels of binding ability.

[0033] For example, the fact that the antibody of the first embodiment of the present disclosure has a higher binding ability to AAV empty capsids than to AAV full capsids under condition (II) can be confirmed by the fact that when a mixture of equal amounts (referring to an amount based on the number of capsids; the same applies hereinafter) of AAV full capsids and AAV empty capsids is subjected to condition (II), the amount of bound empty capsids (referring to an amount based on the number of capsids; the same applies hereinafter) is greater than the amount of bound AAV full capsids (referring to an amount based on the number of capsids; the same applies hereinafter).

[0034] When an equal mixture of AAV full capsids and AAV empty capsids is subjected to condition (II), the ratio of the amount of bound empty capsids to the amount of bound AAV full capsids, taken as 1 (hereinafter also referred to as "empty capsid binding ratio") is, for example, 3 or more, preferably 3.5 or more, more preferably 4 or more, even more preferably 4.5 or more, even more preferably 4.8 or more, and particularly preferably 5 or more.

[0035] More specifically, the binding amount ratio of AAV empty capsids can be calculated based on the following formula (1) from the injected amount of AAV (which refers to the amount based on the number of capsids) and the eluted amount of AAV (which refers to the amount based on the number of capsids) when an affinity solid phase in which the antibody of the first embodiment of the present disclosure is immobilized on a chromatography carrier is used, a mixture of equal amounts of AAV full capsids and AAV empty capsids adjusted to pH 7.4 (25°C) is injected at 4°C, and then a liquid phase satisfying condition (II) is delivered to elute the AAV full capsids.

[0036] In the specific first embodiment, the temperature under condition (II) that confers a higher binding ability to AAV empty capsids than to AAV full capsids may be at least any temperature selected from the range of 2 to 37° C. Furthermore, to establish condition (II) that confers a higher binding ability to AAV empty capsids than to AAV full capsids in the specific first embodiment, a 20 mM citrate buffer solution can be prepared in the presence of 1 M NaCl, adjusted to pH 3.0 at 25° C., and then adjusted to a temperature of 2 to 37° C. as necessary.

[0037] 1-3-2. Second embodiment The antibody of the second embodiment of the present disclosure has a higher binding affinity to AAV empty capsids than to AAV full capsids under condition (I), i.e., under the condition of pH 7.4.

[0038] The fact that the antibody of the second embodiment of the present disclosure has a higher binding ability to AAV empty capsids than to AAV full capsids under condition (I) can be evaluated by determining the binding ability of the antibody of the present disclosure to each of AAV full capsids and AAV empty capsids under condition (I) using any method that can evaluate the level of binding ability between an antigen and an antibody, and comparing the levels of binding ability.

[0039] For example, the fact that the antibody of the second embodiment of the present disclosure has a higher binding ability to AAV empty capsids than to AAV full capsids under condition (I) can be confirmed by the fact that when an equal mixture of AAV full capsids and AAV empty capsids is subjected to condition (I), the amount of empty capsid bound is greater than the amount of AAV full capsid bound.

[0040] When an equal mixture of AAV full capsids and AAV empty capsids is subjected to condition (I), the ratio of the amount of bound empty capsids to the amount of bound AAV full capsids taken as 1 (empty capsid binding ratio) is, for example, 3 or more, preferably 3.5 or more, more preferably 4 or more, even more preferably 4.5 or more, even more preferably 4.8 or more, and particularly preferably 5 or more.

[0041] More specifically, the binding amount ratio of AAV empty capsids can be calculated based on the above formula (1) from the injected amount of AAV (which refers to the amount based on the number of capsids) and the eluted amount of AAV (which refers to the amount based on the number of capsids) when an affinity solid phase in which the antibody of the second embodiment of the present disclosure is immobilized on a chromatography carrier is used, a mixture of equal amounts of AAV full capsids and AAV empty capsids adjusted to pH 7.4 (25°C) is injected at 4°C, and then a liquid phase satisfying condition (I) is delivered to elute the AAV full capsids.

[0042] In the second embodiment, the temperature under condition (I) that confers a higher binding ability to AAV empty capsids than to AAV full capsids may be at least any temperature selected from the range of 2 to 37° C. Furthermore, in the second embodiment, condition (I) that confers a higher binding ability to AAV empty capsids than to AAV full capsids can be established by adding 0.13 M NaCl to a 20 mM citrate buffer, adjusting the pH to 7.4 at 25° C., and then adjusting the temperature to any temperature between 2 and 37° C. as necessary.

[0043] 1-3-3. Selectivity for AAV full capsids and AAV capsids Although not wishing to be bound by theory, the higher binding ability to AAV empty capsids than to AAV full capsids under condition (II) in the first embodiment or condition (I) in the second embodiment is presumed to be due to the difference in hydrophobicity between AAV full capsids and AAV empty capsids. Specifically, as shown in Figure 1, the particle structure of AAV empty capsids is disrupted, causing a disturbance in the icosahedral VP orientation of the capsid proteins (VPs), resulting in the exposure of hydrophobic surfaces such as the interior of the VPs, and the particle as a whole is thought to exhibit stronger hydrophobicity than the AAV full capsid. Under condition (II) in the first embodiment or condition (I) in the second embodiment, by creating hydrophobic interaction chromatography conditions in the presence of a predetermined concentration of NaCl, it is thought that a difference in binding ability will occur based on the difference in hydrophobicity between the AAV full capsid and the AAV empty capsid.

[0044] 1-4. Complementarity Determining Regions In a preferred embodiment, the structural domain of an antibody of the present disclosure comprises three CDRs (Complementarity Determining Regions): CDR1, CDR2, and CDR3. In the structural domain of an antibody of the present invention, the three CDRs are present in the order of CDR1, CDR2, and CDR3 from the N-terminus.

[0045] 1-4-1. In a preferred embodiment, the antibodies of the present disclosure include the antibodies of the first to second embodiments, and specifically include antibodies 1 to 16 having one or more structural domains based on CDR1 to CDR3 shown in [1] to

[16] of Table 1, respectively.

[0046] In a more preferred embodiment, the antibody of the first embodiment of the present disclosure includes antibody 1, and the antibody of the second embodiment of the present disclosure includes antibodies 2 to 10, 12, 14, and 16.

[0047]

[0048] The first antibody has one or more structural domains including 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 (preferably 100%) sequence identity with the amino acid sequence represented by SEQ ID NO:3.

[0049] The second antibody has one or more structural domains including CDR1 consisting of the amino acid sequence represented by SEQ ID NO:4, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:5, and CDR3 consisting of an amino acid sequence having 90% or more (preferably 100%) sequence identity with the amino acid sequence represented by SEQ ID NO:6.

[0050] The third antibody has one or more structural domains including CDR1 consisting of the amino acid sequence represented by SEQ ID NO:7, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:8, and CDR3 consisting of an amino acid sequence having 90% or more (preferably 95% or more, more preferably 100%) sequence identity to the amino acid sequence represented by SEQ ID NO:9.

[0051] The fourth antibody has one or more structural domains including a CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and a CDR3 consisting of an amino acid sequence having 90% or more (preferably 95% or more, more preferably 100%) sequence identity to the amino acid sequence represented by SEQ ID NO: 12.

[0052] The fifth antibody has one or more structural domains including CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 13, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 14, and CDR3 consisting of an amino acid sequence having 90% or more (preferably 100%) sequence identity with the amino acid sequence represented by SEQ ID NO: 15.

[0053] The sixth antibody has one or more structural domains including a CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 16, a CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 17, and a CDR3 consisting of an amino acid sequence having 90% or more (preferably 95% or more, more preferably 100%) sequence identity to the amino acid sequence represented by SEQ ID NO: 18.

[0054] The seventh antibody has one or more structural domains including CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 19, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 20, and CDR3 consisting of an amino acid sequence having 90% or more (preferably 100%) sequence identity with the amino acid sequence represented by SEQ ID NO: 21.

[0055] The eighth antibody has one or more structural domains including CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 22, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 23, and CDR3 consisting of an amino acid sequence having 90% or more (preferably 100%) sequence identity with the amino acid sequence represented by SEQ ID NO: 24.

[0056] The ninth antibody has one or more structural domains including CDR1 consisting of the amino acid sequence represented by SEQ ID NO:25, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:26, and CDR3 consisting of an amino acid sequence having 90% or more (preferably 100%) sequence identity with the amino acid sequence represented by SEQ ID NO:27.

[0057] The tenth antibody has one or more structural domains including CDR1 consisting of the amino acid sequence represented by SEQ ID NO:28, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:29, and CDR3 consisting of an amino acid sequence having 90% or more (preferably 100%) sequence identity with the amino acid sequence represented by SEQ ID NO:30.

[0058] The eleventh antibody has one or more structural domains including a CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 31, a CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 32, and a CDR3 consisting of an amino acid sequence having 90% or more (preferably 100%) sequence identity with the amino acid sequence represented by SEQ ID NO: 33.

[0059] The twelfth antibody has one or more structural domains including a CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 34, a CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 35, and a CDR3 consisting of an amino acid sequence having 90% or more (preferably 100%) sequence identity with the amino acid sequence represented by SEQ ID NO: 36.

[0060] The thirteenth antibody has one or more structural domains including a CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 37, a CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 38, and a CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 39.

[0061] The fourteenth antibody has one or more structural domains including CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 40, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 41, and CDR3 consisting of an amino acid sequence having 90% or more (preferably 100%) sequence identity with the amino acid sequence represented by SEQ ID NO: 42.

[0062] The fifteenth antibody has one or more structural domains including a CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 43, a CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 44, and a CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 45.

[0063] The sixteenth antibody has one or more structural domains including a CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 46, a CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 47, and a CDR3 consisting of an amino acid sequence having 90% or more (preferably 100%) sequence identity with the amino acid sequence represented by SEQ ID NO: 48.

[0064] 1-4-2. The first to sixteenth antibodies include not only those described in "1-4-1" above, but also the following antibodies. That is, the first to sixteenth antibodies also include the first to sixteenth antibodies having one or more CDR-based structural domains consisting of amino acid sequences that share 90% or more sequence identity with the amino acid sequences of the structural domains excluding the framework regions shown in [1] to

[16] of Table 2 in "1-5. Framework Regions" below. That is, in the following embodiments, mismatches are permitted for CDR1 and / or CDR2 with respect to the amino acid sequences shown in the specified SEQ ID NOs.

[0065] The first antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, and even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:49, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 113 to 123.

[0066] The second antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, and even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:50, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 115 to 125.

[0067] The third antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, and even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:51, excluding framework regions consisting of positions 1 to 28, 37 to 53, 62 to 99, and 120 to 130.

[0068] The fourth antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:52, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 118 to 128.

[0069] The fifth antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:53, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 114 to 124.

[0070] The sixth antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:54, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 118 to 128.

[0071] The seventh antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of SEQ ID NO:55, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 116 to 126.

[0072] The eighth antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of SEQ ID NO:56, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 112 to 122.

[0073] The ninth antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:57, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 108 to 117.

[0074] The tenth antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, and even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:58, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 113 to 123.

[0075] The eleventh antibody comprises a CDR consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, and even more preferably 97% or more, 98% or more) sequence identity with the amino acid sequence of a structural domain consisting of the amino acid sequence of SEQ ID NO:59, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 111 to 121;

[0076] The twelfth antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO: 60, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 112 to 122.

[0077] The thirteenth antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:61, excluding framework regions consisting of positions 1 to 22, 31 to 47, 56 to 93, and 100 to 110.

[0078] The fourteenth antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, and even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:62, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 111 to 121.

[0079] The fifteenth antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:63, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 105 to 115.

[0080] The sixteenth antibody has one or more CDR-containing structural domains consisting of an amino acid sequence that has 90% or more (preferably 92% or more, 94% or more, more preferably 95% or more, 96% or more, even more preferably 97% or more, 98% or more) sequence identity to the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:64, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 108 to 118.

[0081] 1-4-3. In the present disclosure, 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.

[0082] Furthermore, in the present invention, when a mismatched amino acid residue in an amino acid sequence having a sequence identity of less than 100% 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).

[0083] 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.

[0084] 1-5. Framework Regions The structural domains of the antibody, 1 to 16, may have framework regions at both ends of CDR1, CDR2, and CDR3. Framework regions are regions in the variable region of an antibody molecule excluding the complementarity-determining regions, and are highly conserved regions. The framework regions are not particularly limited, as long as the antibody of the present disclosure has the ability to bind to AAV capsids under condition (I) (in the first embodiment, the binding affinity to AAV empty capsids is higher than that to AAV full capsids under condition (II) and in the second embodiment, the binding affinity to AAV empty capsids is higher than that to AAV full capsids under condition (I)). Framework regions from antibodies of any animal (human and non-human, preferably alpaca) can be used.

[0085] In a preferred embodiment, the structural domain of an antibody of the present disclosure comprises, in that order, a first framework region (FR1), CDR1, a second framework region (FR2), CDR2, a third framework region (FR3), CDR3, and a fourth framework region (FR4).

[0086] In a preferred embodiment, examples of amino acid sequences of framework regions in structural domains will be described using the amino acid sequences of specific structural domains including CDR1 to CDR3 described above (specifically, SEQ ID NOS: 49 to 64, which are specific examples of the amino acid sequences of the structural domains of each of antibodies 1 to 16). For convenience of the following description, Table 2 is provided. In Table 2, SEQ ID NOS: X [X represents 49 to 64] is an amino acid sequence having, from the N-terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 in this order; the FR1 region is from position "S1" to position "E1" of the corresponding SEQ ID NOS: [for example, the FR1 region of SEQ ID NOS: 49 is from position 1 to position 25 of said SEQ ID NOS:], the FR2 region is from position "S2" to position "E2" of the corresponding SEQ ID NOS: the FR1 region is from position "S3" to position "E3" of the corresponding SEQ ID NO: and the FR4 region is from position "S4" to position "E4" of the corresponding SEQ ID NO:; specific SEQ ID NOs for FR1, FR2, FR3, and FR4 (for example, the amino acid sequence of FR1 in SEQ ID NO:49 is SEQ ID NO:65); and specific SEQ ID NOs for CDR1, CDR2, and CDR3 (for example, the amino acid sequence of CDR1 in SEQ ID NO:49 is SEQ ID NO:1).

[0087]

[0088] In a preferred embodiment, the amino acid sequences of the framework regions in the structural domains include the following: FR1: an amino acid sequence having 50% or more sequence identity with the amino acid sequence represented by SEQ ID NO:65 (corresponding to positions 1 to 25 of SEQ ID NO:49), FR2: an amino acid sequence having 50% or more sequence identity with the amino acid sequence represented by SEQ ID NO:66 (corresponding to positions 34 to 50 of SEQ ID NO:49), FR3: an amino acid sequence having 50% or more sequence identity with the amino acid sequence represented by SEQ ID NO:67 (corresponding to positions 58 to 95 of SEQ ID NO:49), and FR4: an amino acid sequence having 50% or more sequence identity with the amino acid sequence represented by SEQ ID NO:68 (corresponding to positions 113 to 123 of SEQ ID NO:49). The above-mentioned sequence identity of 50% or more may be 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%.

[0089] In a preferred embodiment, the amino acid sequences of the framework regions in the structural domains also include the following: FR1: an amino acid sequence having 70% or more sequence identity with the amino acid sequence from position "S1" to position "E1" of SEQ ID NO: "X" shown in Table 2; FR2: an amino acid sequence having 70% or more sequence identity with the amino acid sequence from position "S2" to position "E2" of SEQ ID NO: "X" shown in Table 2; FR3: an amino acid sequence having 70% or more sequence identity with the amino acid sequence from position "S3" to position "E3" of SEQ ID NO: "X" shown in Table 2; FR4: an amino acid sequence having 70% or more sequence identity with the amino acid sequence from position "S4" to position "E4" of SEQ ID NO: "X" shown in Table 2. Preferred examples of the above-mentioned sequence identity of 70% or more include 75% or more, 80% or more, 85% or more, and more preferably 90% or more, 95% or more, or 100%.

[0090] The above-mentioned "sequence identity," conservative amino acid substitution which is a preferred form of substitution for mismatched amino acid residues in amino acid sequences having a sequence identity of less than 100%, and methods for substituting a predetermined amino acid in an amino acid sequence with another amino acid are as described above in "1-4. Complementarity-Determining Region."

[0091] 1-6. Specific Examples of Structural Domains Antibodies of the present disclosure can be designed by arbitrarily combining FR1 to FR4 described above in "1-5. Framework Regions" with the 16 predetermined combinations of CDR1 to CDR3 described above in "1-4. Complementarity-Determining Regions."

[0092] For example, in the case of the first antibody, FR1 to FR4 in its structural domain to be combined with the CDR shown in row [1] of Table 1 (including those with the above-mentioned sequence identity of 90% or more) may be selected from FR1 to FR4 shown in row [1] of Table 2 (including those with the above-mentioned sequence identity of 50% or more), or at least any or all of FR1 to FR4 may be randomly selected from the group of FR1 to FR4 shown in rows other than row [1] of Table 2 (including those with the above-mentioned sequence identity of 70% or more). (For example, in the structural domain of the first antibody, FR1 shown in row

[13] of Table 2, FR2 shown in row

[16] of Table 2, FR3 shown in row [2] of Table 2, and FR4 shown in row [9] of Table 2 may be selected as FR1 to FR4 to be combined with the CDR shown in row [1] of Table 1.) The same applies to the second to sixteenth antibodies.

[0093] In a preferred embodiment, the structural domains of the antibodies of the present disclosure are configured as follows: First antibody: A combination of the CDRs shown in row [1] of Table 1 (including those with a sequence identity of 90% or more as described above) and FR1 to FR4 shown in row [1] of Table 2 (including those with a sequence identity of 50% or more as described above); Second antibody: A combination of the CDRs shown in row [2] of Table 1 (including those with a sequence identity of 90% or more as described above) and FR1 to FR4 shown in row [2] of Table 2 (including those with a sequence identity of 70% or more as described above); Third antibody: A combination of the CDRs shown in row [3] of Table 1 (including those with a sequence identity of 90% or more as described above) and FR1 shown in row [3] of Table 2 Fourth antibody: a combination of the CDRs shown in row [4] of Table 1 (including those with a sequence identity of 90% or more as described above) and FR1 to FR4 shown in row [4] of Table 2 (including those with a sequence identity of 70% or more as described above). Fifth antibody: a combination of the CDRs shown in row [5] of Table 1 (including those with a sequence identity of 90% or more as described above) and FR1 to FR4 shown in row [5] of Table 2 (including those with a sequence identity of 70% or more as described above). Sixth antibody: a combination of the CDRs shown in row [6] of Table 1 (including those with a sequence identity of 90% or more as described above). Antibody No. 7: A combination of the CDRs shown in row [7] of Table 1 (including those with a sequence identity of 90% or more) and FR1 to FR4 shown in row [6] of Table 2 (including those with a sequence identity of 70% or more as described above). Antibody No. 8: A combination of the CDRs shown in row [8] of Table 1 (including those with a sequence identity of 90% or more as described above) and FR1 to FR4 shown in row [8] of Table 2 (including those with a sequence identity of 70% or more as described above). Ninth antibody: a combination of the CDRs shown in row [9] of Table 1 (including those with a sequence identity of 90% or more as described above) and FR1 to FR4 shown in row [9] of Table 2 (including those with a sequence identity of 70% or more as described above). Tenth antibody: a combination of the CDRs shown in row

[10] of Table 1 (including those with a sequence identity of 90% or more as described above) and FR1 to FR4 shown in row

[10] of Table 2 (including those with a sequence identity of 70% or more as described above). Eleventh antibody: a combination of the CDRs shown in row

[11] of Table 1 (including those with a sequence identity of 90% or more as described above),Antibody No. 12: A combination of the CDRs shown in row

[12] of Table 1 (including those with a sequence identity of 90% or more as described above) and FR1 to FR4 shown in row

[12] of Table 2 (including those with a sequence identity of 70% or more as described above). Antibody No. 13: A combination of the CDRs shown in row

[13] of Table 1 (including those with a sequence identity of 90% or more as described above) and FR1 to FR4 shown in row

[13] of Table 2 (including those with a sequence identity of 70% or more as described above). Antibody No. 14: A combination of the CDRs shown in row

[14] of Table 1 (including those with a sequence identity of 90% or more as described above). Antibody No. 15: A combination of the CDRs shown in row

[15] of Table 1 (including those with a sequence identity of 90% or more) and FR1 to FR4 shown in row

[14] of Table 2 (including those with a sequence identity of 70% or more). Antibody No. 16: A combination of the CDRs shown in row

[16] of Table 1 (including those with a sequence identity of 90% or more) and FR1 to FR4 shown in row

[16] of Table 2 (including those with a sequence identity of 70% or more).

[0094] 1-7. Specific Forms of Antibodies The form of the antibodies of the present disclosure is not limited as long as they have the ability to bind to AAV capsids under condition (I) (in the first embodiment, they also have a higher binding ability to AAV empty capsids than to AAV full capsids under condition (II); in the second embodiment, they also have a higher binding ability to AAV empty capsids than to AAV full capsids under condition (I)). In other words, the antibodies of the present disclosure may be either whole antibodies or small molecular weight antibodies (antibodies with a smaller molecular weight than whole antibodies).

[0095] When the antibody 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.

[0096] When the antibody of the present disclosure is a low molecular weight antibody, specific examples thereof include a Fab antibody (approximately 55 kDa), a Fab' antibody (approximately 55 kDa), and a F(ab') 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), tetrabodies (approximately 100 kDa), heavy chain antibodies (approximately 80 kDa), VHH antibodies (approximately 15 kDa), and VHH antibody multimers. The VHH antibody multimer may be a multimer in which a plurality of the above structural domains are linked, or may be a multimer in which one or more of the above structural domains are linked to one or more structural domains having antigen specificities different from those of the structural domains.

[0097] Furthermore, the antibodies of the present disclosure also encompass multispecific antibodies (e.g., bispecific antibodies) as long as they have the ability to bind to AAV capsids under condition (I) (in the first embodiment, they also have a higher binding ability to AAV empty capsids than to AAV full capsids under condition (II); in the second embodiment, they also have a higher binding ability to AAV empty capsids than to AAV full capsids under condition (I)).

[0098] In preferred embodiments, the antibody of the present disclosure is a heavy chain antibody, a VHH antibody, or a VHH antibody multimer.

[0099] The antibodies of the present disclosure are isolated antibodies, typically monoclonal antibodies.

[0100] 1-8. Other Components The antibodies of the present disclosure may include 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 "1-7. Specific Forms of Antibodies"), so long as they have the ability to bind to AAV capsids under condition (I) (in the first embodiment, they further have a higher binding ability to AAV empty capsids than to AAV full capsids under condition (II); in the second embodiment, they further have a higher binding ability to AAV empty capsids than to AAV full capsids under condition (I)).

[0101] Antibodies of the present disclosure that contain other components include conjugated antibodies to which various compounds such as polyethylene glycol, radioactive substances, and toxins are bound as other components, antibodies to which modified glycans are bound as other components, and fusion antibodies to which another protein is fused as other components.

[0102] Preferred examples of other components include a hinge region, a spacer, a purification tag that enables purification of the antibody of the present disclosure, an immobilization modification group that enables immobilization of the antibody of the present disclosure to a solid phase, etc. These components may be used alone or in combination of two or more.

[0103] 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.

[0104] 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.

[0105] 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+The metal ions are reversibly chelated to a solid phase having a central metal ion such as methyl methacrylate (MM), etc. on its surface. epitope tags such as a Strep tag (a tag consisting of WSHPQFEK, which reversibly binds to a solid phase having streptavidin on its surface); a 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), a Spot tag (a tag consisting of PDRVRAVSHWSS, which reversibly binds to a solid phase having a Spot tag recognition site on its surface), and a C tag (a tag consisting of EPEA, which reversibly binds to a solid phase having a C tag recognition site on its surface); and protein tags such as a glutathione-S-transferase tag (which reversibly binds to a solid phase having a glutathione-S-transferase recognition site or glutathione on its surface) and a maltose-binding protein tag (which reversibly binds to a solid phase having a maltose-binding protein recognition site on its surface). In another embodiment, these purification tags may be used alone or in combination of two or more. In a preferred embodiment, the purification tag is bound to the structural domain via the hinge region, preferably at the C-terminus. In a preferred embodiment, a spacer may be inserted between the purification tag and the hinge region.

[0106] 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).

[0107] The above-mentioned purification tag or immobilization modifying group can be introduced into the constituent elements of the antibody by conventional methods, thereby synthesizing the antibody of the present disclosure.

[0108] 2. Affinity Solid Phase The affinity solid phase of the present disclosure includes the above-mentioned "1. Antibody" and a solid phase material to which the antibody is immobilized (also referred to herein simply as "solid phase").

[0109] 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.

[0110] 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 of the present disclosure. Examples of the shape of the solid phase include plate-like, particulate, and fibrous shapes.

[0111] 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.

[0112] In the affinity solid phase of the present disclosure, the mode of antibody immobilization is as described above in the description of the immobilization modifying group in "1-8. Other Configurations."

[0113] The immobilization of antibodies on the affinity solid phase of the present disclosure is not particularly limited. However, under the condition (I) of pH 7.4, for example, 10 10 Capsid or more, 10 11 Capsid or more, or 10 12 Capsids or more, preferably 10 13 Capsids or more, more preferably 10 14 The degree of capturing AAV capsids or more, more specifically, 10 per mL of affinity solid phase 10 ~10 16 Capsid, 10 11 ~10 16 Capsid, 10 12 ~10 16 Capsid, 10 13 ~10 16 Capsid, 10 14 ~10 16 Capsid, or 10 14 ~10 15 The degree to which the capsid captures AAV is an example.

[0114] The affinity solid phase of the present disclosure can be used to purify AAV capsids from a mixture containing AAV capsids and impurities (e.g., free viral proteins and / or other impurities). Specific methods for use are described in "3. Method for producing a purified AAV sample."

[0115] Furthermore, the affinity solid phase of the present disclosure, on which the antibody of the first embodiment or the antibody of the second embodiment is immobilized, can preferably be used to purify full capsids from a mixture containing full capsids and empty capsids. Specific methods for use are as described in "4. Method for producing a sample from which AAV full capsids have been purified."

[0116] 3. Method for Producing a Sample from Which AAV Has Been Purified The method for producing a sample from Which AAV has been purified according to the present disclosure comprises Step 1 of contacting a sample containing AAV and impurities with the affinity solid phase described above in "2. Affinity Solid Phase" (a solid phase to which an antibody capable of binding to an AAV capsid under the condition (I) of at least pH 7.4 has been immobilized), and Step 2 of eluting the AAV.

[0117] Note that a preferred embodiment of the method for producing a sample from purified AAV of the present disclosure is included in "4. Method for producing a sample from purified AAV full capsid," a preferred example of step 1 is included in the details described in "4-1. Step 1'," and a preferred example of step 2 is included in the details described in "4-2. Step 2'."

[0118] 3-1. Step 1 In step 1, a sample containing AAV and impurities is contacted with an affinity solid phase. In a sample containing AAV and impurities, the impurities may be components other than AAV that are unnecessary for the storage or use of purified AAV, and specifically include at least one of free viral proteins, free DNA, and host cell proteins (HCPs). The sample containing AAV and impurities may further include a water-soluble salt. Preferred examples of a sample containing AAV and impurities include a sample containing AAV empty capsids and AAV full capsids as AAV, as described in "4-1. Step 1'".

[0119] The method for preparing a sample containing AAV and impurities is not particularly limited. Examples include a culture medium of cells producing recombinant AAV or a crude product thereof (from which impurities have been insufficiently removed). The recombinant AAV may be used, for example, in a gene therapy drug, a vaccine, or a regenerative medicine drug.

[0120] The conditions for contact are not particularly limited, and can be set depending on whether the antibody immobilized on the affinity solid phase is of the first embodiment or the second embodiment.

[0121] For example, when the antibody of the first embodiment is used, the pH conditions include, for example, 6.5 to 8.5, and preferably 7 to 8; the temperature conditions include, for example, 2 to 37°C, preferably 2 to 30°C, 2 to 28°C, and more preferably 3 to 26°C; and the concentration of the water-soluble salt is, for example, 0.13 to 4 M, preferably 0.25 to 4 M, more preferably 0.5 to 4 M, even more preferably 0.5 to 4 M, even more preferably 0.8 to 4 M, and particularly preferably 0.9 to 4 M.

[0122] Furthermore, when the antibody of the second embodiment is used, the pH conditions include, for example, 6.5 to 8.5, and preferably 7 to 8; the temperature conditions include, for example, 2 to 37°C, preferably 2 to 30°C, 2 to 28°C, and more preferably 3 to 26°C; and the concentration of the water-soluble salt is, for example, 2.5 to 4 M, preferably 3 to 4 M, and more preferably 0.5 to 4 M.

[0123] Preferred examples of the conditions for the contact include the conditions detailed in "4-1. Step 1'".

[0124] The water-soluble salt is not particularly limited, but preferably includes normal salt, more preferably includes salts of strong acids and strong bases, and even more preferably includes sodium chloride.

[0125] When the affinity solid phase is used in a form packed in a column, for example, a sample containing AAV and impurities (regardless of whether the above-mentioned conditions for contact are met) can be injected, and then an aqueous solution that satisfies the above-mentioned conditions for contact can be passed through in step 1. Alternatively, when the affinity solid phase is used in a form packed in a column, a sample containing AAV and impurities and that satisfies the above-mentioned conditions for contact can be injected into the column in step 1.

[0126] The amount of AVV captured in step 1 is not particularly limited, but may be, for example, 10 per mL of affinity solid phase. 10 Capsid or more, 10 11Capsid or more, or 10 12 Capsids or more, preferably 10 13 Capsids or more, more preferably 10 14 More specifically, the amount of AVV captured in step 1 can be, for example, 10 capsids per mL of the affinity solid phase. 10 ~10 16 Capsid, 10 11 ~10 16 Capsid, 10 12 ~10 16 Capsid, 10 13 ~10 16 Capsid, 10 14 ~10 16 Capsid, or 10 14 ~10 15 Capsids are included.

[0127] 3-2. Step 2 In step 2, AAV is eluted. In step 2, AAV captured on the affinity solid phase can be eluted with an aqueous solution (elution solution) that satisfies the conditions for elution. When the affinity solid phase is used in a form packed in a column, the elution solution can be passed through as a mobile phase.

[0128] The conditions for elution can be selected to be different from the conditions used for capturing AAV in step 1. Preferred examples of step 2, including preferred examples of conditions for elution, include those described in detail in "4-2. Step 2'".

[0129] 3-3. Other Steps The method for producing a purified AAV sample according to the present disclosure may or may not include any other steps in addition to Step 1 and Step 2, as long as a purified AAV sample can be obtained.

[0130] Other steps include desalting and / or solvent exchange steps. The method for desalting and / or solvent exchange is not particularly limited, but examples include membrane filtration, preferably ultrafiltration, etc.

[0131] Other steps include other AAV full capsid purification steps. Other AAV full capsid purification steps are not particularly limited, and examples thereof include ultracentrifugation, gradient elution on an ion exchange column, a combination of ion exchange chromatography and gel filtration chromatography, etc. In a preferred embodiment, the method for producing a sample from purified AAV of the present disclosure does not include other AAV full capsid purification steps.

[0132] Other steps include a concentration or drying step. In the concentration or drying step, part or all of the water is removed from the sample obtained in step 2 or, if necessary, from the sample that has been subjected to the above-mentioned desalting step, solvent exchange step, and / or other AAV full capsid purification step. Examples of concentration methods include vacuum concentration, membrane concentration, freeze concentration, etc., and examples of drying methods include freeze drying, vacuum drying, spray drying, etc.

[0133] Preferred examples of the other steps include those detailed in "4-3. Other steps."

[0134] 4. Method for Producing a Sample from Which AAV Full Capsids Are Purified The method for producing a sample from Which AAV full capsids are purified according to the present disclosure comprises: Step 1' of contacting a sample containing AAV empty capsids and AAV full capsids with one of the affinity solid phases described above in "2. Affinity Solid Phase" to which is immobilized an antibody that has binding ability to AAV capsids under condition (I) at pH 7.4 and has higher binding ability to AAV empty capsids than to AAV full capsids under condition (II) at pH 3.0; and Step 2' of eluting the AAV full capsids.

[0135] The method for producing a sample from which AAV full capsid has been purified according to the present disclosure includes a preferred embodiment of "3. Method for producing a sample from which AAV has been purified," in which step 1' includes a preferred example of "3-1. Step 1," and step 2' includes a preferred example of "3-2. Step 2."

[0136] Step 1' and step 2' may be performed sequentially by applying different conditions (hereinafter also referred to as "first mode"), or may be performed simultaneously by applying the same requirements (hereinafter also referred to as "second mode"). In the first mode, both AAV empty capsids and AAV full capsids are captured on the affinity solid phase, and then the AAV full capsids can be eluted. In the second mode, the AAV full capsids can be recovered in a flow-through mode without first being captured.

[0137] 4-1. Step 1' In step 1', a sample containing AAV empty capsids and AAV full capsids is contacted with an affinity solid phase.

[0138] The sample containing AAV empty capsids and AAV full capsids may or may not contain impurities as described in "3. Method for producing a purified AAV sample."

[0139] The method for preparing a sample containing AAV empty capsids and AAV full capsids is not particularly limited. Examples include a culture medium of cells producing recombinant AAV, a crude product thereof (from which impurities have been insufficiently or sufficiently removed), etc. The recombinant AAV may be used, for example, in a gene therapy drug, a vaccine, or a regenerative medicine drug.

[0140] The conditions for contact are not particularly limited, and can be set depending on whether the antibody immobilized on the affinity solid phase is the first embodiment or the second embodiment, and whether it is the first mode or the second mode.

[0141] For example, in the first mode using the antibody of the first embodiment, pH conditions include, for example, 6.5 to 8.5, and preferably 7 to 8; temperature conditions include, for example, 2 to 37°C, preferably 2 to 30°C, 2 to 28°C, and more preferably 3 to 26°C; and the concentration of the water-soluble salt includes, for example, 0.13 to 4 M, preferably 0.25 to 4 M, more preferably 0.5 to 4 M, even more preferably 0.5 to 4 M, even more preferably 0.8 to 4 M, and particularly preferably 0.9 to 4 M. Conditions in the second mode using the antibody of the first embodiment include the elution conditions described in "4-2. Step 2'".

[0142] In the first mode using the antibody of the second embodiment, the pH conditions include, for example, 6.5 to 8.5, and preferably 7 to 8; the temperature conditions include, for example, 2 to 37°C, preferably 2 to 30°C, 2 to 28°C, and more preferably 3 to 26°C; and the concentration of the water-soluble salt is, for example, 2.5 to 4 M, preferably 3 to 4 M, and more preferably 0.5 to 4 M. In the second mode using the antibody of the second embodiment, the conditions for elution include those described in "4-2. Step 2'".

[0143] The water-soluble salt is not particularly limited, but preferably includes normal salt, more preferably includes salts of strong acids and strong bases, and even more preferably includes sodium chloride.

[0144] When the affinity solid phase is used in a form packed in a column, in step 1', for example, a sample containing AAV empty capsids and AAV full capsids (regardless of whether the above-mentioned conditions for contact are met) can be injected, and then an aqueous solution satisfying the above-mentioned conditions for contact can be passed through. Alternatively, when the affinity solid phase is used in a form packed in a column, in step 1', a sample containing AAV empty capsids and AAV full capsids and satisfying the above-mentioned conditions for contact can be injected into the column.

[0145] The amount of AAV captured in step 1 (the total amount of AAV empty capsids and AAV full capsids) is not particularly limited, but may be, for example, 10 10Capsid or more, 10 11 Capsid or more, or 10 12 Capsids or more, preferably 10 13 Capsids or more, more preferably 10 14 More specifically, the amount of AVV captured in step 1 can be, for example, 10 capsids per mL of the affinity solid phase. 10 ~10 16 Capsid, 10 11 ~10 16 Capsid, 10 12 ~10 16 Capsid, 10 13 ~10 16 Capsid, 10 14 ~10 16 Capsid, or 10 14 ~10 15 These examples of the amount of capture are preferably applicable to the first mode.

[0146] 4-2. Step 2' In step 2', AAV full capsids are eluted. By utilizing the property of the antibody immobilized on the affinity solid phase (the antibody described in "1. Antibody" above), which has a higher binding affinity for AAV empty capsids than for AAV full capsids, in step 2', AAV empty capsids can be captured on the affinity solid phase while AAV full capsids can be eluted using an aqueous solution (elution solution) that satisfies the elution conditions. When the affinity solid phase is used in a form packed in a column, the elution solution can be passed through as the mobile phase.

[0147] The conditions for elution are not particularly limited, as long as they are conditions under which the antibody immobilized on the affinity solid phase exhibits a higher binding ability to AAV empty capsids than to AAV full capsids, depending on whether the antibody is of the first embodiment or the second embodiment.

[0148] When the antibody of the first embodiment is used, the pH conditions can be, for example, 1 to 5, 2 to 5, or 2 to 4, preferably 2.4 to 3.5, and more preferably 2.4 to 3.1. When the first antibody is used as the antibody immobilized on the affinity solid phase, the pH conditions can be, for example, 2.4 to 3.1 or 2.5 to 3. In this case, the means for establishing the pH conditions is not particularly limited, and either a pH adjuster or a buffer solution can be used, preferably a buffer solution, specifically a citrate buffer or a glycine buffer solution, more preferably a citrate buffer solution. Furthermore, when the antibody of the second embodiment is used, the pH conditions can be, for example, 6.5 to 8.5, preferably 7 to 8. In this case, the means for establishing the pH conditions is not particularly limited, and either a pH adjuster or a buffer solution can be used, preferably a buffer solution, specifically a phosphate buffer solution.

[0149] The temperature conditions include, for example, 2 to 37°C, preferably 2 to 30°C, 2 to 28°C, and more preferably 3 to 26°C.

[0150] The elution conditions may or may not include the presence of a water-soluble salt, but preferably an elution solution containing a water-soluble salt is used. The concentration of the water-soluble salt in the elution solution is, for example, 0.13 to 2 M, preferably 0.25 to 2 M, more preferably 0.5 to 2 M, even more preferably 0.5 to 1.5 M, even more preferably 0.8 to 1.5 M, and particularly preferably 0.9 to 1.2 M or 1 M. The water-soluble salt is not particularly limited, but preferably includes normal salts, more preferably salts of strong acids and strong bases, and even more preferably sodium chloride.

[0151] The eluate obtained in step 2' has a higher ratio of the AAV full capsid content to the AAV empty capsid content (hereinafter also referred to as "AAV full capsid ratio") than the sample subjected to step 1'. In other words, a sample in which AAV full capsids are purified is obtained. In the present invention, "purification" of AAV full capsids refers to increasing the AAV full capsid ratio, and the degree to which the AAV full capsid ratio is increased is not particularly limited, as it can vary depending on the ratio of the binding ability for AAV full capsids to the binding ability for AAV empty capsids under the conditions of step 2'.

[0152] 4-3. Other Steps The method for producing a sample from purified AAV full capsid of the present disclosure may or may not include any other steps in addition to Step 1' and Step 2', as long as a sample from purified AAV full capsid is obtained.

[0153] Other steps include desalting and / or solvent exchange steps. The method for desalting and / or solvent exchange is not particularly limited, but examples include membrane filtration, preferably ultrafiltration, etc.

[0154] Other steps include other AAV full capsid purification steps. The other AAV full capsid purification steps are not particularly limited, and examples thereof include ultracentrifugation, gradient elution on an ion exchange column, a combination of ion exchange chromatography and gel filtration chromatography, etc. In a preferred embodiment, the method for producing a sample from which AAV full capsid has been purified according to the present disclosure does not include other AAV full capsid purification steps.

[0155] Other steps include a concentration or drying step. In the concentration or drying step, part or all of the water is removed from the sample of purified AAV full capsid obtained in step 2 or, if necessary, from the sample of purified AAV full capsid that has been subjected to the above-mentioned desalting step, solvent exchange step, and / or other AAV full capsid purification steps. Examples of concentration methods include reduced pressure concentration, membrane concentration, freeze concentration, etc., and examples of drying methods include freeze drying, vacuum drying, spray drying, etc.

[0156] 5. Nucleic Acids Nucleic acids encoding the antibodies described above in "1. Antibodies," "1-4. Complementarity Determining Regions" and subsequent sections (hereinafter also referred to as "nucleic acids of the present disclosure") can be appropriately prepared and designed by those skilled in the art based on the amino acid sequences of the antibodies of the present disclosure. The nucleic acids of the present disclosure may be DNA or RNA.

[0157] The base sequence of the nucleic acid of the present disclosure can be appropriately designed by a person skilled in the art according to the amino acid sequences of the complementarity determining regions described above in "1-4. Complementarity determining regions," the amino acid sequences of the framework regions described above in "1-5. Framework regions," the amino acid sequences of the framework regions described above in "1-6. Specific examples of structural domains," and "1-7. Specific forms of antibodies," or further, the amino acid sequences of those composed of multiple amino acid residues in "1-8. Other configurations."

[0158] For example, examples of nucleotide sequences encoding SEQ ID NOs: 49 to 64, which are the amino acid sequences of the structural domains of each of antibodies 1 to 16, include SEQ ID NOs: 69 to 84, respectively, and nucleotide sequences of nucleic acids that hybridize under stringent conditions with nucleic acids consisting of nucleotide sequences complementary to these nucleotide sequences. Thus, the structural domains of the nucleic acid nucleotide sequences of the present disclosure can be designed by combining the nucleotide sequences of the regions encoding CDR1 to CDR3 in each of SEQ ID NOs: 69 to 84 (or the nucleotide sequences of nucleic acids that hybridize under stringent conditions with nucleic acids consisting of nucleotide sequences complementary to these nucleotide sequences) with the nucleotide sequences of the regions encoding FR1 to FR4 (or the nucleotide sequences of nucleic acids that hybridize under stringent conditions with nucleic acids consisting of nucleotide sequences complementary to these nucleotide sequences), as described above in "1-6. Specific examples of structural domains."

[0159] 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.

[0160] The nucleic acid of the present disclosure can also be obtained by using the above-described nucleic acid encoding the antibody as a template to obtain at least the region encoding the antibody by PCR, etc. The nucleic acid of the present disclosure can also be artificially synthesized by gene synthesis methods.

[0161] 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.

[0162] 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.

[0163] 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).

[0164] 6. Expression Cassette or Recombinant Vector An expression cassette or recombinant vector containing the nucleic acid described above in "5. 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 7. 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 8. Method for Producing Antibodies The present disclosure also provides a method for producing the antibodies described above in "1. Antibodies." Antibodies having the ability to bind to AAV capsids under condition (I), preferably antibodies having the ability to bind to AAV capsids under condition (I) and having higher binding affinity for AAV empty capsids than for AAV full capsids under condition (II), can be appropriately produced by limited mechanical operations.

[0174] 8-1. The antibodies of the present disclosure can be confirmed to have a predetermined binding ability by obtaining antibodies that have the property of binding via hydrophobic interactions from an antibody library such as that described in Test Example 1 [1] by a selection method such as that described in Test Example 2 [3], particularly a selection method using a solution containing approximately 137 mmol / L (or alternatively 0.13 to 4 M) NaCl and no surfactant, and further confirming that the antibodies bind to AAV capsids under condition (I) (preferably, in the case of the antibody of the first embodiment, binding to AAV capsids under condition (I) and binding more easily to AAV empty capsids than to AAV full capsids under condition (II); in the case of the antibody of the second embodiment, binding more easily to AAV empty capsids than to AAV full capsids under condition (I)).

[0175] 8-2. The antibodies of the present disclosure can be produced not only by limited mechanical operations but also by appropriately producing CDRs having an amino acid sequence represented by a specific SEQ ID NO., but also by producing CDRs having a sequence identity of 90% or more but less than 100% to an amino acid sequence represented by a specific SEQ ID NO. Specifically, an antibody library is prepared by introducing into the CDR region on the structural domain a very limited variation in sequence (preferably a variation in sequence due to conservative amino acid substitution) that has 90% or more but less than 100% sequence identity with the amino acid sequence represented by a specific SEQ ID NO:; and antibodies that have the property of binding via hydrophobic interactions are obtained by a selection method such as that described in Test Example 2, [3], particularly a selection method using a solution containing approximately 137 mmol / L (or 0.13 to 4 M) NaCl and no surfactant. Furthermore, the predetermined binding ability can be confirmed by confirming that the antibody binds to the AAV capsid under condition (I) (preferably, in the case of the antibody of the first embodiment, it binds to the AAV capsid under condition (I) and binds more easily to the AAV empty capsid than to the AAV full capsid under condition (II); in the case of the antibody of the second embodiment, it binds more easily to the AAV empty capsid than to the AAV full capsid under condition (I)).

[0176] 8-3. In a preferred embodiment, the above-described antibody can be produced by culturing the transformant of the present disclosure described above in "7. Transformant."

[0177] 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 may be used.

[0178] 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 ultrasonic waves, a mechanical method such as a French press, or 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 antibody of the present disclosure.

[0179] Furthermore, by selecting an appropriate expression vector and host, the expressed antibody of the present disclosure may be secreted into the culture medium.

[0180] The culture medium, water-soluble fraction, or protease-treated product containing the antibody of the present disclosure obtained as described above may be subjected to a purification treatment as is, or the antibody 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.

[0181] 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 acetone), or the like.

[0182] The antibody of the present disclosure can be purified by, for example, appropriately combining methods such as gel filtration, hydrophobic chromatography, ion exchange chromatography, and affinity chromatography corresponding to a purification tag.

[0183] The antibody of the present disclosure purified in this manner may be powdered by freeze-drying, vacuum drying, spray drying, or the like, if necessary.

[0184] Each feature disclosed herein may be combined with any other feature disclosed herein.

[0185] 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.

[0186] [Test Example 1] (1) Preparation of VHH phage library An alpaca-derived VHH-displaying M13 phage library was prepared according to the experimental section "Construction of alpaca VHH phage library" in the literature Monoclon Antib Immunodiagn Immunother. (2019) 38, 190-200. doi: 10.1089 / mab.2019.0027., except that RNA was extracted and used from peripheral blood mononuclear cells (PBMCs) derived from multiple alpacas.

[0187] (2) Preparation of AAV8 full capsid sample, AAV9 full capsid sample, AAV8 empty capsid sample, and AAV9 empty capsid sample An AAV8 or AAV9 sample (containing an AAV8 or AAV9 vector encapsulating DNA comprising a WPRE (Woodchuck hepatitis virus posttranscriptional regulatory element) sequence as a regulatory element) was subjected to ultracentrifugation, and a fraction in which 90% or more of the field of view was recognized as full capsids in TEM observation stained with uranyl acetate was prepared as an AAV8 or AAV9 full capsid sample, and a fraction in which 90% or more of the field of view was recognized as empty capsids was prepared as an AAV8 or AAV9 empty capsid sample.

[0188] (3) Biopanning: AAV8 full capsid sample, AAV9 full capsid sample, AAV8 empty capsid sample, and AAV9 empty capsid sample were separately physically adsorbed to a 96-well microtiter plate in buffer (137 mmol / L NaCl, 8.1 mmol / L NaHPO, 2.68 mmol / L KCl, 1.47 mmol / L KHPO, pH 7.4). Each well was then blocked with a solution of 3% skim milk dissolved and dispersed in buffer (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 plates were washed five times with a buffer solution containing 0.1% Tween 20 (137 mmol / L NaCl, 8.1 mmol / L NaHPO, 2.68 mmol / L KCl, 1.47 mmol / L KHPO, pH 7.4). 11 pfu) dissolved and dispersed in a buffer solution containing 3% skim milk (137 mmol / L NaCl, 8.1 mmol / L NaHPO, 2.68 mmol / L KCl, 1.47 mmol / L KHPO, pH 7.4) was added to each well and incubated for 1.5 hours at 25°C with shaking. After incubation, the solution was removed, and the wells were washed 5 to 10 times with a buffer solution containing 0.1% Tween 20 (137 mmol / L NaCl, 8.1 mmol / L NaHPO, 2.68 mmol / L KCl, 1.47 mmol / L KHPO, pH 7.4). 0.1 M glycine hydrochloride (HCl) (pH 2.2) was added, and the plate was incubated for 6 minutes at 25°C with shaking. The solution from each well was then recovered, neutralized, and used to infect E. coli TG-1. The above biopanning procedure was repeated 3 to 5 times for each sample.

[0189] (4) Identification of VHHs From the sequences of the clones in each round, 400,000 to 900,000 sequences were confirmed by next-generation sequencing, and from the top 50 clones that were clearly enriched with each round, 16 types of VHH sequences were selected: [1] a first VHH having the amino acid sequence shown in SEQ ID NO: 49; [2] a second VHH having the amino acid sequence shown in SEQ ID NO: 50; [3] a third VHH having the amino acid sequence shown in SEQ ID NO: 51; [4] a fourth VHH having the amino acid sequence shown in SEQ ID NO: 52; [5] a fifth VHH having the amino acid sequence shown in SEQ ID NO: 53; [6] a sixth VHH having the amino acid sequence shown in SEQ ID NO: 54; and [7] an amino acid sequence shown in SEQ ID NO: 55. [8] a seventh VHH having the amino acid sequence set forth in SEQ ID NO:56; [9] a ninth VHH having the amino acid sequence set forth in SEQ ID NO:57;

[10] a tenth VHH having the amino acid sequence set forth in SEQ ID NO:58;

[11] an eleventh VHH having the amino acid sequence set forth in SEQ ID NO:59;

[12] a twelfth VHH having the amino acid sequence set forth in SEQ ID NO:60;

[13] a thirteenth VHH having the amino acid sequence set forth in SEQ ID NO:61;

[14] a fourteenth VHH having the amino acid sequence set forth in SEQ ID NO:62;

[15] a fifteenth VHH having the amino acid sequence set forth in SEQ ID NO:63; and

[16] a sixteenth VHH having the amino acid sequence set forth in SEQ ID NO:64.

[0190] Considering that the solution used during incubation in the above biopanning had a relatively high salt concentration and was a surfactant, these 16 types of VHHs [1] to

[16] are presumed to have been enriched as VHHs that have the property of binding to AVV capsids by utilizing hydrophobic interactions, specifically as VHHs that bind to AVV capsids under conditions of pH 7.4.

[0191] Of these 16 VHHs, the first VHH bound to AAV capsids at pH 7.4 and, as shown in Test Examples 2 to 6, showed higher binding ability to AAV empty capsids than to AAV full capsids under acidic conditions including pH 3.0.

[0192] Of these 16 VHHs, for VHHs 2 to 10, 12, 14, and 16, the ratio of the frequency of appearance after panning, based on the sequencing results before and after panning, where the frequency of appearance before panning is set to 1, is shown in Tables 3A and 3B below for each AAV capsid sample. A higher frequency of appearance after panning indicates a higher frequency of binding to AAV capsids.

[0193]

[0194] As shown in Tables 3A and 3B, VHHs 2 to 10, 12, 14, and 16 showed higher binding affinity to AAV empty capsid than to AAV full capsid under the condition of pH 7.4.

[0195] [Test Example 2] (1) Preparation of antibody (first VHH) As the gene for the first VHH, DNA was synthesized in which a base sequence encoding a hinge region (EPKTPKPQ)-TSA-Hisx6 tag was added to the base sequence of SEQ ID NO: 69, which encodes the amino acid sequence of SEQ ID NO: 49, and each was inserted into the pET17b plasmid. C43 Escherichia coli was transformed with the inserted plasmid by heat shock. The transformed Escherichia coli was cultured in liquid culture in 2xYT medium supplemented with ampicillin, and the OD 600 IPTG was added (final concentration: 1 mM) when the pH reached 0.8, and shaking culture was continued for 12 to 16 hours to express VHH. The cells and culture supernatant were separated by centrifugation (12,100 rpm, 10 minutes, 4°C), and the fraction containing VHH was purified using a Ni-NTA column (HisTrap FF crude, Cytiva). The solvent in the resulting VHH solution was replaced with an aqueous solution (pH 8.3) containing 0.2 M sodium bicarbonate and 0.5 M NaCl by ultrafiltration (Amicon15 MWCO 10k, Merck Millipore).

[0196] (2) Preparation of a column packed with an affinity solid phase. The VHH solution after substitution obtained in (1) above was immobilized on an NHS-activated column (HiTrap NHS-activated HP (1 mL), Cytiva) while adjusting the concentration, followed by washing and capping according to the manufacturer's instructions. The resulting column was connected to a system capable of controlling the flow rate (low-pressure / medium-pressure chromatography system (AKTA start, Cytiva) and a syringe pump).

[0197] Test Example 3 (1) Sample Containing AAV and Impurities 3 mL of DNA solution (30 μg each of Rap / Cap plasmid, pHelper, and expression plasmid diluted with D-MEM) and 3 mL of PEI solution (180 μL of 1 mg / mL PEI solution diluted with D-MEM) were mixed and incubated at room temperature for 30 minutes. This was added to 60 mL of medium in a 24.5 cm square dish seeded with HEK245T cells and incubated. The next day, the medium was replaced with 42 mL of FES-free D-MEM. After five days of incubation, the entire medium was collected and centrifuged to remove dead cells and cell debris. This procedure was repeated four times, and the resulting AAV8 culture supernatant (pH approximately 7.4, 25°C) was subjected to the purification procedure described below in (2).

[0198] (2) AAV Purification Step 1: The column (1 mL) obtained in Test Example 2 was equilibrated with 10 mL of 1x PBS buffer (pH 7.4, 25°C), and 143 mL (1.53 x 10 14 The column was washed with 10 mL of 1x PBS buffer (pH 7.4, 25°C) at 25°C. The solution obtained by washing was collected as the "wash" fraction.

[0199] Step 2: AAV8 was eluted by passing 10 mL of 20 mM citrate buffer (pH 2.0) through the column at a rate of 1 mL / min at 25° C. The eluate was collected as an “Elution” fraction.

[0200] (3) Quantification of AAV The amounts of AAV (number of capsids), HCP (host cell protein) (ng), and DNA (ng) contained in the applied AAV8 culture supernatant ("Apply"), "PT" fraction, "wash" fraction, and "Elution" fraction were quantified, and the results are shown in Figures 2A, 2B, and 2C, respectively. Table 4 also shows the percentages (%) of the contents of each component contained in the PT fraction, wash fraction, and Elution fraction, relative to the amounts of AAV (number of capsids), HCP (host cell protein) (ng), and DNA (ng) contained in the applied AAV8 culture supernatant, respectively, taken as 100%.

[0201]

[0202] As shown in Figure 2A and Table 4, most of the AAV contained in the applied culture supernatant (10 per mL of affinity solid phase) was 14 The amount of AAV (exceeding the capsid) was captured on the affinity solid phase, and 92.55% of the applied AAV was recovered.

[0203] As shown in FIG. 2B and Table 4, most of the HCP contained in the applied culture supernatant was eluted in the PT fraction and the wash fraction, but not at all in the elution fraction.

[0204] As shown in FIG. 2C and Table 4, most of the DNA contained in the applied culture supernatant was eluted into the PT fraction and the wash fraction, and almost none was eluted into the elution fraction.

[0205] Test Example 4 (1) Equal Mixture Sample of AAV Full Capsids and AAV Empty Capsids An AAV8 sample (containing an AAV8 vector encapsulating DNA comprising a WPRE (Woodchuck hepatitis virus posttranscriptional regulatory element) sequence as a regulatory element) was subjected to ultracentrifugation, and the fraction in which 90% or more of the field of view was recognized as full capsids in TEM observation stained with uranyl acetate was prepared as the AAV full capsid sample, and the fraction in which 90% or more of the field of view was recognized as empty capsids was prepared as the AAV8 empty capsid sample. The AAV full capsid sample and the AAV empty capsid sample were mixed to obtain an equal mixture sample (pH approximately 7.4, 25°C) containing equal numbers of AAV full capsids and AAV empty capsids.

[0206] (2) Purification of AAV full capsids Step 1: The column (1 mL) obtained in Test Example 2 was loaded with the equal amounts of the mixed sample (pH about 7.4, 25°C) obtained in (1) above, with 7.5 x 10 AAV full capsids and 7.5 x 10 AAV empty capsids. 10 An amount sufficient to form capsids was injected at 25°C, and 1x PBS (pH 7.4, 25°C) was passed through for 5 CV at 1.5 mL / min at 4°C (passage was carried out under condition (I)). When passed through under condition (I), all AAV was captured by the affinity solid phase.

[0207] Step 2: Thereafter, as the mobile phase for elution, 20 mM citrate buffer (pH 3.0, 25°C) containing 1 M NaCl was passed through the column at 0.5 mL / min for 5 CV (under condition (II)) at 4°C. The eluate was collected.

[0208] (3) AAV quantification: The total amount of AAV in the injected equal-volume mixed sample or eluate was measured by ELISA (AAV8 Titration ELISA, progen). The amount of AAV full capsid in the injected equal-volume mixed sample or eluate was measured by qPCR using the WPRE sequence contained in the encapsulated DNA as an index. The amount of AAV empty capsid was calculated by subtracting the amount of AAV full capsid from the total amount of AAV.

[0209] (4) AAV empty capsid binding ratio The AAV empty capsid binding ratio of the antibody immobilized on the affinity solid phase was calculated based on the following formula (1) using the amount of AAV empty capsid in the injected equal-volume mixed sample (empty capsid injection amount), the amount of AAV full capsid in the injected equal-volume mixed sample (full capsid injection amount), the amount of AAV empty capsid in the eluate (empty capsid elution amount), and the amount of AAV full capsid in the eluate (full capsid elution amount).

[0210]

[0211] As a result, the ratio of the amount of AAV empty capsid bound to the first VHH under condition (II) was 5.3.

[0212] (5) Full Capsid Ratio in Eluate To examine the degree of purification using the affinity solid phase, the full capsid ratio in the eluate was calculated. Specifically, it was calculated based on the amount of AAV empty capsid in the eluate (elution amount of empty capsid) and the amount of AAV full capsid in the eluate (elution amount of full capsid) according to the following formula (2):

[0213]

[0214] As a result, the full capsid ratio in the eluate obtained by affinity chromatography using the first VHH-immobilized solid phase was 86.5%. The full capsid ratio of 50.0% in a mixed sample of equal amounts of AAV full capsids and AAV empty capsids was increased to 86.5% by affinity chromatography alone.

[0215] Test Example 5 (1) AAV full capsid sample or AAV empty capsid sample An AAV8 sample (containing an AAV8 vector encapsulating DNA comprising a WPRE (Woodchuck hepatitis virus posttranscriptional regulatory element) sequence as a regulatory element) was subjected to ultracentrifugation, and a fraction in which 90% or more of the field of view was recognized as full capsids in TEM observation stained with uranyl acetate was prepared as an AAV full capsid sample, and a fraction in which 90% or more of the field of view was recognized as empty capsids was prepared as an AAV8 empty capsid sample.

[0216] (2) Affinity chromatography step 1: A solution containing the AAV full capsid sample or AAV empty capsid sample described in [1] above (pH 7.4, 25°C) was added to the column (1 mL) obtained in Test Example 2 at 1.5 x 10 11 The solution was injected at 25°C so as to achieve the capsid amount, and 1x PBS (pH 7.4, 25°C) was passed through for 5 CV at 1.5 mL / min at 25°C (passage was carried out under condition (I)). When the solution was passed through under condition (I), all of the AAV was captured by the affinity solid phase.

[0217] Step 2: Thereafter, as the mobile phase for elution, 20 mM citrate buffer (pH 2.5, 25° C.) was passed through the column for 5 CV at 0.5 mL / min at 25° C. The eluate was collected.

[0218] (3) Results The elution amount of each capsid at each pH is shown in Figure 3. As shown in Figure 3, the binding ability was higher for AAV empty capsids than for AAV full capsids.

[0219] Test Example 6 (1) AAV full capsid sample or AAV empty capsid sample The same samples as in Test Example 5 (1) were prepared.

[0220] (2) Affinity chromatography step 1: The column (1 mL) obtained in Test Example 2 was loaded with the AAV full capsid sample or the AAV empty capsid sample obtained in (1) above at 1.5 × 10 11The capsid amount was injected at 25°C, and 1x PBS (pH 7.4, 25°C) was passed through for 5 CV at 1.5 mL / min at 25°C (condition (I)). Under condition (I), all AAV was captured by the affinity solid phase. Step 2: Subsequently, 20 mM citrate buffer (pH 3.0, 25°C) containing 2.0 M NaCl was passed through for 5 CV at 0.5 mL / min at 25°C as the mobile phase for elution, and the eluate was collected. Subsequently, 20 mM citrate buffer (pH 3.0, 25°C) containing 1.5 M NaCl was passed through under the same conditions, and the eluate was collected. Subsequently, 20 mM citrate buffer (pH 3.0, 25°C) containing 1.0 M NaCl and 20 mM citrate buffer (pH 3.0, 25°C) containing 0.5 M NaCl were passed through the column under the same conditions, and the eluate was collected for each mobile phase.

[0221] (3) Results The elution amounts of each capsid at each NaCl concentration are shown in Figure 4. As shown in Figure 4, at all NaCl concentrations, the antibody had a higher binding affinity to AAV empty capsids than to AAV full capsids. Furthermore, as shown in Figure 4, the antibody immobilized on the column bound both AAV capsids more strongly with increasing salt concentration, i.e., a hydrophobic interaction chromatography (HIC)-like phenomenon was observed. This suggests that the antibody immobilized on the column has the property of utilizing hydrophobic interactions to bind. In particular, a NaCl concentration of 1.0 M exhibited excellent compatibility between the separation ability from empty capsids and the elution amount of full capsids. In other words, a salt concentration of 1.0 M was found to be optimal, being a concentration high enough to maximize the difference in binding affinity between the full capsid and empty capsid, yet low enough to allow elution without significant binding to full capsids.

[0222] Test Example 7 (1) AAV full capsid sample or AAV empty capsid sample The same samples as in Test Example 5 (1) were prepared.

[0223] (2) Affinity chromatography step 1: The column (1 mL) obtained in Test Example 2 was loaded with the AAV full capsid sample or the AAV empty capsid sample obtained in (1) above at 1.5 × 10 11 The solution was injected at 25°C so as to achieve the capsid amount, and 1x PBS (pH 7.4, 25°C) was passed through for 5 CV at 1.5 mL / min at 25°C (passage was carried out under condition (I)). When the solution was passed through under condition (I), all of the AAV was captured by the affinity solid phase.

[0224] Step 2: Then, as the mobile phase for elution, 20 mM citrate buffer (pH 3.5, 25° C.) containing 250 mM NaCl was passed through the column at 0.5 mL / min for 5 CV at 4° C. or 25° C. The eluate was collected.

[0225] (3) Results The elution amounts of each capsid at each temperature are shown in Figures 5A and 5B. As shown in Figures 5A and 5B, although the salt concentration in this test example was low, the binding ability to AAV empty capsids was higher than that to AAV full capsids at all temperatures. In particular, at a temperature of 4°C, both the separation ability from empty capsids and the elution amount of full capsids were superior. Considering that higher temperatures are expected to increase the likelihood of hydrophobic surface exposure on the capsid particle surface, for the samples subjected to affinity chromatography in this test example, a temperature condition of 4°C is considered to have better suppressed the exposure of the hydrophobic surface on full capsids, thereby maximizing the difference in hydrophobicity between full capsids and empty capsids as a difference in binding ability for the antibody immobilized on the column. This also suggests that the antibody has the property of binding via hydrophobic interactions.

[0226] On the other hand, when a sample containing many impurities other than AAV capsids was used instead of the sample in this test example (pure AAV capsid), the compatibility of the ability to separate from empty capsids and the amount of eluted full capsid was better at 25° C. than at 4° C. This is thought to be because, when a sample containing many impurities other than AAV capsids was used, the nonspecific binding of the impurities to the AAV capsid competed with each other, offsetting the effect of the temperature-induced exposure of the hydrophobic surface, and the rate of nonspecific binding and dissociation of the impurities was faster at 25° C. than at 4° C., increasing the opportunity for antibody contact and improving the binding efficiency of the AAV empty capsid.

Claims

1. An antibody that has binding ability to AAV capsids under a condition of pH 7.4 (I) and has higher binding ability to AAV empty capsids than to AAV full capsids under a condition of pH 3.0 (II).

2. The antibody described in claim 1, wherein, when an equal mixture of the AAV full capsid and the AAV empty capsid is subjected to the condition (II), the ratio of the amount of bound empty capsid to the amount of bound AAV full capsid is 3 or more when the amount of bound AAV full capsid is 1.

3. An antibody having a higher binding affinity to AAV empty capsid than to AAV full capsid under the condition (I) of pH 7.

4.

4. An antibody having one or more structural domains comprising CDR1 to CDR3 of the following [1] to [16], and having the ability to bind to an AAV capsid under the condition (I) at pH 7.4: [1] 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; [2] CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, 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: 6; [3] CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, 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: 9; [4] CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 10, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 11, 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: 12; [5] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 13, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 14, 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; [6] 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; [7] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 19, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 20, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 21; [8] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 22, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 23, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24;[9] CDR1 consisting of the amino acid sequence represented by SEQ ID NO:25, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:26, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:27; [10] CDR1 consisting of the amino acid sequence represented by SEQ ID NO:28, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:29, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:30; [11] CDR1 consisting of the amino acid sequence represented by SEQ ID NO:31, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:32, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:33; [12] CDR1 consisting of the amino acid sequence represented by SEQ ID NO:34, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:35, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:36; [13] CDR1 consisting of the amino acid sequence represented by SEQ ID NO:37, CDR2 consisting of the amino acid sequence represented by SEQ ID NO:38, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO:

39. [14] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 40, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 41, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 42; [15] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 43, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 44, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO:

45. [16] CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 46, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 47, and CDR3 consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 48.; 5. An antibody having one or more structural domains containing CDRs of any one of [1] to [16] below, and having the ability to bind to an AAV capsid under condition (I) at pH 7.4: [1] A CDR consisting of an amino acid sequence that has 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:49, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 113 to 123; [2] A CDR consisting of an amino acid sequence that has 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence of SEQ ID NO:50, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 115 to 125; [3] [4] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 51, excluding framework regions consisting of positions 1 to 28, 37 to 53, 62 to 99, and 120 to 130; [4] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 52, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 118 to 128; [5] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 53, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 114 to 124; [6] [6] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 54, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 118 to 128; [7] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 55, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 116 to 126;[8] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 56, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 112 to 122; [9] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 57, excluding framework regions consisting of positions 1 to 25, 34 to 50, 59 to 96, and 108 to 117; [10] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 58, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 113 to 123; [11] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 59, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 111 to 121; [12] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 60, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 112 to 122; [13] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 61, excluding framework regions consisting of positions 1 to 22, 31 to 47, 56 to 93, and 100 to 110; [14] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a structural domain consisting of the amino acid sequence represented by SEQ ID NO: 62, excluding framework regions consisting of positions 1 to 25, positions 34 to 50, positions 58 to 95, and positions 111 to 121;[15] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 63, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 105 to 115; [16] A CDR consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the structural domain consisting of the amino acid sequence represented by SEQ ID NO: 64, excluding framework regions consisting of positions 1 to 25, 34 to 50, 58 to 95, and 108 to 118; 6. The antibody described in claim 4 or 5, which has a higher binding ability to AAV empty capsid than to AAV full capsid under condition (II) of pH 3.

0.

7. The antibody described in claim 4 or 5, which has a higher binding ability to AAV empty capsid than to AAV full capsid under condition (I) of pH 7.

4.

8. An antibody according to any one of claims 1, 3, 4 and 5, which is a heavy chain antibody, a VHH antibody or a VHH antibody multimer.

9. An affinity solid phase comprising an antibody according to any one of claims 1, 3, 4 and 5 and a solid phase material on which the antibody is immobilized.

10. An affinity solid phase comprising the antibody according to any one of claims 1, 3, 6 and 7 and a solid phase material to which the antibody is immobilized, and used for purifying AAV full capsids from a sample containing AAV empty capsids and AAV full capsids.

11. A method for producing a sample from which AAV has been purified, comprising step 1 of contacting a sample containing AAV and impurities with the affinity solid phase described in claim 9, and step 2 of eluting the AAV.

12. In step 1, 10 10 The method of claim 11, wherein the AAV is captured in the capsid or larger form.

13. The method of claim 11, wherein step 1 is carried out at a pH of 6.5 to 8.5 and at 2 to 37°C, and step 2 is carried out at a pH of 1 to 5 and at 2 to 37°C.

14. A method for producing a sample from which AAV full capsids have been purified, comprising step 1' of contacting a sample containing AAV empty capsids and AAV full capsids with the affinity solid phase described in claim 10, and step 2' of eluting the AAV full capsids.

15. The method of claim 14, wherein step 1' is carried out at a pH of 6.5 to 8.5 and at 2 to 37°C, and step 2' is carried out at a pH of 1 to 5 and at 2 to 37°C.

16. The method of claim 14, wherein steps 1' and 2' are carried out at a pH of 1 to 5 and a temperature of 2 to 37°C.

17. The method of claim 14, wherein steps 1' and 2' are carried out at a pH of 6.5 to 8.5 and at 2 to 37°C.

18. The method of claim 14, wherein the elution solution in step 2' contains 0.13 to 2 M of a water-soluble salt.

19. A nucleic acid encoding the antibody of claim 4 or 5.

20. An expression cassette or recombinant vector comprising the nucleic acid of claim 19.

21. A transformant obtained by transforming a host with the expression cassette or recombinant vector according to claim 20.

22. A method for producing an antibody, comprising the step of culturing the transformant according to claim 21.

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