Adeno-associated virus antibodies and fragments thereof

Abstract

The present disclosure relates to isolated anti-AAV (adeno-associated virus) antibodies or antigen-binding fragments thereof capable of specifically binding to an epitope of the AAVrh74 capsid protein, and uses thereof. The present disclosure relates to murine, humanized, or chimeric antibodies that bind to an epitope of the AAV capsid protein. More specifically, the present disclosure provides isolated anti-AAV (adeno-associated virus) antibodies or antigen-binding fragments thereof capable of specifically binding to an epitope of the AAV capsid protein, wherein the epitope comprises the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45) or a portion thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 038,957, filed June 15, 2020, the contents of which are hereby incorporated by reference. Reference to electronically submitted sequence listing.

[0002] The contents of the electronically submitted Sequence Listing (Name: 8176WO00_SL; Size: 54 KB; and Creation Date: May 25, 2021) filed with this application as an ASCII text file are incorporated herein by reference in their entirety. [Background technology]

[0003] Adeno-associated viruses (AAVs) are a type of non-enveloped, single-stranded DNA parvovirus (approximately 25 nm). AAVs have become attractive vehicles for gene transfer due to their unique characteristics, such as their ability to transduce different types of dividing and non-dividing cells in different tissues and their ability to establish stable, long-term transgene expression. Furthermore, AAVs are not naturally pathogenic in humans. Over 100 human and non-human primate AAVs have been identified, including 12 serotypes with capsid amino acid sequence identity ranging from 51% to 99%.

[0004] AAV capsid proteins determine tissue tropism and are thus the primary interface between target tissues and AAV vectors. However, due to similarities in the structures and amino acid sequences of capsid proteins from some AAV serotypes used in gene therapy, antibodies that recognize capsid proteins of one AAV serotype cross-react with capsid proteins from other AAV serotypes. In particular, there is a need for antibodies that can distinguish or specifically identify AAVrh74 from other serotypes of AAV, such as AAV8 and / or AAV9. There is also a need for reliable methods for measuring or quantitating the level of AAVrh74-specific antibodies in the serum of patients undergoing gene therapy with AAVrh74. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure relates to a murine, humanized, or chimeric antibody that binds to an epitope of an AAV capsid protein. More specifically, the present disclosure provides an isolated anti-AAV (adeno-associated virus) antibody or antigen-binding fragment thereof capable of specifically binding to an epitope of an AAV capsid protein, wherein the epitope comprises the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45) or a portion thereof. In another aspect, the present invention relates to a murine, humanized, or chimeric antibody that binds to an epitope of an AAV rh74 capsid protein, wherein the epitope comprises the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45) or a portion thereof. In one embodiment, the antibody is a monoclonal or polyclonal antibody. In another embodiment, the capsid protein is an AAV rh74 capsid protein. In one embodiment, the antibody is an anti-AAV antibody.

[0006] The present disclosure further provides an isolated anti-AAV antibody, or antigen-binding fragment thereof, that specifically binds to an epitope within an AAVrh74 capsid protein, wherein the antibody competes with a reference antibody for binding to the epitope, wherein (a) the heavy chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:9, and SEQ ID NO:17; and (b) the light chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:10, and SEQ ID NO:18.

[0007] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a VH CDR1, a VH CDR2, and a VH CDR3 domain, and a light chain variable region comprising a VL CDR1, a VL CDR2, and a VL CDR3 domain, wherein (a) the VH CDR1 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO:58; (b) the VH CDR2 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:47, SEQ ID NO:53, and SEQ ID NO:59; (c) the VH CDR3 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:48, SEQ ID NO:54, and SEQ ID NO:60; (d) the VL CDR1 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:49, SEQ ID NO:55, and SEQ ID NO:61; and (e) the VL CDR1 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:49, SEQ ID NO:55, and SEQ ID NO:61. (f) the CDR2 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:56, and SEQ ID NO:62; or (f) the VL CDR3 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:51, SEQ ID NO:57, and SEQ ID NO:63.

[0008] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:9, and SEQ ID NO:17; and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:10, and SEQ ID NO:18.

[0009] In some aspects of the present disclosure, the isolated antibody or antigen-binding fragment thereof binds to a capsid protein of AAV8 and / or AAV9 with less affinity than the equivalent protein or capsid protein of AAVrh74.

[0010] In some aspects, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VH CDR3 selected from the group consisting of GVAHYSDSRFAFDY (SEQ ID NO: 35), GNAHPGGSAFVY (SEQ ID NO: 41), RGSYYYDSSPAWFAY (SEQ ID NO: 48), RGVDSSGYGAFAY (SEQ ID NO: 54), and TRGTSTMISTFAFVY (SEQ ID NO: 60).

[0011] In some aspects, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof further comprises a VH CDR1 selected from the group consisting of NYGMN (SEQ ID NO: 33), DYGMN (SEQ ID NO: 39), YTFTNYGMN (SEQ ID NO: 46), YTFTKYGMN (SEQ ID NO: 52), and YTFTNYGMN (SEQ ID NO: 58).

[0012] In some aspects, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof further comprises a VH CDR2 selected from the group consisting of WINTYTGEPTYADDFKG (SEQ ID NO: 34), WINTNTGEPTYGDDFKG (SEQ ID NO: 40), WMGWINTYTGEPTY (SEQ ID NO: 47), WMGWINTYTGEPTY (SEQ ID NO: 53), and WMGWINTYTGEPTY (SEQ ID NO: 59).

[0013] In some aspects, the light chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VL CDR1 selected from the group consisting of SVSSVSSYMH (SEQ ID NO: 36), SASSGVTYMH (SEQ ID NO: 42), SSVSSYMH (SEQ ID NO: 49), SSVSSYMH (SEQ ID NO: 55), and SSVRYMH (SEQ ID NO: 61).

[0014] In some aspects, the light chain variable region of the isolated antibody or antigen-binding fragment thereof further comprises a VL CDR2 selected from the group consisting of YTSNLAS (SEQ ID NO: 37), RTSNLAS (SEQ ID NO: 43), LWIYSTSNLAS (SEQ ID NO: 50), LWIYSTSNLAS (SEQ ID NO: 56), and VWIYSTSNLAS (SEQ ID NO: 62).

[0015] In some aspects, the light chain variable region of the isolated antibody or antigen-binding fragment thereof further comprises a VL CDR3 selected from the group consisting of QQRSSSYPFT (SEQ ID NO: 38), QQRSSSYPFT (SEQ ID NO: 44), QQRSTYPF (SEQ ID NO: 51), QQRSSFYPF (SEQ ID NO: 57), and QQRRTYYPF (SEQ ID NO: 63).

[0016] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises: a. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 33, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 34, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 35, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 36, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 37, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 38; b. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 39, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 40, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 41, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 42, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 43, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 44; c. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 46, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 47, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 48, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 49, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 50, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 51; d. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 52, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 53, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 54, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 55, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 56, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 57; and e. A heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3 domains, and a light chain variable region comprising VL CDR1, VL CDR2, and VL CDR3 domains, selected from the group consisting of VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 58, VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 59, VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 60, VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 61, VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 62, and VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 63.

[0017] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:1; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:2; b. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:5; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:6; c. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 13; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 14; d. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:9; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:10; or e. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 17; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 18.

[0018] In some aspects of the disclosure, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:9, and SEQ ID NO:17.

[0019] In some aspects, the light chain variable region of the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:10, and SEQ ID NO:18.

[0020] In some aspects of the disclosure, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:9, and SEQ ID NO:17; and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:10, and SEQ ID NO:18.

[0021] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2.

[0022] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6.

[0023] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14.

[0024] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10.

[0025] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:17 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:18.

[0026] In some aspects of the disclosure, the isolated antibody or antigen-binding fragment thereof is labeled with a radioactive, enzymatic, or fluorescent group.

[0027] In some aspects, the isolated antibody is a full-length antibody or an antibody fragment selected from the group consisting of a Fab, Fab', Fab'-SH, Fd, Fv, dAb, F(ab')2, scFv, bispecific single-chain Fv dimer, diabody, triabody, and sxFv genetically fused to the same or a different antibody.

[0028] In some aspects, the isolated antibody is a murine antibody, a chimeric murine / human antibody, a human antibody, an engineered antibody, or a humanized antibody.

[0029] In some aspects, the isolated antibody is a chimeric antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:21 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:22.

[0030] In some aspects, the isolated antibody is a chimeric antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:23 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:24.

[0031] In some aspects, the isolated antibody is a chimeric antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:25 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:26.

[0032] In some aspects, the isolated antibody is a chimeric antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:27 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:28.

[0033] In some aspects, the isolated antibody or antigen-binding fragment thereof is a bispecific antibody.

[0034] In some aspects, the isolated antibody or antigen-binding fragment thereof is a multispecific antibody.

[0035] The present disclosure also provides an isolated polynucleotide comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof described herein.

[0036] In some aspects, the polynucleotides of the present disclosure comprise nucleic acid sequences encoding: (a) a VH CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO:58; (b) a VH CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:47, SEQ ID NO:53, and SEQ ID NO:59; (c) a VH CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:48, SEQ ID NO:54, and SEQ ID NO:60; (d) a VL CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:49, SEQ ID NO:55, and SEQ ID NO:61; (e) a VL CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:56, and SEQ ID NO:62; and (f) a VL CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:51, SEQ ID NO:57, and SEQ ID NO:63.

[0037] In some aspects of the present disclosure, the nucleic acid sequence encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:11, and SEQ ID NO:19; and the nucleic acid sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:12, and SEQ ID NO:20.

[0038] The present disclosure also provides vectors comprising the polynucleotides described herein.

[0039] The present disclosure also provides host cells comprising the vectors described herein.

[0040] The present disclosure also provides in vitro detection kits comprising the isolated antibodies or antigen-binding fragments thereof described herein.

[0041] In some embodiments, the kit further comprises a second antibody or antigen-binding fragment thereof labeled with a radioactive, enzymatic, and / or fluorescent group.

[0042] The present disclosure further provides a method for detecting the presence of AAVrh74 capsid protein in a sample, comprising contacting the sample with a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0043] In some aspects, the presence of AAVrh74 capsid protein in a sample is indicated by detecting the presence of antibodies or antigen-binding fragments thereof.

[0044] In some aspects, the presence of the antibody or antigen-binding fragment thereof is detected by immunoassay.

[0045] In some aspects, the immunoassay comprises one or more of an immunofluorescence assay, an immunohistochemistry assay, a Western blot, a direct enzyme-linked immunosorbent assay (ELISA), an indirect ELISA, a sandwich ELISA, a competitive ELISA, a chemiluminescence assay, a radioimmunoassay, and an immunoprecipitation assay.

[0046] In some aspects, the method has a lower sensitivity for detecting AAV8 capsid protein and / or AAV9 capsid protein in a sample compared to AAVrh74 capsid protein. In some aspects, the isolated antibody or antigen-binding fragment thereof specifically binds to AAVrh74 capsid protein but does not bind to AAV8 capsid protein and / or AAV9 capsid protein in a sample.

[0047] The present disclosure also provides a method for producing an anti-AAV antibody or antigen-binding fragment thereof, comprising: (a) administering to a non-human vertebrate an immunogenic amount of a polypeptide comprising the amino acid sequence QGAGKDNVDYSS (SEQ ID NO:45); (b) recovering spleen cells from the animal; (c) fusing the recovered spleen cells with myeloma cells to generate hybridomas; (d) screening the hybridomas for those that produce an antibody that specifically binds to an AAV capsid protein; and (e) recovering the antibody. In another embodiment, the capsid protein is an AAVrh74 capsid protein. In one embodiment, the antibody is an anti-AAV antibody.

[0048] In some aspects, the non-human vertebrate is a transgenic animal, wherein the transgenic animal expresses human immunoglobulin genes.

[0049] In some aspects, the methods further comprise administering to the non-human vertebrate one or more immune adjuvants.

[0050] In some aspects, the non-human vertebrate is selected from a mouse, rat, hamster, guinea pig, rabbit, chicken, non-human primate, pig, goat, cow, and horse.

[0051] In some aspects, the antibody or antigen-binding fragment thereof specifically binds to AAVrh74 capsid protein with greater affinity than to other serotype o AAV capsids, e.g., AAV8 capsid protein and / or AAV9 capsid protein.

[0052] The present disclosure also provides a method for producing an anti-AAV antibody or antigen-binding fragment thereof, comprising: (a) immobilizing an antigen comprising the amino acid sequence QGAGKDNVDYSS (SEQ ID NO:45) on a solid support; (b) applying a phage antibody library to the immobilized antigen; (c) screening the library for phage that bind to the antigen; and (d) recovering the antigen-binding phage. In another embodiment, the capsid protein is an AAVrh74 capsid protein. In one embodiment, the antibody is an anti-AAV antibody.

[0053] In some aspects, the solid support is selected from the group consisting of a microtiter plate well, a polyvinylidene fluoride (PVDF) membrane, a column matrix, an immunotube, and a magnetic bead.

[0054] In some embodiments, the phage display antibody library is derived from a non-human vertebrate that has previously been immunized with a composition comprising an immunogenic amount of a polypeptide comprising the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45).

[0055] In some aspects, the non-human vertebrate is selected from a mouse, rat, hamster, guinea pig, rabbit, chicken, non-human primate, pig, goat, cow, and horse.

[0056] In some aspects, the antibody or antigen-binding fragment thereof specifically binds to AAVrh74 capsid protein, but does not bind to AAV8 capsid protein and / or AAV9 capsid protein.

[0057] The present disclosure also provides an in silico method for producing an anti-AAVrh74 antibody or antigen-binding fragment thereof, comprising: (a) in silico designing CDRs that specifically bind to an epitope on an AAV capsid protein; (b) grafting the CDRs onto single-chain variable fragments (scFvs); (c) using antibody phage display to screen the scFvs for binding to the target polypeptide; and (d) selecting scFvs that bind to the target polypeptide, wherein the epitope on the AAV capsid protein and the target polypeptide each comprise the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45). In another embodiment, the capsid protein is an AAVrh74 capsid protein. In one embodiment, the antibody is an anti-AAV antibody.

[0058] In some aspects, the antibody or antigen-binding fragment thereof specifically binds to AAVrh74 capsid protein with greater affinity than to AAV8 capsid protein and / or AAV9 capsid protein. In an embodiment of the present invention, for example, the following items are provided: (Item 1) An isolated anti-AAV (adeno-associated virus) antibody or antigen-binding fragment thereof capable of specifically binding to an epitope of an AAV capsid protein, wherein the epitope comprises the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45) or a portion thereof. (Item 2) An isolated anti-AAV antibody, or antigen-binding fragment thereof, that specifically binds to an epitope within an AAV capsid protein, wherein the antibody competes with a reference antibody for binding to the epitope; a. the heavy chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:9, and SEQ ID NO:17; and b. An isolated anti-AAV antibody or antigen-binding fragment thereof, wherein the light chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:10, and SEQ ID NO:18. (Item 3) 1. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a VH CDR1, a VH CDR2, and a VH CDR3 domain, and a light chain variable region comprising a VL CDR1, a VL CDR2, and a VL CDR3 domain, a. the VH CDR1 Domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO:58; b. the VH CDR2 Domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:47, SEQ ID NO:53, and SEQ ID NO:59; c. the VH CDR3 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:48, SEQ ID NO:54, and SEQ ID NO:60; d. the VL CDR1 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:49, SEQ ID NO:55, and SEQ ID NO:61; e. the VL CDR2 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:56, and SEQ ID NO:62; or f. An isolated antibody or antigen-binding fragment thereof, wherein the VL CDR3 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:51, SEQ ID NO:57, and SEQ ID NO:63. (Item 4) a. a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:9, and SEQ ID NO:17; and b. An isolated antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:10, and SEQ ID NO:18. (Item 5) 5. The isolated antibody or antigen-binding fragment thereof of any one of items 1 to 4, wherein the antibody binds to the capsid protein of AAV8 and / or AAV9 with less affinity than the capsid protein of AAVrh74. (Item 6) 6. The isolated antibody or antigen-binding fragment thereof of any one of items 1 to 5, wherein the heavy chain variable region comprises a VH CDR3 selected from the group consisting of GVAHYSDSRFAFDY (SEQ ID NO: 35), GNAHPGGSAFVY (SEQ ID NO: 41), RGSYYYDSSPAWFAY (SEQ ID NO: 48), RGVDSSGYGAFAY (SEQ ID NO: 54), and TRGTSTMISTFAFVY (SEQ ID NO: 60). (Item 7) 7. The isolated antibody or antigen-binding fragment thereof of any one of items 1 to 6, wherein the heavy chain variable region further comprises a VH CDR1 selected from the group consisting of NYGMN (SEQ ID NO: 33), DYGMN (SEQ ID NO: 39), YTFTNYGMN (SEQ ID NO: 46), YTFTKYGMN (SEQ ID NO: 52), and YTFTNYGMN (SEQ ID NO: 58). (Item 8) 8. The isolated antibody or antigen-binding fragment thereof of any one of items 1 to 7, wherein the heavy chain variable region further comprises a VH CDR2 selected from the group consisting of WINTYTGEPTYADDFKG (SEQ ID NO: 34), WINTNTGEPTYGDDFKG (SEQ ID NO: 40), WMGWINTYTGEPTY (SEQ ID NO: 47), WMGWINTYTGEPTY (SEQ ID NO: 53), and WMGWINTYTGEPTY (SEQ ID NO: 59). (Item 9) 9. The isolated antibody or antigen-binding fragment thereof of any one of items 1 to 8, wherein the light chain variable region comprises a VL CDR1 selected from the group consisting of SVSSSVSYMH (SEQ ID NO: 36), SASSGVTYMH (SEQ ID NO: 42), SSVSYMH (SEQ ID NO: 49), SSVSYMH (SEQ ID NO: 55), and SSVRYMH (SEQ ID NO: 61). (Item 10) 10. The antibody or antigen-binding fragment thereof according to any one of items 1 to 9, wherein the light chain variable region further comprises a VL CDR2 selected from the group consisting of YTSNLAS (SEQ ID NO: 37), RTSNLAS (SEQ ID NO: 43), LWIYSTSNLAS (SEQ ID NO: 50), LWIYSTSNLAS (SEQ ID NO: 56), and VWIYSTSNLAS (SEQ ID NO: 62). (Item 11) 11. The antibody or antigen-binding fragment thereof according to any one of items 1 to 10, wherein the light chain variable region further comprises a VL CDR3 selected from the group consisting of QQRSSYPFT (SEQ ID NO: 38), QQRSSYPFT (SEQ ID NO: 44), QQRSTYPF (SEQ ID NO: 51), QQRSFYPF (SEQ ID NO: 57), and QQRTYYPF (SEQ ID NO: 63). (Item 12) The antibody a. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 33, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 34, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 35, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 36, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 37, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 38; b. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 39, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 40, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 41, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 42, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 43, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 44; c. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 46, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 47, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 48, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 49, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 50, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 51; d. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 52, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 53, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 54, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 55, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 56, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 57; and e. VH CDR1 having the amino acid sequence shown in SEQ ID NO: 58, VH CDR2 having the amino acid sequence shown in SEQ ID NO: 59, and VH CDR3 having the amino acid sequence shown in SEQ ID NO: 60. 12. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 11, comprising a heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3 domains selected from the group consisting of a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 61, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 62, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 63. (Item 13) the antibody or antigen-binding fragment thereof a. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:1; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:2; b. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:5; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:6; c. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 13; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 14; d. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:9; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:10; or e. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 17; and 13. The isolated antibody or antigen-binding fragment thereof of claim 12, comprising a light chain variable region comprising an amino acid sequence that is about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the antibody or antigen-binding fragment thereof. (Item 14) 14. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 13, wherein the heavy chain variable region of the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 9, and SEQ ID NO: 17. (Item 15) 15. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 14, wherein the light chain variable region of the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 10, and SEQ ID NO: 18. (Item 16) a. the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:9, and SEQ ID NO:17; and b. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 15, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 10, and SEQ ID NO: 18. (Item 17) 17. The isolated antibody or antigen-binding fragment thereof according to any one of Items 1 to 16, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2. (Item 18) 17. The isolated antibody or antigen-binding fragment thereof according to any one of Items 1 to 16, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 6. (Item 19) 17. The isolated antibody or antigen-binding fragment thereof according to any one of Items 1 to 16, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14. (Item 20) 17. The isolated antibody or antigen-binding fragment thereof according to any one of Items 1 to 16, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10. (Item 21) 17. The isolated antibody or antigen-binding fragment thereof according to any one of Items 1 to 16, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18. (Item 22) 22. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 21, wherein the antibody or antigen-binding fragment thereof is labeled with a radioactive group, an enzymatic group, or a fluorescent group. (Item 23) 23. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 22, wherein the antibody is a full-length antibody or an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fd, Fv, dAb, F(ab')2, scFv, bispecific single-chain Fv dimers, diabodies, triabodies, and sxFv genetically fused to the same or a different antibody. (Item 24) 6. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 5, wherein the antibody is a murine antibody, a chimeric mouse / human antibody, a human antibody, an engineered antibody, or a humanized antibody. (Item 25) 25. The isolated antibody of item 24, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 22. (Item 26) 25. The isolated antibody of item 24, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24. (Item 27) 25. The isolated antibody of claim 24, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 25 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 26. (Item 28) 25. The isolated antibody of item 24, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 27 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28. (Item 29) 29. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 28, wherein the antibody or antigen-binding fragment thereof is a bispecific antibody. (Item 30) 30. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 29, wherein the antibody or antigen-binding fragment thereof is a multispecific antibody. (Item 31) 31. An isolated polynucleotide comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof according to any one of items 1 to 30. (Item 32) the nucleic acid sequence a. A VH CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 46, SEQ ID NO: 52, and SEQ ID NO: 58; b. A VH CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 53, and SEQ ID NO: 59; c. A VH CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 48, SEQ ID NO: 54, and SEQ ID NO: 60; d. A VL CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:49, SEQ ID NO:55, and SEQ ID NO:61; e. a VL CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO: 56, and SEQ ID NO: 62; or f. The polynucleotide of paragraph 31, encoding a VL CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 57, and SEQ ID NO: 63. (Item 33) a. the nucleic acid encoding the heavy chain variable region comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:11, and SEQ ID NO:19; and b. The polynucleotide of item 31 or 32, wherein the nucleic acid encoding the light chain variable region comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:12, and SEQ ID NO:20. (Item 34) A vector comprising the polynucleotide according to any one of Items 31 to 33. (Item 35) A host cell containing the vector described in item 34. (Item 36) 31. An in vitro detection kit comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 30. (Item 37) 37. The kit of item 36, further comprising a second antibody or antigen-binding fragment thereof labeled with a radioactive group, an enzymatic group, and / or a fluorescent group. (Item 38) 31. A method for detecting the presence of AAVrh74 capsid protein in a sample, comprising contacting the sample with a composition comprising the isolated antibody or antigen-binding fragment thereof of any one of paragraphs 1 to 30. (Item 39) 39. The method of claim 38, wherein the presence of AAVrh74 capsid protein in the sample is indicated by detecting the presence of the antibody or antigen-binding fragment thereof. (Item 40) 40. The method of claim 39, wherein the presence of the antibody or antigen-binding fragment thereof is detected by immunoassay. (Item 41) 41. The method of claim 40, wherein the immunoassay comprises one or more of an immunofluorescence assay, an immunohistochemical assay, a Western blot, a direct enzyme-linked immunosorbent assay (ELISA), an indirect ELISA, a sandwich ELISA, a competitive ELISA, a reverse ELISA, a chemiluminescence assay, a radioimmunoassay, or an immunoprecipitation assay. (Item 42) 42. The method of any one of items 38 to 41, wherein the method has a lower sensitivity for detecting AAV8 capsid protein and / or AAV9 capsid protein in the sample compared to AAVrh74 capsid protein. (Item 43) 43. The method of claim 42, wherein the isolated antibody or antigen-binding fragment thereof specifically binds to AAVrh74 capsid protein but does not bind to AAV8 capsid protein and / or AAV9 capsid protein in the sample. (Item 44) A method for producing an anti-AAV antibody or antigen-binding fragment thereof, comprising: (a) administering to a non-human vertebrate an immunogenic amount of a polypeptide comprising the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45); (b) recovering spleen cells from the animal; (c) fusing the recovered spleen cells with myeloma cells to produce hybridomas; (d) screening for hybridomas that produce antibodies that specifically bind to AAVrh74 capsid protein; and (e) recovering the antibodies. (Item 45) 45. The method of claim 44, wherein the non-human vertebrate is a transgenic animal, and the transgenic animal expresses human immunoglobulin genes. (Item 46) 46. ​​The method of claim 44 or 45, further comprising administering to the non-human vertebrate one or more immune adjuvants. (Item 47) 47. The method according to any one of items 44 to 46, wherein the non-human vertebrate is selected from a mouse, a rat, a hamster, a guinea pig, a rabbit, a chicken, a non-human primate, a pig, a goat, a cow, and a horse. (Item 48) 48. The method of any one of items 44 to 47, wherein the antibody or antigen-binding fragment thereof specifically binds to AAVrh74 capsid protein but does not bind to AAV8 capsid protein and / or AAV9 capsid protein. (Item 49) A method for producing an anti-AAV antibody or antigen-binding fragment thereof, comprising: (a) immobilizing an antigen comprising the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45) on a solid support; (b) applying a phage display antibody library to the immobilized antigen; (c) screening the library for phage that bind to the antigen; and (d) recovering antigen-binding phage. (Item 50) 50. The method of claim 49, wherein the solid support is selected from the group consisting of a microtiter plate well, a polyvinylidene fluoride (PVDF) membrane, a column matrix, an immunotube, and a magnetic bead. (Item 51) 51. The method of claim 49 or 50, wherein the phage display antibody library is derived from a non-human vertebrate previously immunized with a composition comprising an immunogenic amount of a polypeptide comprising the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45). (Item 52) 52. The method of claim 51, wherein the non-human vertebrate is selected from a mouse, a rat, a hamster, a guinea pig, a rabbit, a chicken, a non-human primate, a pig, a goat, a cow, and a horse. (Item 53) 53. The method of any one of items 49 to 52, wherein the antibody or antigen-binding fragment thereof specifically binds to AAVrh74 capsid protein but does not bind to AAV8 capsid protein and / or AAV9 capsid protein. (Item 54) An in silico method for producing an anti-AAV antibody or antigen-binding fragment thereof, comprising: (a) in silico designing CDRs that specifically bind to an epitope on an AAV capsid protein; (b) grafting the CDRs onto single-chain variable fragments (scFvs); (c) screening the scFvs for binding to the target polypeptide using antibody phage display; and (d) selecting scFvs that bind to the target polypeptide, wherein the epitope on the AAVrh74 capsid protein and the target polypeptide each comprise the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45). (Item 55) 55. The method of claim 54, wherein the antibody or antigen-binding fragment thereof specifically binds to AAVrh74 capsid protein with higher affinity than to AAV8 capsid protein and / or AAV9 capsid protein. (Item 56) 31. A method for detecting a pre-existing antibody against an AAV capsid in a sample from a subject, the method comprising subjecting the sample to an assay, wherein the antibody or antigen-binding fragment thereof according to any one of items 1 to 30 is used as a positive control. (Item 57) 57. The method of claim 56, wherein the assay is an immunoassay. (Item 58) 57. The method of claim 56, wherein the assay is an immunofluorescence assay, an immunohistochemical assay, a Western blot, a direct enzyme-linked immunosorbent assay (ELISA), an indirect ELISA, a sandwich ELISA, a competitive ELISA, a reverse ELISA, a chemiluminescence assay, a radioimmunoassay, or an immunoprecipitation assay. (Item 59) 57. The method of claim 56, wherein the assay is an electrochemiluminescence immunoassay (ECLIA). (Item 60) 57. The method of claim 56, further comprising quantifying the pre-existing antibody through absorbance readings. [Brief explanation of the drawings]

[0059] [Figure 1] FIG. 1 shows ELISA data for screening antibodies that bind to AAVrh74.

[0060] [Figure 2] FIG. 2 shows ELISA data to screen for antibodies that bind AAVrh74 but have less, limited, or no binding to AAV8 and / or AAV9.

[0061] [Figure 3A] Figures 3A-3C show ELISA data for the titration curves of chimeric IgG1 10D2 AAVrh74 antibody and chimeric IgG 28D8 AAVrh74 antibody that bind to AAVrh74. Figure 3A shows ELISA data for the titration curve of chimeric IgG1 10D2. [Figure 3B] Panel B shows the ELISA data for the titration curve of chimeric IgG1 28D8. [Figure 3C]FIG. 3C shows an overlay graph for the titration curves of chimeric IgG 10D2 and chimeric IgG1 28D8.

[0062] [Figure 4A] Figures 4A-4D show ELISA data for the titration curve of chimeric IgG1 10D2 AAVrh74 antibody that binds to AAVrh74 but has little or no binding to AAV8 and / or AAV9. Figure 4A shows ELISA data for the titration curve of chimeric IgG1 10D2 that binds to AAVrh74. [Figure 4B] Figure 4B shows ELISA data for the titration curve of chimeric IgG1 10D2 binding to AAV8. [Figure 4C] Figure 4C shows ELISA data for the titration curve of chimeric IgG1 10D2 binding to AAV9. [Figure 4D] FIG. 4D shows the ELISA data for the positive and negative controls.

[0063] [Figure 5A] Figures 5A-5D show ELISA data for the titration curve of chimeric IgG1 28D8 AAVrh74 antibody that binds to AAVrh74 but has little or no binding to AAV8 or AAV9. Figure 5A shows ELISA data for the titration curve of chimeric IgG1 28D8 that binds to AAVrh74. [Figure 5B] Figure 5B shows ELISA data for the titration curve of chimeric IgG1 28D8 binding to AAV8. [Figure 5C] Figure 5C shows ELISA data for the titration curve of chimeric IgG1 28D8 binding to AAV9. [Figure 5D] FIG. 5D shows the ELISA data for the positive and negative controls.

[0064] [Figure 6A]6A-6C show ELISA data for the titration curves of chimeric IgA 10D2 AAVrh74 antibody and chimeric IgA 28D8 AAVrh74 antibody that bind to AAVrh74. Figure 6A shows ELISA data for the titration curve of chimeric IgA 10D2. [Figure 6B] FIG. 6B shows ELISA data for the titration curve of chimeric IgA 28D8. [Figure 6C] FIG. 6C shows a graph of the overlay of titration curves for chimeric IgA 10D2 and chimeric IgA 28D8.

[0065] [Figure 7A] Figures 7A-7D show ELISA data for the titration curve of chimeric IgA 10D2 AAVrh74 antibody that binds to AAVrh74 but has little or no binding to AAV8 or AAV9. Figure 7A shows ELISA data for the titration curve of chimeric IgA 10D2 binding to AAVrh74. [Figure 7B] FIG. 7B shows ELISA data for the titration curve of chimeric IgA 10D2 binding to AAV8. [Figure 7C] FIG. 7C shows ELISA data for the titration curve of chimeric IgA 10D2 binding to AAV9. [Figure 7D] FIG. 7D shows the ELISA data for the positive and negative controls.

[0066] [Figure 8A] Figures 8A-8D show ELISA data for the titration curve of chimeric IgA 28D8 AAVrh74 antibody that binds to AAVrh74 but has little or no binding to AAV8 or AAV9. Figure 8A shows ELISA data for the titration curve of chimeric IgA 28D8 binding to AAVrh74. [Figure 8B] Figure 8B shows ELISA data for the titration curve of chimeric IgA 28D8 binding to AAV8. [Figure 8C]FIG. 8C shows ELISA data for the titration curve of chimeric IgA 28D8 binding to AAV9. [Figure 8D] FIG. 8D shows the ELISA data for the positive and negative controls.

[0067] [Figure 9A] Figures 9A-9E show ELISA data for titration curves of monoclonal antibodies 7D4B9, 1C4F4, and 6E10B5, which bind to AAVrh74 with much higher affinity than AAV8 or AAV9. Figure 9A shows the titration curve and cross-reactivity for 7D4B9. [Figure 9B] FIG. 9B shows the titration curve and cross-reactivity for 1C4F4. [Figure 9C] FIG. 9C shows the titration curve and cross-reactivity for 6E10B5. [Figure 9D] FIG. 9D shows the data for the positive and negative controls used in the assay. [Figure 9E] Figure 9E shows an overlay of titration curves for 7D4B9, 1C4F4, and 6E10B5. All three antibodies show serotype-specific binding to AAVrh74 with relatively low cross-reactivity with AAV8 and / or AAV9. DETAILED DESCRIPTION OF THE INVENTION

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including definitions, will control. Unless otherwise required by context, the singular shall include the plural and the plural shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety and for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0069] Throughout this disclosure, the terms "a" or "an" entity refer to one or more of that entity; for example, a polynucleotide is understood to refer to one or more polynucleotides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0070] Furthermore, "and / or," as used herein, is to be understood as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" when used in a phrase, such as "A and / or B" herein, is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" when used in a phrase, such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0071] The term "about" is used herein to mean approximately, roughly, around, or within that range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. Generally, the term "about" is used herein to modify a numerical value above and below the stated value by above or below (higher or lower) a variance of 10 percent.

[0072] As used herein, "antibody" refers to an intact immunoglobulin, an antigen-binding fragment thereof, or an antigen-binding molecule. The antibodies of the present disclosure can be of any isotype or class (e.g., IgM, IgD, IgG, IgE, and IgA), or any subclass (e.g., IgG1-4, IgA1-2), or any type of subclass (e.g., IgG2a, IgG2b), and can have either a kappa (κ) or lambda (λ) light chain. Antigen-binding fragments of antibodies include, for example, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies (scAbs), single-domain antibodies (dAbs), single-domain heavy-chain antibodies, single-domain light-chain antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins comprising the antigen-binding portions of an antibody and a non-antibody protein. The antibodies of the present disclosure can be labeled, for example, with a radioactive, enzymatic, or fluorescent group. The antibodies of the disclosure can also be conjugated to other moieties, such as members of specific binding pairs, such as biotin (a member of the biotin-avidin specific binding pair). The antibodies of the disclosure can also be bound to a solid support, such as a polystyrene plate or beads.

[0073] As used herein, a "monoclonal antibody" is an antibody produced by a group of identical cells, all of which have been produced from a single cell by repeated cell replication. That is, a clone of cells only produces a single antibody species. Monoclonal antibodies can be produced using hybridoma production techniques or using other production methods known to those skilled in the art, including, for example, antibody phage display libraries.

[0074] As used herein, the term "heavy chain" refers to an antibody heavy chain consisting of a variable region and a constant region. As used herein, the term "light chain" refers to an antibody light chain consisting of a variable region and a constant region.

[0075] The term "full-length antibody" refers to an antibody comprising two full-length antibody heavy chains and two full-length antibody light chains. For example, a full-length IgG antibody heavy chain is a polypeptide consisting of, from N- to C-terminus, an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH-HR-CH2-CH3. A full-length antibody light chain is a polypeptide consisting of, from N- to C-terminus, an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated as VL-CL. The antibody light chain constant domain (CL) can be κ (kappa) or λ (lambda). The two full-length antibody domains are linked together via interpolypeptide disulfide bonds between the CL and CH1 domains and between the hinge regions of the full-length antibody heavy chain. Full-length antibodies can be of any isotype or class (e.g., IgM, IgD, IgG, IgE, and IgA) or any subclass (e.g., IgG1-4, IgA1-2) or any type of subclass (e.g., IgG2a, IgG2b).

[0076] As used herein, as mentioned above, the term "capsid" or "capsid protein" refers to the proteinaceous shell or coat of a viral particle. The capsid functions to encapsidate, protect, transport, and release the viral genome into the host cell. Capsids are generally composed of protein oligomeric structural subunits ("capsid proteins"), such as VP1, VP2, and VP3. As used herein, the term "encapsidated" means enclosed within a viral capsid. For example, the AAVrh74 capsid sequence is disclosed in U.S. Patent No. 9,434,928, the contents of which are incorporated by reference in its entirety.

[0077] As used herein, and as mentioned above, "complementarity determining region" (CDR) describes the non-contiguous antigen binding sites (also known as antigen binding regions) found within the variable regions of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Department of Health and Human Services, "Sequences of proteins of Immunological interest" (1991) (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared against each other. However, application of either definition to refer to a CDR of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers encompassing a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine which residues comprise a particular CDR, given the amino acid sequence of the variable region of an antibody. As used herein, the terms "CDRL1," "CDRL2," and "CDRL3" refer to the first, second, and third CDRs, respectively, in a light chain variable region. As used herein, the terms "CDRH1," "CDRH2," and "CDRH3" refer to the first, second, and third CDRs, respectively, in a heavy chain variable region.

[0078] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. In some aspects, the term "isolated" is used interchangeably with the term "recombinant." As used herein, the terms "isolated" and "recombinant" refer to a polypeptide or nucleotide formed by laboratory methods, such as molecular cloning. In some aspects, the antibody is purified (a) to greater than 90%, 95%, or 98%, e.g., greater than 99% by weight of the antibody as determined by the Lowry method; (b) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer; or (c) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells because at least one component of the antibody's natural environment will not be present. In some aspects, isolated antibody is prepared by at least one purification step.

[0079] As used herein, the term "percent (%) identity" is defined as the percentage of nucleotide or amino acid residues in a sequence that are identical to those in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. For example, the percent (%) amino acid sequence identity of an antibody refers to the antibody sequence that is identical to the amino acid residues in a reference antibody sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA program package. In one embodiment, sequence comparison is performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are available from the European Bioinformatics Institute (EBI) at ebi.ac.uk / Tools / psa. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0080] Those skilled in the art will understand that the generation of sequence alignments for the calculation of percent sequence identity is not limited to binary sequence-to-sequence comparisons driven solely by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One suitable program for generating multiple sequence alignments is ClustalW2, available from clustal.org. Another suitable program is MUSCLE, available from drive5.com / muscle / . ClustalW2 and MUSCLE are alternatively available, for example, from EBI.

[0081] As used herein, the term "80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical" as used in this disclosure means that all such sequences are at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, This means either about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or 100% identity (completely identical).

[0082] The term "epitope" refers to the region of an antigen bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0083] The term "binding" refers to a direct association between two molecules, e.g., due to covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including, for example, salt bridges and water bridges. The subject anti-AAV antibodies specifically bind to an epitope within the AAVrh74 capsid protein.

[0084] As used herein, the term "murine antibody" includes antibodies whose variable and constant region sequences are derived from a mouse.

[0085] As used herein, the term "chimeric antibody" includes antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., antibodies in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0086] As used herein, the term "humanized antibody" includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences as well as within the CDR sequences derived from the germline of the other mammalian species.

[0087] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human and / or that has been made using any of the techniques for making human antibodies known in the art or disclosed herein. This definition of a human antibody includes antibodies that comprise at least one human heavy chain polypeptide or at least one human light chain polypeptide.

[0088] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Preferably, the subject is a human. Anti-AAVrh74 antibody

[0089] The present disclosure provides an isolated anti-AAV (adeno-associated virus) antibody or antigen-binding fragment thereof capable of specifically binding to an epitope of an AAVrh74 capsid protein or a recombinant AAV vector on the AAVrh74 capsid protein. In certain embodiments, the epitope within the AAVrh74 capsid protein comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45) or a portion thereof. In some embodiments, the amino acid sequence of the epitope within the AAVrh74 capsid protein is QGAGKDNVDYSS (SEQ ID NO: 45).

[0090] In some aspects, the present disclosure provides an isolated anti-AAV antibody or antigen-binding fragment thereof that specifically binds to an epitope within the AAVrh74 capsid protein, where the antibody competes with a standard antibody for binding to the epitope. Competitive antibodies or antigen-binding fragments thereof can be identified, for example, using an antibody competition assay. Details of procedures for conducting such competition assays are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pages 567-569. For purposes of this disclosure, an anti-AAVrh74 antibody or fragment thereof that competes with a reference antibody is one that reduces binding of the reference antibody to a target polypeptide by at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0091] In some aspects, the heavy chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 9, and SEQ ID NO: 17. In some aspects, the light chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 10, and SEQ ID NO: 18. In some aspects, the heavy chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 9, and SEQ ID NO: 17, and the light chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 10, and SEQ ID NO: 18.

[0092] In a particular embodiment, the heavy chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 1, and the light chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the heavy chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 5, and the light chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 6. In another embodiment, the heavy chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 13, and the light chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 14. In another embodiment, the heavy chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 10. In another embodiment, the heavy chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 17, and the light chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0093] The present disclosure further provides an isolated anti-AAV antibody or antigen-binding fragment thereof that binds to the same epitope within the AAVrh74 capsid protein as a reference antibody. Assays for identifying antibodies that bind to the same epitope within a particular protein are known to those skilled in the art. For example, such assays are described in detail in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, Chapter 14.

[0094] In some aspects, the heavy chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 9, and SEQ ID NO: 17. In some aspects, the light chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 10, and SEQ ID NO: 18. In some aspects, the heavy chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 9, and SEQ ID NO: 17, and the light chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 10, and SEQ ID NO: 18.

[0095] In a particular embodiment, the heavy chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 1, and the light chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the heavy chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 5, and the light chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 6. In another embodiment, the heavy chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 13, and the light chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 14. In another embodiment, the heavy chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 10. In another embodiment, the heavy chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 17, and the light chain variable region of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0096] The isolated anti-AAVrh74 antibody or antigen-binding fragment thereof of the present disclosure specifically binds to an epitope within the AAVrh74 capsid protein. In one embodiment, the antibody or antigen-binding fragment thereof binds to VP1, VP2, and / or VP3 of the AAVrh74 capsid protein. In some embodiments, the isolated antibody or antigen-binding fragment thereof has higher affinity for binding to the AAVrh74 capsid compared to AAV8 or AAV9 capsid. In some embodiments, the isolated antibody or antigen-binding fragment thereof does not bind to the AAV8 capsid protein. In some embodiments, the isolated antibody or antigen-binding fragment thereof does not bind to the AAV9 capsid protein. In some aspects, the isolated antibody or antigen-binding fragment thereof does not bind to AAV8 and / or AAV9.

[0097] In some aspects of the disclosure, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VH CDR3 selected from the group consisting of GVAHYSDSRFAFDY (SEQ ID NO: 35), GNAHPGGSAFVY (SEQ ID NO: 41), RGSYYYDSSPAWFAY (SEQ ID NO: 48), RGVDSSGYGAFAY (SEQ ID NO: 54), and TRGTSTMISTFAFVY (SEQ ID NO: 60).

[0098] In some aspects, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VH CDR1 selected from the group consisting of NYGMN (SEQ ID NO: 33), DYGMN (SEQ ID NO: 39), YTFTNYGMN (SEQ ID NO: 46), YTFTKYGMN (SEQ ID NO: 52), and YTFTNYGMN (SEQ ID NO: 58).

[0099] In some aspects, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VH CDR2 selected from the group consisting of WINTYTGEPTYADDFKG (SEQ ID NO: 34), WINTNTGEPTYGDDFKG (SEQ ID NO: 40), WMGWINTYTGEPTY (SEQ ID NO: 47), WMGWINTYTGEPTY (SEQ ID NO: 53), and WMGWINTYTGEPTY (SEQ ID NO: 59).

[0100] In some aspects, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VH CDR3 having the amino acid sequence GVAHYSDSRFAFDY (SEQ ID NO: 35), a VH CDR1 having the amino acid sequence NYGMN (SEQ ID NO: 33), and a VH CDR2 having the amino acid sequence WINTYTGEPTYADDFKG (SEQ ID NO: 34).

[0101] In some aspects, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VH CDR3 having the amino acid sequence GNAHPGGSAFVY (SEQ ID NO: 41), a VH CDR1 having the amino acid sequence DYGMN (SEQ ID NO: 39), and a VH CDR2 having the amino acid sequence WINTNTGEPTYGDDFKG (SEQ ID NO: 40).

[0102] In some aspects, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VH CDR3 having the amino acid sequence RGSYYYDSSPAWFAY (SEQ ID NO: 48), a VH CDR1 having the amino acid sequence YTFTNYGMN (SEQ ID NO: 46), and a VH CDR2 having the amino acid sequence WMGWINTYTGEPT_ (SEQ ID NO: 47).

[0103] In some aspects, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VH CDR3 having the amino acid sequence RGVDSSGYGAFAY (SEQ ID NO: 54), a VH CDR1 having the amino acid sequence YTFTKYGMN (SEQ ID NO: 52), and a VH CDR2 having the amino acid sequence WMGWINTYTGEPTY (SEQ ID NO: 53).

[0104] In some aspects, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VH CDR3 having the amino acid sequence TRGTSTMISTFAFVY (SEQ ID NO: 60), a VH CDR1 having the amino acid sequence YTFTNYGMN (SEQ ID NO: 58), and a VH CDR2 having the amino acid sequence WMGWINTYTGEPTY (SEQ ID NO: 59).

[0105] In some aspects of the disclosure, the light chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VL CDR1 selected from the group consisting of SVSSSVSYMH (SEQ ID NO: 36), SASSGVTYMH (SEQ ID NO: 42), SSVSSYMH (SEQ ID NO: 49), SSVSSYMH (SEQ ID NO: 55), and SSVRYMH (SEQ ID NO: 61).

[0106] In some aspects, the light chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VL CDR2 selected from the group consisting of YTSNLAS (SEQ ID NO: 37), RTSNLAS (SEQ ID NO: 43), LWIYSTSNLAS (SEQ ID NO: 50), LWIYSTSNLAS (SEQ ID NO: 56), and VWIYSTSNLAS (SEQ ID NO: 62).

[0107] In some aspects, the light chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VL CDR3 selected from the group consisting of QQRSSSYPFT (SEQ ID NO: 38), QQRSSSYPFT (SEQ ID NO: 44), QQRSTYPF (SEQ ID NO: 51), QQRSSFYPF (SEQ ID NO: 57), and QQRRTYYPF (SEQ ID NO: 63).

[0108] In some aspects, the light chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VL CDR1 having the amino acid sequence SVSSVSSYMH (SEQ ID NO: 36), a VL CDR2 having the amino acid sequence YTSNLAS (SEQ ID NO: 37), and a VL CDR3 having the amino acid sequence QQRSSSYPFT (SEQ ID NO: 38).

[0109] In some aspects, the light chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VL CDR1 having the amino acid sequence SASSGVTYMH (SEQ ID NO: 42), a VL CDR2 having the amino acid sequence RTSNLAS (SEQ ID NO: 43), and a VL CDR3 having the amino acid sequence QQRSSSYPFT (SEQ ID NO: 44).

[0110] In some aspects, the light chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VL CDR1 having the amino acid sequence SSVSSYMH (SEQ ID NO: 49), a VL CDR2 having the amino acid sequence LWIYSTSNLAS (SEQ ID NO: 50), and a VL CDR3 having the amino acid sequence QQRSTYPF (SEQ ID NO: 51).

[0111] In some aspects, the light chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VL CDR1 having the amino acid sequence SSVSSYMH (SEQ ID NO: 55), a VL CDR2 having the amino acid sequence LWIYSTSNLAS (SEQ ID NO: 56), and a VL CDR3 having the amino acid sequence QQRSFYPF (SEQ ID NO: 57).

[0112] In some aspects, the light chain variable region of the isolated antibody or antigen-binding fragment thereof comprises a VL CDR1 having the amino acid sequence SSVRYMH (SEQ ID NO: 61), a VL CDR2 having the amino acid sequence VWIYSTSNLAS (SEQ ID NO: 62), and a VL CDR3 having the amino acid sequence QQRTYYPF (SEQ ID NO: 63).

[0113] In some aspects of the disclosure, the isolated antibody or antigen-binding fragment thereof comprises: a. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 33, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 34, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 35, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 36, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 37, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 38; b. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 39, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 40, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 41, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 42, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 43, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 44; c. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 46, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 47, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 48, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 49, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 50, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 51; d. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 52, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 53, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 54, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 55, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 56, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 57; and e. A heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3 domains, and a light chain variable region comprising VL CDR1, VL CDR2, and VL CDR3 domains, selected from the group consisting of VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 58, VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 59, VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 60, VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 61, VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 62, and VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 63.

[0114] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:1; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:2; b. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:5; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:6; c. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 13; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 14; d. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:9; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:10; or e. a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 17; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 18.

[0115] In some embodiments of the present disclosure, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof has an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:9, and SEQ ID NO:17; and further comprises a VH CDR1, a VH CDR2, and a VH CDR3 from an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:9, and SEQ ID NO:17.

[0116] In some aspects of the disclosure, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:9, and SEQ ID NO:17.

[0117] In some embodiments of the present disclosure, the light chain variable region of the isolated antibody or antigen-binding fragment thereof is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:10, and SEQ ID NO:18; and further comprises a VL CDR1, VL CDR2, and / or VL CDR3 from an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:10, and SEQ ID NO:18.

[0118] In some aspects, the light chain variable region of the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:10, and SEQ ID NO:18.

[0119] In some aspects, the heavy chain variable region of the isolated antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:9, and SEQ ID NO:17; and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:10, and SEQ ID NO:18.

[0120] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2.

[0121] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6.

[0122] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14.

[0123] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10.

[0124] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:17 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:18.

[0125] In some embodiments, the isolated antibodies of the disclosure are those presented in Table 1 below. [Table 1]

[0126] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1, a VH CDR2, and a VH CDR3 domain, and a light chain variable region comprising a VL CDR1, a VL CDR2, and a VL CDR3 domain, selected from the sequences presented in Table 2. [Table 2]

[0127] Standard assays for assessing the binding ability of an antibody to AAVrh74 or an AAVrh74 antibody are known in the art and include, for example, ELISA, BIAcore®, Western blot, RIA, and flow cytometry analysis. The binding kinetics (e.g., binding affinity, such as KD) of an antibody can also be assessed by standard assays known in the art, such as Scatchard or BIAcore® system analysis. Relative binding affinity, K i can be assessed by standard competition assays known in the art.

[0128] In some aspects, the isolated antibody or antigen-binding fragment thereof is labeled with a radioactive, enzymatic, or fluorescent group. Examples of groups for use in labeling antibodies include various enzymes, binding pairs, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable binding pairs include, but are not limited to, streptavidin / biotin and avidin / biotin; examples of suitable fluorescent groups include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, danyl chloride, or phycoerythrin; examples of luminescent materials include, but are not limited to, luminol; examples of bioluminescent materials include, but are not limited to, luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include, but are not limited to, 125 I, 131 I, 35 S, or 3 Including, but not limited to, H.

[0129] In some embodiments, the isolated antibody is a full-length antibody or an antibody fragment selected from the group consisting of a Fab, Fab', Fab'-SH, Fd, Fv, dAb, F(ab')2, scFv, bispecific single-chain Fv dimer, diabody, triabody, and sxFv genetically fused to the same or a different antibody.

[0130] In some embodiments, the isolated antibody is a murine antibody, a chimeric antibody, a human antibody, an engineered antibody, or a humanized antibody. In some aspects, the chimeric antibody is a chimeric mouse / human antibody.

[0131] In some aspects, the isolated antibody is a chimeric antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21. In some aspects, the isolated antibody is a chimeric antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 22. In some aspects, the isolated antibody is a chimeric antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 22.

[0132] In some aspects, the isolated antibody is a chimeric antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23. In some aspects, the isolated antibody is a chimeric antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24. In some aspects, the isolated antibody is a chimeric antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0133] In other aspects, the isolated antibody is a chimeric antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 25. In some aspects, the isolated antibody is a chimeric antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 26. In some aspects, the isolated antibody is a chimeric antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 25 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0134] In other aspects, the isolated antibody is a chimeric antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 27. In some aspects, the isolated antibody is a chimeric antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28. In some aspects, the isolated antibody is a chimeric antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 27 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 28.

[0135] In some embodiments, the isolated antibody is a chimeric antibody presented in Table 3: [Table 3-1] [Table 3-2]

[0136] In some embodiments, the isolated antibody or antigen-binding fragment thereof is a bispecific antibody. In some embodiments, the isolated antibody or antigen-binding fragment thereof is a multispecific antibody.

[0137] Nucleic acids, vectors, and host cells The present disclosure also provides isolated polynucleotides, including nucleic acids encoding antibodies and fragments thereof that bind to AAVrh74, vectors, and host cells containing the polynucleotide or vector. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it has been purified away from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other standard techniques well known in the art. See, e.g., F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Nucleic acids of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0138] Nucleic acids of the invention can be obtained using standard molecular biology techniques, e.g., cDNAs encoding the light and / or heavy chains of an antibody, or encoding the VH and / or VL segments, can be obtained using standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), one or more nucleic acids encoding the antibody can be recovered from the library. Methods for introducing exogenous nucleic acids into host cells are well known in the art and vary depending on the host cell used. Techniques include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, calcium chloride treatment, polyethyleneimine-mediated transfection, polybrene-mediated transfection, protoplast fusion, electroporation, virus or phage infection, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. In the case of mammalian cells, transfection can be either transient or stable.

[0139] In some aspects, a polynucleotide of the disclosure comprises a nucleic acid sequence encoding a VH CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO:58.

[0140] In some aspects, a polynucleotide of the disclosure comprises a nucleic acid sequence encoding a VH CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:47, SEQ ID NO:53, and SEQ ID NO:59.

[0141] In some aspects, a polynucleotide of the disclosure comprises a nucleic acid sequence encoding a VH CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:48, SEQ ID NO:54, and SEQ ID NO:60.

[0142] In some aspects, a polynucleotide of the disclosure comprises a nucleic acid sequence encoding a VL CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:49, SEQ ID NO:55, and SEQ ID NO:61.

[0143] In some aspects, a polynucleotide of the disclosure comprises a nucleic acid sequence encoding a VL CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:56, and SEQ ID NO:62.

[0144] In some aspects, a polynucleotide of the disclosure comprises a nucleic acid sequence encoding a VL CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:51, SEQ ID NO:57, and SEQ ID NO:63.

[0145] In some aspects, the polynucleotides of the disclosure comprise: (a) a nucleic acid sequence encoding a VH CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO:58; (b) a VH CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:47, SEQ ID NO:53, and SEQ ID NO:59; (c) a VH CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:48, SEQ ID NO:54, and SEQ ID NO:60; (d) a VL CDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:49, SEQ ID NO:55, and SEQ ID NO:61; (e) a VL CDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:56, and SEQ ID NO:62; and (f) a nucleic acid sequence encoding a VL CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:51, SEQ ID NO:57, and SEQ ID NO:63.

[0146] In some aspects of the present disclosure, the nucleic acid encoding the heavy chain variable region of the antibody of the fragment comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:11, and SEQ ID NO:19.

[0147] In some aspects of the present disclosure, the nucleic acid encoding the light chain variable region comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:12, and SEQ ID NO:20.

[0148] In some embodiments of the present disclosure, the nucleic acid encoding the heavy chain variable region of the antibody of the fragment comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:11, and SEQ ID NO:19; and the nucleic acid encoding the light chain variable region comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:12, and SEQ ID NO:20.

[0149] In some embodiments, the nucleic acid encoding the heavy chain variable region of the antibody of the fragment comprises the nucleotide sequence of SEQ ID NO:3, and the nucleic acid encoding the light chain variable region comprises the nucleotide sequence of SEQ ID NO:4.

[0150] In some embodiments, the nucleic acid encoding the heavy chain variable region of the antibody of the fragment comprises the nucleotide sequence of SEQ ID NO:7, and the nucleic acid encoding the light chain variable region comprises the nucleotide sequence of SEQ ID NO:8.

[0151] In some aspects, the nucleic acid encoding the heavy chain variable region of the antibody of the fragment comprises the nucleotide sequence of SEQ ID NO:11, and the nucleic acid encoding the light chain variable region comprises the nucleotide sequence of SEQ ID NO:12.

[0152] In some aspects, the nucleic acid encoding the heavy chain variable region of the antibody of the fragment comprises the nucleotide sequence of SEQ ID NO:15, and the nucleic acid encoding the light chain variable region comprises the nucleotide sequence of SEQ ID NO:16.

[0153] In some aspects, the nucleic acid encoding the heavy chain variable region of the antibody of the fragment comprises the nucleotide sequence of SEQ ID NO:19, and the nucleic acid encoding the light chain variable region comprises the nucleotide sequence of SEQ ID NO:20.

[0154] In some embodiments, the nucleic acids of the disclosure are sequences shown in Table 4. [Table 4-1] [Table 4-2]

[0155] Once DNA fragments encoding the VH and / or VL segments have been obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert heavy chain variable region DNA sequences into sequences encoding full-length heavy chain variable and constant region sequences, or fragments corresponding to the fragments described herein, such as Fab or scFv. In these manipulations, the VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked," as used in this context, is intended to mean that two DNA fragments are joined so that the amino acid sequences encoded by the two DNA fragments remain in frame. Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2, CH3). Sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat EA et al., supra), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1 (IGHG1), IgG2 (IGHG2), IgG3 (IGHG3), IgG4 (IGHG4), IgA1 (IGHA1), IgA2 (IGHA2), IgM (IGHM), IgD (IGHD), or IgE (IGHE) constant region.

[0156] For nucleic acids encoding a Fab fragment heavy chain, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region. Isolated DNA encoding the VL region can be converted to a full-length light chain (as well as a Fab light chain) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat EA et al., supra), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In some embodiments, the light chain constant region can be a kappa or lambda constant region, preferably a kappa constant region.

[0157] For nucleic acids encoding scFvs, the DNA fragments encoding the VH and VL are operatively linked to another fragment encoding a flexible linker, e.g., an amino acid linker sequence, so that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions being connected by the flexible linker (see, e.g., Bird RE et al., (1988) Science, 242:423-426; Huston JS et al., (1988) Proc. Natl. Acad. Sci. USA, 85:5879-83; McCafferty J et al., (1990) Nature, 348:552-554). Various techniques have been developed for the production of antibody fragments of antibodies. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto K et al., (1992) J. Biochem. & Biophysical Methods, 24:107-117 and Brennan M et al., (1985) Science, 229:81-3). However, these fragments can also be produced directly by recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter P et al., (1992) Bio / Technology, 10:163-167). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In some embodiments, the antibody of choice is a single-chain Fv fragment (scFv).

[0158] The nucleic acid encoding the antibody of the present disclosure may be incorporated into a vector, preferably an expression vector, to express the protein. Various expression vectors may be utilized for protein expression. Expression vectors may include self-replicating extrachromosomal vectors or vectors that integrate into the host genome. Expression vectors are constructed to be compatible with the host cell type. Thus, vectors, preferably expression vectors, find use in the present invention and include, but are not limited to, vectors that enable protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As is known in the art, various expression vectors are commercially or otherwise available that may find use in the present disclosure for expressing antibodies. An example of a suitable expression vector is the pcDNA3.1 expression vector (Thermo Fisher Scientific).

[0159] Expression vectors typically contain a protein operably linked to control or regulatory sequences, selectable markers, optional fusion partners, and / or additional elements. As used herein, "operably linked" means that a nucleic acid is placed into a functional relationship with another nucleic acid sequence. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Generally, these expression vectors contain transcriptional and translational regulatory nucleic acid operably linked to the antibody-encoding nucleic acid and are typically appropriate for the host cell used to express the protein. Generally, transcriptional and translational regulatory sequences may include promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. As is also known in the art, expression vectors typically contain a selection gene or marker that allows for the selection of transformed host cells containing the expression vector. Selection genes are well known in the art and vary depending on the host cell used. For example, typically, the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0160] In some embodiments, a vector of the present disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a vector comprises the nucleic acid sequence set forth in SEQ ID NO: 30. In some embodiments, a vector comprises the nucleic acid sequence set forth in SEQ ID NO: 31. In some embodiments, a vector comprises the nucleic acid sequence set forth in SEQ ID NO: 32.

[0161] Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryotic cells, yeast cells, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, Gram-negative or Gram-positive bacteria, such as Enterobacteriaceae, such as Escherichia, for example, E. coli, Enterobacter, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescans, and Shigella, as well as Bacillus, for example, Bacillus subtilis and B. licheniformis, Pseudomonas, for example, Pseudomonas aeruginosa, and Streptomyces. Suitable E. coli cloning hosts include E. coli 294 (ATCC 31,446), E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325).

[0162] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, numerous other genera, species, and strains are commonly available and useful, such as Schizosaccharolis pombe; Kluyveromyces hosts (K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickerami (ATCC 24,178), K. waItH (ATCC 56,500), K. drosopumarum (ATCC 24,178); and others). 36,906), K. thermotolerans, or K. marcianuschalowia (EP 402226); Pichia pastoris (EP 183070); Candida; Trichoderma lecia (EP 244234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi (including Neurospora, Penicillium, Tolypocladium, or Aspergillus hosts, such as A. nidulans or A. niger).

[0163] Suitable host cells for expression of the antibodies of the present invention are derived from multicellular organisms. Examples of invertebrate cells include pratlyl and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (catellia), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0164] Host cells for expressing the recombinant antibodies of the invention also include mammalian host cells, including, but not limited to, Chinese hamster ovary (CHO cells), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is grown. Host cells useful for producing antibodies that bind to the AAVrh74 capsid protein may be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland), Minimal Essential Medium (MEM; Sigma-Aldrich Chemie GmbH), RPMI-1640 (Sigma-Aldrich Chemie GmbH, Basel, Switzerland), and Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich Chemie GmbH), are suitable for culturing the host cells. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0165] Antibodies may be operatively linked to fusion partners to enable targeting of the expressed protein, purification, screening, display, etc. The fusion partner may be linked to the antibody sequence via a linker sequence. Linker sequences generally contain a small number of amino acids, typically less than 10, although longer linkers may also be used. Typically, linker sequences are selected to be flexible and resistant to degradation. As will be appreciated by those skilled in the art, any of a wide variety of sequences may be used as a linker. For example, a common linker sequence includes the amino acid sequence G4S. The fusion partner may be a targeting or signal sequence that directs the antibody and any associated fusion partner to a desired cellular location or to the extracellular medium. As is known in the art, certain signaling sequences can target proteins located between the inner and outer membranes of a cell, either secreted into the growth medium or into the periplasmic space. The fusion partner may also be a sequence encoding a peptide or protein that allows for purification and / or screening. Such fusion partners include polyhistidine tags (His-tags) (e.g., H6 and H10), or immobilized metal affinity chromatography (IMAC) systems (e.g., Ni +2 Other tags that may be used include, but are not limited to, GST fusions, MBP fusions, Strep-tags, the BSP biotinylation target sequence of the bacterial enzyme BirA, and epitope tags targeted by antibodies (e.g., c-myc tags, Flag tags, etc.). As will be appreciated by those of skill in the art, such tags may be useful for purification, for screening, or both.

[0166] Construction and production of antibodies The present disclosure further provides methods for producing anti-AAV antibodies or antigen-binding fragments thereof. In some aspects, the methods of the present disclosure include producing antibodies using hybridoma technology. Techniques for producing hybridoma cells are well known to those of skill in the art. In some aspects, the present disclosure provides methods for producing antibodies, including: (a) administering to a non-human vertebrate an immunogenic amount of a polypeptide comprising the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45); (b) recovering spleen cells from the animal; (c) fusing the recovered spleen cells with myeloma cells to generate hybridomas; (d) screening the hybridomas for those that produce antibodies that specifically bind to the AAVrh74 capsid protein; and (e) recovering the antibodies. Administration of the polypeptide to an animal can be carried out using well-known, routine protocols. See, for example, Handbook of Experimental Immunology (Weir DM (ed.), Vol. 4, Blackwell Scientific Publishers, Oxford, England, 1986).

[0167] In some embodiments, the non-human vertebrate is a transgenic animal, and the transgenic animal expresses human immunoglobulin genes. In such aspects, the transgenic animal produces human antibodies directed against AAVrh74.

[0168] In some embodiments, the methods of the present disclosure further comprise administering to the non-human vertebrate one or more immune adjuvants.

[0169] In some embodiments, the non-human vertebrate is selected from a mouse, a rat, a hamster, a guinea pig, a rabbit, a chicken, a non-human primate, a pig, a goat, a cow, and a horse. In a particular aspect, the non-human vertebrate is a mouse. In another particular aspect, the non-human vertebrate is a rat.

[0170] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to AAVrh74 capsid protein, but does not bind to AAV8 capsid protein and / or AAV9 capsid protein.

[0171] In some aspects, the hybridoma expressing an antibody of the disclosure is a murine hybridoma, hi some aspects, the hybridoma is a hybridoma selected from the group consisting of 10D2-1, 28D8-1, 1C4F4, 6E10B5, and 7D4B9.

[0172] The present disclosure further provides a method for producing an anti-AAV antibody or antigen-binding fragment thereof using a phage display antibody library. Techniques for producing antibodies using a phage display antibody library are well known to those skilled in the art. In some embodiments of the present disclosure, the method includes: (a) immobilizing an antigen comprising the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45) on a solid support; (b) applying a phage display antibody library to the immobilized antigen; (c) screening the library for phages that bind to the antigen; and (d) recovering the antigen-binding phages.

[0173] Suitable supports are well known in the art and include, among others, commercially available column material, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, Duracyte, wells of reaction trays (e.g., multiwell plates), plastic tubes, and the like. Solid supports can comprise any of a variety of materials, including, for example, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose, and magnetite. Suitable methods for immobilizing antibodies of interest on solid supports are well known and include, but are not limited to, ionic, hydrophobic, covalent interactions, and the like. Solid supports can be soluble or insoluble, for example, in aqueous solutions. In some aspects, suitable solid supports are generally insoluble in aqueous solutions. In some embodiments, the solid support is selected from the group consisting of a microtiter plate well, a polyvinylidene fluoride (PVDF) membrane, a column matrix, an immunotube, and a magnetic bead.

[0174] In some embodiments, the phage display antibody library is derived from a non-human vertebrate that has previously been immunized with a composition comprising an immunogen. In some aspects, the immunogen comprises an immunogenic amount of a polypeptide comprising the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45).

[0175] In some embodiments, the non-human vertebrate is selected from a mouse, rat, hamster, guinea pig, rabbit, chicken, non-human primate, pig, goat, cow, and horse.

[0176] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to AAVrh74 capsid protein, but does not bind to AAV8 capsid protein and / or AAV9 capsid protein.

[0177] The present disclosure further provides an in silico method for producing an anti-AAV antibody or antigen-binding fragment thereof. In silico techniques for designing antibodies are known to those skilled in the art. In some aspects, the method includes: (a) in silico designing CDRs that specifically bind to an epitope on an AAVrh74 capsid protein; (b) grafting the CDRs onto single-chain variable fragments (scFvs); (c) screening the scFvs for binding to the target polypeptide using antibody phage display; and (d) selecting scFvs that bind to the target polypeptide, wherein the epitope on the AAVrh74 capsid protein and the target polypeptide each comprise the amino acid sequence QGAGKDNVDYSS (SEQ ID NO: 45).

[0178] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to AAVrh74 capsid protein, but does not bind to AAV8 capsid protein and / or AAV9 capsid protein.

[0179] Humanized antibodies of the present disclosure can be constructed by transferring one or more CDRs, or portions thereof, from a VH and / or VL region from a non-human animal (e.g., mouse) into one or more framework regions from a human VH and / or VL region. Optionally, human framework residues thus present in the VH and / or VL regions can be substituted with corresponding non-human (e.g., mouse) residues, as needed or desired to reduce immunogenicity of the antibody and / or maintain binding affinity. Optionally, non-human amino acid residues present in the CDRs can be substituted with human residues. Chimeric or humanized antibodies of the present invention can be prepared based on the sequences of non-human monoclonal antibodies prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the non-human hybridoma of interest and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, murine variable regions can be linked to human constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). To create a humanized antibody, murine CDR regions can be inserted into a human framework using methods known in the art (see, e.g., U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.).

[0180] The present invention provides a method for producing an antibody or fragment thereof that binds to AAVrh74, comprising culturing a host cell containing an isolated nucleic acid encoding an antibody or fragment thereof that binds to AAVrh74, or a vector containing an isolated nucleic acid encoding an antibody or fragment thereof that binds to AAVrh74, so that the nucleic acid is expressed to produce the antibody. Preferably, the antibody is isolated. The host cells, nucleic acids, and vectors described above can be used. Expression of the nucleic acid can be achieved, for example, by a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison S (1985) Science 229:1202) and as further outlined above. For example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the antibody of interest), and the DNA can be inserted into a vector, such as an expression vector. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to generate full-length antibody genes of any antibody isotype by inserting them into an expression vector already encoding heavy and light chain constant regions of the desired isotype, such that the VH segment is operably linked to the CH1 segment in the vector and the VK segment is operably linked to the CK segment in the vector.

[0181] Purification of anti-AAVrh74 antibody Screening for antibodies can be performed using antibody binding assays known to those skilled in the art. Detailed methods for performing antibody screening assays can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Such assays can be used to measure antibody binding to AAVrh74 capsid protein. An example of a binding assay is enzyme-linked immunosorbent assay (ELISA). In some aspects, the ELISA comprises a fusion protein of AAVrh74 capsid protein and human Fc, which is immobilized on a solid support, and a conjugated secondary antibody is used to detect anti-AAVrh74 antibodies bound to the fusion protein. The antibodies of the present invention can be used as a positive control for measuring anti-AAVrh74 antibodies in a sample (e.g., blood). In one embodiment, the ELISA is a direct ELISA, an indirect ELISA, a sandwich ELISA, a reverse ELISA, or a competitive ELISA. In one embodiment, the AAVrh74 antibodies of the present disclosure can be used in assays to detect pre-existing antibodies to AAVrh74 in serum or plasma from subjects, including mice, non-human primates, and humans. In one aspect, for any embodiment directed to detecting pre-existing antibodies, the pre-existing antibodies are contained in the serum or plasma of such subjects treated with AAV-based gene therapy. In one embodiment, the gene therapy is AAVrh74-based gene therapy. In another aspect of any embodiment directed to detecting pre-existing antibodies, the subject has not been treated with such gene therapy. The assay can be an ELISA or electrochemiluminescence immunoassay (ECLIA). In such assays, the AAVrh74 antibody serves as a positive control or capture. The ELISA approach optionally utilizes either serum or plasma known to have antibodies to AAVrh74 as an optional positive control.The assay involves detection of antibodies found in test serum or plasma using a substrate to bind to the antigen (AAVrh74 capsid), followed by quantification of the antibodies through absorbance readings. The mean optical density (OD) of wells receiving antigen is calculated relative to the OD of uncoated wells to determine the antibody endpoint titer. The ECLIA assay follows the same concept as indirect ELISA, identifying samples with or without anti-rh74 antibodies, determining the specificity of positive samples through competitive binding, and can determine antibody levels through titration methods.

[0182] Additionally, anti-AAVrh74 antibodies can be used to quantify capsids in gene therapy products through a sandwich ELISA, where the AAVrh74 antibody binds to a capture antibody, followed by the addition of a detector antibody, enzyme, and substrate, and quantification through absorbance readings.

[0183] In one aspect, the present disclosure provides a method for detecting pre-existing antibodies against AAV capsids in a sample from a subject, comprising subjecting the sample to an assay, wherein the antibody or antigen-binding fragment thereof described in any one of claims 1 to 30 is used as a positive control capture. In one embodiment, the assay is an immunoassay. In another embodiment, the assay is an immunofluorescence assay, an immunohistochemistry assay, a Western blot, a direct enzyme-linked immunosorbent assay (ELISA), an indirect ELISA, a sandwich ELISA, a competitive ELISA, a reverse ELISA, a chemiluminescence assay, a radioimmunoassay, or an immunoprecipitation assay. In another embodiment, the assay is an electrochemiluminescence immunoassay (ECLIA). In another embodiment, the method further comprises quantifying pre-existing antibodies through absorbance readings.

[0184] The antibodies of the present invention can be isolated or purified by various methods known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, size exclusion, or gel filtration, and reversed-phase, performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques are also useful in conjunction with protein concentration. To purify AAVrh74 antibodies, selected host cells can be grown, for example, in spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated before affinity chromatography using protein A-Sepharose (Pharmacia, Piscataway, NJ). Eluted antibodies can be checked by gel electrophoresis and high-performance liquid chromatography to ensure purity. Preferred antibodies of the present invention are thus isolated and / or purified antibodies that bind to the AAVrh74 capsid protein.

[0185] Immunological assays The present disclosure also provides a method for detecting the presence of AAVrh74 capsid protein in a sample, comprising contacting the sample with a composition comprising an antibody or antigen-binding fragment thereof described herein. In some aspects, the sample is an environmental sample. In some aspects, the sample is a biological sample, including, but not limited to, blood (e.g., whole blood), urine, saliva, plasma, lung lavage fluid, or lymphatic fluid. In some embodiments, the presence of AAVrh74 capsid protein in the sample is indicated by detecting the presence of the antibody or antigen-binding fragment thereof.

[0186] In some aspects of the present disclosure, the presence of an antibody or antigen-binding fragment thereof is detected by immunoassay. Suitable immunoassays for detecting the presence of an antibody or antigen-binding fragment are known to those skilled in the art. Examples of suitable immunoassays include, but are not limited to, immunofluorescence assays, immunohistochemistry assays, Western blots, direct enzyme-linked immunosorbent assays (ELISAs), indirect ELISAs, sandwich ELISAs, competitive ELISAs, chemiluminescence assays, radioimmunoassays, and immunoprecipitation assays. Methods for performing exemplary suitable immunoassays of the present disclosure can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

[0187] Immunofluorescence assays detect the expression and / or cellular location of proteins of interest. In direct immunofluorescence assays, a primary detection antibody is conjugated to a fluorophore. The labeled antibody bound to the protein of interest is detected using a fluorescence microscope. In indirect immunofluorescence assays, a secondary antibody is conjugated to a fluorophore and specifically binds to the primary antibody. The secondary antibody is then detected using a fluorescence microscope.

[0188] In some embodiments, the anti-AAVrh74 antibodies disclosed herein comprise a primary antibody in an immunofluorescence assay to detect the presence of AAVrh74 capsid protein in a sample.

[0189] Immunohistochemistry (IHC) assays detect the expression and / or cellular location of a protein of interest. In direct immunohistochemistry assays, a primary detection antibody conjugated to an enzyme binds to the protein of interest. Detection is achieved by assessing the presence of the conjugated enzyme via incubation with a substrate to produce a measurable product, such as color. In indirect IHC assays, a secondary antibody is conjugated to an enzyme and specifically binds to the primary antibody. Detection of the secondary antibody is achieved by assessing the presence of the conjugated enzyme via incubation with a substrate to produce a measurable product, such as color.

[0190] In some embodiments, the anti-AAVrh74 antibodies disclosed herein comprise a primary antibody in an immunohistochemistry assay to detect the presence of AAVrh74 capsid protein in a sample.

[0191] Western blots are used to detect a specific protein or proteins in a sample. In a Western blot, a protein sample is treated with detergent to unfold the proteins. Linear proteins are separated by size via gel electrophoresis (e.g., polyacrylamide gel electrophoresis, or PAGE) and then transferred to a blotting membrane (e.g., polyvinylidene difluoride-PVDF). The membrane is then incubated with a primary antibody that binds to the protein of interest. The primary antibody is then bound by a labeled secondary antibody. The labeled secondary antibody is linked to a reporter enzyme. Detection is achieved by assessing the presence of the conjugated enzyme on the secondary antibody via incubation with a substrate to produce a measurable product, such as color or light.

[0192] In some embodiments, the anti-AAVrh74 antibodies disclosed herein comprise a primary antibody in a Western blot to detect the presence of AAVrh74 capsid protein in a sample.

[0193] Pre-existing neutralizing antibody titers against AAV capsids and / or AAV serotypes, as well as their cross-reactivity, or those induced by administration, have emerged as concerns and challenges for the clinical application of AAV vector-mediated gene therapy and gene vaccines. Pre-existing Nabs that recognize viral capsids may inhibit AAV entry into host cells and transgene delivery, thereby hindering long-term treatment in humans. Determining the presence and cross-reactivity of neutralizing antibodies in subjects is essential for the clinical application of AAV vectors.

[0194] Thus, in another aspect, the present disclosure relates to a method for detecting the presence of an AAV antibody in a sample, comprising detecting the AAV antibody by immunoassay, wherein the immunoassay uses a composition comprising an isolated antibody or antigen-binding fragment thereof of the present disclosure. In one embodiment, the AAV antibody is an AAVrh74 antibody. In one embodiment, the sample is a biological sample from a subject. In one embodiment, the sample is blood, serum, plasma, body fluid, urine, or tissue from the subject. In one embodiment, the subject is a mammal carrying a genetic disorder. In one embodiment, the mammal is a human, pig, horse, cow, sheep, goat, monkey, rat, mouse, cat, or dog. In another embodiment, the subject is a patient. In another embodiment, the patient suffers from heart disease, muscular dystrophy, autoimmune disease, metabolic disorder, diabetes, eye disease, and / or kidney disease. In another embodiment, the patient suffers from Duchenne muscular dystrophy (DMD) or limb-girdle muscular dystrophy (LGMD). LGMD refers to a group of LGMD types classified by their associated genetic defect. Non-limiting examples of LGMD include LGMD1A, LGMD1B, LGMD1C, LGMD1D, LGMD1E, LGMD1F, LGMD1G, LGMD2A, LGMD2B, LGMD2C, LGMD2D, LGMD2E, LGMD2F, LGMD2H, LGMD2I, LGMD2J, LGMD2K, and LGMD2L.

[0195] In another embodiment, the immunoassay includes one or more of enzyme immunoassay (EIA), radioimmunoassay (MA), neutralization assay, fluoroimmunoassay (FIA) using fluorescent materials, chemiluminescent immunoassay (CLIA) using chemiluminescent materials, and counting immunoassay (CIA) using particle counting technology, as well as other modified assays such as Western blot, immunohistochemistry (IHC), and agglutination. One of the most common enzyme immunoassays is the enzyme-linked immunosorbent assay (ELISA). In one embodiment, the immunoassay includes one or more of an immunofluorescence assay, immunohistochemistry assay, Western blot, direct enzyme-linked immunosorbent assay (ELISA), indirect ELISA, sandwich ELISA, competitive ELISA, reverse ELISA, ECLIA, chemiluminescence assay, radioimmunoassay, or immunoprecipitation assay.

[0196] The terms "neutralization assay" and "serum virus neutralization assay" refer to serological tests for detecting the presence of systemic antibodies that can prevent viral infectivity. Such assays can also qualitatively or quantitatively identify the binding capacity (e.g., magnitude) or efficiency of antibodies to neutralize a target. The term "patient" includes humans and other mammalian subjects receiving either prophylactic or therapeutic treatment. Neutralization assays are specific immunoassays adapted to quantify the titer of virus-neutralizing antibodies. Typically, a sample, e.g., a serum sample and antibody solution, is diluted and mixed with a virus suspension. After incubation, the mixture is added to a confluent monolayer of host cells to allow the neutralizing antibodies to react with the virus. The infectivity of the virus to the host cells is quantified. The most common assay is called the plaque reduction neutralization test (PRNT). In this assay, the concentration of plaque-forming units is estimated by the number of plaques (areas of infected cells) formed in the culture after a period of incubation (typically several days). Depending on the virus, plaque-forming units are measured by microscopy, fluorescent antibodies, or specific dyes that react with infected cells. The concentration of serum that reduces the number of plaques by 50% compared to free virus in the serum provides a measure of the presence or effectiveness of antibodies. Virus neutralization assays are also widely used to detect and measure neutralizing antibodies against AAV. Several assays have been proposed in the art to detect neutralizing antibodies against different AAV serotypes. Some of these methods detect total binding antibodies against the AAV capsid, while others detect antibodies that neutralize AAV vector transduction in vitro or in vivo. Early methods for assessing total antibody responses to AAV vectors included ELISA and Western blot (Blacklow et al., J Natl Cancer Inst, 1968, 40(2):319; Mayor et al., Am J Obstet Gynecol, 1976, 126(1):100; and Parks et al., Infect Immun, 1970;2(6):716).ELISA can detect the total amount of antibodies in serum that bind to AAV serotypes, including non-neutralizing and neutralizing antibodies (Chirmule et al., Gene Ther., 1999, 6:1574-1583; Erles et al., J Med. Virol., 1999, 59:406-411; and Boutin et al., Hum. Gene Ther., 2010, 21:704-712). ELISA-based assays are easy to set up and provide a relatively sensitive measurement of total antibodies that bind to AAV, but the results do not necessarily reflect their neutralizing activity.

[0197] In another embodiment, the AAVrh74 antibody is a neutralizing antibody. In another aspect of any embodiment directed to the detection of pre-existing antibodies, the subject has not been treated with such gene therapy. The assay can be an ELISA or electrochemiluminescence immunoassay (ECLIA). In such an assay, the AAVrh74 antibody serves as a positive control or capture. The ELISA approach optionally utilizes either serum or plasma known to have antibodies against AAVrh74 as an optional positive control. The assay involves detecting antibodies found in the test serum or plasma using a substrate to bind to the antigen (AAVrh74 capsid), followed by quantification of the antibodies through absorbance readings. The average optical density (OD) of wells receiving the antigen is calculated relative to the OD of uncoated wells to determine the antibody endpoint titer. The ECLIA assay follows the same concept as indirect ELISA, identifying samples with or without anti-rh74 antibodies, determining the specificity of positive samples through competitive binding, and determining antibody levels through titration methods.

[0198] ELISA is an assay designed to detect and quantify substances such as peptides, proteins, and antibodies. In ELISA, an antigen is immobilized on a solid surface and then complexed with an antibody linked to an enzyme. Detection is achieved by assessing the presence of the conjugated enzyme via incubation with a substrate to produce a measurable product, such as color. The detection enzyme can be directly linked to the primary antibody (direct ELISA) or can be introduced through a secondary antibody that recognizes the primary antibody (indirect ELISA).

[0199] In some aspects, the anti-AAV antibodies disclosed herein comprise a primary antibody in a direct or indirect ELISA for detecting the presence of an AAV capsid protein in a sample. In one embodiment, the anti-AAV antibody is an anti-AAVrh74 antibody. The AAV capsid protein is an AAVrh74 capsid protein.

[0200] A sandwich ELISA is an ELISA in which a capture antibody is first immobilized on a solid support, followed by the addition of the protein of interest and a secondary antibody. In a sandwich ELISA, the protein of interest binds between the capture antibody immobilized on a solid surface and a detection antibody that binds to the protein of interest.

[0201] In some aspects, the anti-AAVrh74 antibodies disclosed herein comprise a capture antibody or a detection antibody in a sandwich ELISA to detect the presence of AAVrh74 capsid protein in a sample.

[0202] Competitive ELISA, also known as inhibition ELISA or competitive immunoassay, measures antigen concentration by detecting signal interference. The sample antigen competes with a reference antigen to bind to a specific amount of labeled antibody. The reference antigen is pre-coated on a multi-well plate. The sample is pre-incubated with the labeled antibody and added to the wells. Depending on the amount of antigen in the sample, more or less free antibody may be available to bind the reference antigen. Therefore, the more antigen present in the sample, the less reference antigen is detected, resulting in a weaker signal.

[0203] In some embodiments, the anti-AAVrh74 antibodies disclosed herein comprise a labeled antibody for detecting the presence of AAVrh74 capsid protein in a sample in a competitive ELISA.

[0204] Chemiluminescent assays utilize a luminescent chemical as a substrate instead of a chromogen. Common enzymes conjugated to detection antibodies include, but are not limited to, horseradish peroxidase (HRP) and alkaline phosphatase (AP). Luminol is a common chemiluminescent substrate used for the detection of HRP.

[0205] In some embodiments, the anti-AAVrh74 antibodies disclosed herein are conjugated to an enzyme that catalyzes a chemiluminescent reaction to detect the presence of AAVrh74 capsid protein in a sample.

[0206] In a radioimmunoassay, a known amount of antigen is radiolabeled and mixed with a known amount of antibody for that antigen. A sample containing an unknown amount of unlabeled antigen is then added. The unlabeled and radiolabeled antigen compete for antibody binding sites. As the concentration of unlabeled antigen increases, more radiolabeled antigen is displaced, and the ratio of antibody-bound to free radiolabeled antigen decreases. The bound radiolabeled antigen is then separated, and the radioactivity of the free (unbound) radiolabeled antigen remaining in the supernatant is measured using a gamma counter.

[0207] In some embodiments, the anti-AAVrh74 antibodies disclosed herein are used in radioimmunoassays to detect the presence of AAVrh74 capsid protein in a sample.

[0208] In an immunoprecipitation assay, an antibody specific for a protein of interest is used to isolate that protein from a solution containing many different proteins, such as a crude lysate of plant or animal cells or tissues, or a body fluid. In a direct immunoprecipitation assay, an antibody specific for the protein of interest is immobilized on a solid substrate, such as superparamagnetic microbeads or microscopic agarose beads. The beads with the bound antibody are then added to the solution, and the protein of interest binds to the antibody and is captured by the beads. The protein of interest is then eluted from the beads.

[0209] In an indirect immunoprecipitation assay, an antibody specific for the protein of interest is added directly to the solution. The antibody is not already attached to a solid support. Beads coated in Protein A / G are added to the mixture, where the antibody binds to the protein of interest and binds to the beads. The protein of interest is then eluted from the beads.

[0210] In some embodiments, the anti-AAVrh74 antibodies disclosed herein can be used to isolate AAVrh74 from a sample.

[0211] The immunoassays and methods described above are intended to be exemplary and non-limiting.

[0212] The methods of the present disclosure specifically detect the presence of AAVrh74 capsid protein in a sample. In some embodiments of the present disclosure, the methods do not detect AAV8 capsid protein. In some embodiments, the methods do not detect AAV9 capsid protein. In some embodiments, the methods do not detect AAV8 and / or AAV9 capsid protein in a sample.

[0213] kit Embodiments of the present disclosure are directed to in vitro detection kits comprising the antibodies or antigen-binding fragments thereof described herein as active agents, and instructions for use. Kits of the present disclosure may include, but are not limited to, kits for use in immunofluorescence assays, immunohistochemistry assays, Western blots, direct enzyme-linked immunosorbent assays (ELISAs), indirect ELISAs, sandwich ELISAs, reverse ELISAs, competitive ELISAs, chemiluminescence assays, radioimmunoassays, and immunoprecipitation assays. In some aspects, the kits may further comprise a secondary antibody or antigen-binding fragment thereof labeled with a radioactive, enzymatic, or fluorescent group. In some aspects, the kits include additional reagents, such as buffers, enzymes, and / or substrates, as well as a user manual for the kit.

[0214] In one embodiment, the present disclosure provides a method for detecting the presence of an AAV antibody in a sample, comprising detecting the AAV antibody by immunoassay, wherein the immunoassay uses a composition comprising an isolated antibody or antigen-binding fragment thereof of the present disclosure. In another embodiment, the immunoassay comprises an enzyme immunoassay (EIA), radioimmunoassay (MA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), enumeration immunoassay (CIA), neutralization assay, or immunohistochemistry (IHC). In another embodiment, the immunoassay comprises one or more of an immunofluorescence assay, an immunohistochemistry assay, a Western blot, a direct enzyme-linked immunosorbent assay (ELISA), an indirect ELISA, a sandwich ELISA, a competitive ELISA, a reverse ELISA, an ECLIA, a chemiluminescent assay, a radioimmunoassay, or an immunoprecipitation assay. In another embodiment, the immunoassay is a neutralization assay. In another embodiment, the AAV antibody is an AAVrh74 antibody. In another embodiment, the AAV antibody is a neutralizing antibody. In another embodiment, the sample is a biological sample from a subject. In one embodiment, the subject is a mammal carrying a genetic disorder. In one embodiment, the mammal is a human, pig, horse, cow, sheep, goat, monkey, rat, mouse, cat, or dog. In one embodiment, the subject is a human patient. In one embodiment, the patient suffers from heart disease, muscular dystrophy, autoimmune disease, metabolic disorder, diabetes, eye disease, and / or kidney disease. In one embodiment, the patient suffers from Duchenne muscular dystrophy (DMD) or limb-girdle muscular dystrophy (LGMD). In one embodiment, a composition comprising an isolated antibody or antigen-binding fragment thereof of the present disclosure serves as a positive control or capture. [Example]

[0215] The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0216] Example 1 Binding of anti-AAVrh74 antibodies to AAVrh74 capsid proteins. Antibodies generated according to the methods of the present disclosure were tested for their specific binding to AAVrh74.

[0217] An enzyme-linked immunosorbent assay was performed to measure antibody binding to AAVrh.74 capsid. Briefly, Immulon-4 HBX 96-well plates were filled with 100 μl per well of 2×10 mAb in carbonate buffer (pH 9.4). 10Plates were coated with 100 μl of AAVrh.74, AAV8, and AAV9 virus stocks at 37°C for 1 hour. Plates were sealed and stored at 4°C overnight. Plates were blocked with 100 μl per well of 5% nonfat dry milk and 1% normal goat serum in PBS for 1 hour at 37°C. Antibody stocks or samples were serially diluted (2-fold) in blocking solution, and 100 μl was added in duplicate to wells coated with AAV particles in carbonate buffer and wells coated with carbonate buffer alone. Nonhuman primate serum diluted 1:50 was used as a positive control for AAV8 and AAV9, while mouse anti-AAVrh74 serum was used as a positive control for mouse primary antibodies. Plates were incubated at room temperature for 1 hour before and after incubation, and wells were washed five times with 200 μl of PBS-T (0.05% Tween®). Blocking solution was again used to dilute the secondary antibody, goat anti-mouse IgG-HRP, at a 1:20,000 dilution. Wells received 100 μl of secondary antibody, washed five times, and incubated for 30 minutes at room temperature before being blotted dry. Tetramethybenzidine substrate (100 μl / well) was added and incubated in the dark for 2-3 minutes at room temperature before the reaction was stopped by adding 100 μl of 1N H2SO4. Absorbance at 450 nm was measured using an ELISA plate reader. Absorbance data for the anti-AAVrh74 antibody are shown in Figures 1 and 2.

[0218] Figure 1 shows the screening of antibodies for binding to AAVrh74. Figure 2 shows the testing of antibodies for those that specifically bind to AAVrh74 with much higher affinity compared to either AAV8 or AAV9. The results demonstrate serotype-specific binding to AAVrh74 with little or no cross-reactivity with AAV8 and / or AAV9.

[0219] Example 2 Binding of chimeric anti-AAVrh74 antibodies to AAVrh74 capsid proteins. Enzyme-linked immunosorbent assays were performed as above to measure binding of chimeric antibodies to AAVrh74 capsids at different antibody concentrations.

[0220] Figures 3A-3C show titration curves for chimeric IgG1 10D2 and chimeric IgG1 28D8. Binding of the chimeric antibodies to AAVrh74 was compared to the background signal at various antibody concentrations. Figure 3A shows the titration curve for the chimeric IgG1 10D2 antibody, with an endpoint titer of 1:1,638,400. Figure 3B shows the titration curve for the chimeric IgG1 28D8 antibody, with an endpoint titer of 1:638,400. Figure 3C shows an overlay of the curves for chimeric IgG1 10D2 and chimeric IgG1 28D8.

[0221] Cross-reactivity for chimeric IgG1 10D2 (Figures 4A-4D) and chimeric IgG1 28D8 (Figures 5A-5D) was tested at different antibody concentrations. Figures 4A-4C show that chimeric IgG1 10D2 specifically binds to AAVrh74 with much higher affinity than either AAV8 (Figure 4B) or AAV9 (Figure 4C) (Figure 4A). Positive and negative controls are shown in Figure 4D. The results demonstrate serotype-specific binding to AAVrh74 with little or no cross-reactivity with AAV8 and / or AAV9. Figures 5A-5D show that chimeric IgG1 28D8 specifically binds to AAVrh74 with much higher affinity than either AAV8 (Figure 5B) or AAV9 (Figure 5C) (Figure 5A). Positive and negative controls are shown in Figure 5D. The results demonstrate serotype-specific binding to AAVrh74 with little or no cross-reactivity with AAV8 and / or AAV9.

[0222] Figures 6A-6C show titration curves for chimeric IgA 10D2 and chimeric IgA 28D8. Binding of the chimeric antibodies to AAVrh74 was compared to background signal at various antibody concentrations. Figure 6A shows the titration curve for chimeric IgA 10D2, with an endpoint titer of 1:409600. Figure 6B shows the titration curve for chimeric IgA 28D8, with an endpoint titer of 1:819200. Figure 6C shows an overlay of the titration curves for chimeric IgA 10D2 and chimeric IgA 28D8.

[0223] Cross-reactivity for chimeric IgA 10D2 (Figures 7A-7D) and chimeric IgA 28D8 (Figures 8A-8D) was tested at different antibody concentrations. Figures 7A-7C show that chimeric IgA 10D2 specifically binds to AAVrh74 with much higher affinity than either AAV8 (Figure 7B) or AAV9 (Figure 7C) (Figure 7A). Positive and negative controls are shown in Figure 7D. The results demonstrate serotype-specific binding to AAVrh74 with little or no cross-reactivity with AAV8 and / or AAV9. Figures 8A-8C show that chimeric IgA 28D8 specifically binds to AAVrh74 with much higher affinity than either AAV8 (Figure 8B) or AAV9 (Figure 8C) (Figure 8A). Positive and negative controls are shown in Figure 8D. The results demonstrate serotype-specific binding to AAVrh74 with little or no cross-reactivity with AAV8 and / or AAV9.

[0224] Cross-reactivity for monoclonal antibodies 7D4B9 (Figure 9A), 1C4F4 (Figure 9B), and 6E10B5 (Figure 9C) was tested at different antibody concentrations. Enzyme-linked immunosorbent assays were performed to measure antibody binding to AAVrh.74 capsids. Briefly, Immulon-4 HBX 96-well plates were filled with 100 μl per well of 2×10 mAb in carbonate buffer (pH 9.4). 10Plates were coated with 100 μl of AAVrh.74, AAV8, and AAV9 virus stocks at 37°C for 1 hour. Plates were sealed and stored at 4°C overnight. Plates were blocked with 100 μl per well of 5% nonfat dry milk and 1% normal goat serum in PBS for 1 hour at 37°C. Antibody stocks or samples were serially diluted (10-fold) in blocking solution, and 100 μl was added in duplicate to wells coated with AAV particles in carbonate buffer and wells coated with carbonate buffer alone. Nonhuman primate serum diluted 1:50 was used as a positive control for AAV8 and AAV9, while mouse anti-AAVrh74 serum was used as a positive control for mouse primary antibodies. Plates were incubated at room temperature for 1 hour before and after incubation, and wells were washed five times with 200 μl of PBS-T (0.05% Tween®). Blocking solution was again used, and the secondary antibody, goat anti-mouse IgG-HRP, was diluted at a 1:10,000 dilution. Wells received 100 μl of secondary antibody, washed five times, and incubated for 30 minutes at room temperature before being blotted dry. Tetramethybenzidine substrate (100 μl / well) was added and incubated in the dark for 2-3 minutes at room temperature before the reaction was stopped by adding 100 μl of 1N H2SO4. Absorbance at 450 nm was measured using an ELISA plate reader.

[0225] Figures 9A-9C show that all three monoclonal antibodies (7D4B9, 1C4F4, 6E10B5) specifically bound to AAVrh74 with higher affinity than either AAV8 or AAV9. Positive and negative controls are shown in Figure 9D. Figure 9E shows an overlay of titration curves for 7D4B9, 1C4F4, and 6E10B5. All three antibodies demonstrate serotype-specific binding to AAVrh74 with relatively low cross-reactivity with AAV8 and / or AAV9.

[0226] Example 3 Sandwich ELISA: Determination of antibodies in serum material Capture antibody = anti-AAVrh74 mAb of the present invention

[0227] Test sample = serum or plasma is useful in detection.

[0228] Antigen = AAVrh74 capsid

[0229] Blocking solution = 5% dry milk, 1% goat serum, 100mL PBS

[0230] Wash buffer = 0.05% PBS-Tween®

[0231] Positive control = serum known to have anti-AAVrh74 antibodies

[0232] Secondary antibody = anti-human-HRP conjugated antibody

[0233] Substrate=TMB

[0234] Stop solution = sulfuric acid

[0235] method All wells of a 96-well plate are coated overnight at 4°C with capture antibody diluted in carbonate buffer. The contents are discarded, and the plate is blocked with blocking solution for 1 hour at 37°C. The blocking solution is discarded, and AAVrh74 capsids are added in duplicate to the capture antibody-coated wells. In addition, carbonate buffer is added to duplicate wells to determine background values. Unbound capsid is discarded, and test serum is added at a starting dilution of 1:25 in blocking solution and serially diluted. A positive control is diluted at a 1:400 dilution in blocking solution. The plate is washed with wash buffer, followed by a secondary incubation at a 1:10,000 dilution in blocking solution. The plate is washed, the buffer is discarded, and substrate is added, followed by termination of the assay with sulfuric acid. Plate absorbance is read at 450 nm. Analysis and Results

[0236] The absorbance ratio is determined by subtracting the mean optical density (OD) of the non-antigen coated wells from the mean OD of the antigen coated wells and dividing by the mean (OD) of the non-antigen coated wells. A ratio of ≥ 2.00 is considered a positive antibody response. The endpoint titer is determined by identifying the last serum dilution that results in a ratio of ≥ 2.00. The antibody cutoff is defined as a serum dilution of > 1:400. Example 4 Sandwich ELISA: Determination of vectors present in a sample OR vector inventory. material

[0237] Capture Ab: ADK8 clone

[0238] Test sample or antigen: Anti-AAVrh74 containing sample

[0239] Blocking solution: 5% dry milk, 1% goat serum or BSA, 100 mL PBS

[0240] Wash buffer = 0.05% PBS-Tween®

[0241] Detection antibody: anti-AAVrh74 mAb of the present invention (biotin conjugate)

[0242] Secondary antibody: HRP-conjugated streptavidin

[0243] Substrate: TMB

[0244] Stop solution: sulfuric acid

[0245] method The wells of a 96-well plate are coated with the desired amount of diluted capture antibody in carbonate buffer at 4°C. AAVrh74 capsid standard samples and test samples are serially diluted in assay buffer and added in duplicate to the corresponding capture antibody-coated wells. The plate is incubated at 37°C for 1 hour. The plate is washed before adding diluted 28D8 biotin antibody (mAb of the present invention) to all designated wells and incubating for 1 hour. The plate is washed, followed by a secondary incubation of diluted Strep-HRP (1:5000-1:10,000) for 1 hour. After washing the plate, pre-made TMB is added to each well and incubated at room temperature for 10-15 minutes. The reaction is stopped by adding sulfuric acid to each well, and the color intensity is measured in a photometer at a wavelength of 450 nm. Analysis and Results

[0246] The amount of capsid present in any given sample is extrapolated using the OD at 450 and the four parameter logistic curve (4PL) equation obtained from the standard curve.

[0247] Example 5 Electrochemiluminescence Immunoassay (ECLIA) ELISA: Determination of antibodies in serum material Capture reagent = AAVrh74 capsid or virus-like particle

[0248] Detection antibody = Sulfo-TAG-labeled anti-human IgG antibody

[0249] Test sample = serum or plasma

[0250] Positive control = AAVrh74 mAb of the present invention

[0251] Blocking solution = 5% dry milk, 1% goat serum or BSA, 100mL PBS

[0252] Wash buffer = 0.05% PBS-Tween®

[0253] Read buffer = MSD buffer or equivalent

[0254] Signal reactor = tripropylamine or equivalent

[0255] Method / Principle: ECL ELISA uses an electrochemiluminescent label conjugated to a detection antibody. The label is called SULFO-TAG and allows for ultrasensitive detection. Electricity is applied to the plate electrodes by specialized equipment, leading to light emission by the SULFO-TAG label. The light intensity is then measured to quantify the analyte in the sample.

[0256] The wells are coated with the capture reagent (AAVrh74 capsid) overnight at 4°C. Non-specific binding to the plate is blocked by adding blocking buffer for 1 hour. The blocking is discarded, followed by the addition of diluted test serum, negative, and control samples to designated wells. Diluted anti-AAVrh74 mAb of the present invention is used as a positive control. The wells are washed, and a detection antibody (diluted anti-human IgG sulfo-TAG) is added. The plate is washed with wash buffer, followed by the addition of read buffer to each well. After adding the read buffer, the plate is read or scanned immediately.

[0257] Analysis and Results The current is used to determine the presence of a signal reactant, the signal being related to the amount of antibody in the serum.

[0258] Surface plasmon resonance Introduction / Principles: Surface plasmon resonance (SPR) is a label-free detection method and a reliable platform for clinical analysis of biomolecular interactions. This technique allows for high sensitivity, label-free, and real-time interaction measurement. Surface plasmon resonance occurs when photons of incident light strike a metal surface (e.g., a gold surface). At a specific angle of incidence, a portion of the light energy couples through the metal coating to electrons in the metal surface layer, which then migrate due to excitation. The electrons, now called plasmons, propagate parallel to the metal surface. The plasmon oscillations then generate an electric field from the boundary between the metal surface and the sample solution. The defined SPR angle, at which resonance occurs under conditions of a constant light source wavelength and thin metal surface, depends on the refractive index of the material near the metal surface. Consequently, plasmons are not formed when there is a small change in the reflectivity of the sensing medium (e.g., through biomolecule attachment). Detection is thus achieved by measuring the resulting change in reflected light on a detector. The amount of surface concentration can also be quantified by monitoring the reflected light intensity or by tracking the resonance angle shift. Typically, SPR biosensors have detection limits on the order of 10 pg / mL.

[0259] Basic method: Surface plasmon resonance is performed using a HEPES buffer using an instrument such as a BIAcore™ T200. The mAb of the present invention is mixed with AAVrh74 at the desired concentration and immobilized on a sensor chip by amide coupling. The test analyte (serum sample) is run through the chip and different dilutions are recorded. The change in refractive index due to binding of AAVrh74-specific antibodies present in the serum sample to the immobilized AAV is recorded. Data and graphs are generated using BIAcore software. A standard curve can be generated, and the amount of antibody present in the serum can be extrapolated using the graph equation.

[0260] The foregoing descriptions of specific embodiments fully reveal the general nature of the present invention, such that others, by applying knowledge within the skill of the art, can readily modify and / or adapt such specific embodiments for various applications without departing from the general concepts of the disclosure and without undue experimentation. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It should be understood that the words and terms used herein are for the purpose of description, not limitation, as the words and terms used herein would be interpreted by one of ordinary skill in the art in light of the teaching and guidance.

Claims

1. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to an AAV capsid protein; a. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 33, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 34, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 35, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 36, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 37, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 38; b. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO:39, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:40, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:41, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:42, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:43, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:44; c. A VH CDR1 having the amino acid sequence set forth in SEQ ID NO:46, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:47, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:48, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:49, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:50, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:51; d. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO:52, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:53, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:54, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:55, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:56, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:57; and e. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO:58, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:59, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:60, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:61, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:62, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:63; a heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3 domains and a light chain variable region comprising VL CDR1, VL CDR2, and VL CDR3 domains selected from the group consisting of: An isolated antibody or antigen-binding fragment thereof.

2. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region of the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 9, and SEQ ID NO:

17.

3. 3. The isolated antibody or antigen-binding fragment thereof of claim 2, wherein the light chain variable region of the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 10, and SEQ ID NO:

18.

4. the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:9, and SEQ ID NO:17; and b) The isolated antibody or antigen-binding fragment thereof of claim 3, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:10, and SEQ ID NO:

18.

5. 5. The isolated antibody or antigen-binding fragment thereof of claim 4, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

2.

6. 5. The isolated antibody or antigen-binding fragment thereof of claim 4, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

6.

7. 5. The isolated antibody or antigen-binding fragment thereof of claim 4, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

14.

8. 5. The isolated antibody or antigen-binding fragment thereof of claim 4, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

10.

9. 5. The isolated antibody or antigen-binding fragment thereof of claim 4, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

18.

10. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds to the capsid protein of AAV8 and / or AAV9 with less affinity than the capsid protein of AAVrh74.

11. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is labeled with a radioactive group, an enzymatic group, or a fluorescent group.

12. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a full-length antibody or antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fd, Fv, dAb, F(ab')2, scFv, bispecific single-chain Fv dimers, diabodies, triabodies, and sxFv genetically fused to the same or a different antibody.

13. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a murine antibody, a chimeric mouse / human antibody, an engineered antibody, or a humanized antibody.

14. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is a bispecific antibody.

15. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is a multispecific antibody.

16. 10. A polynucleotide comprising a nucleic acid sequence encoding the isolated antibody or antigen-binding fragment thereof of claim 1, wherein the polynucleotide comprises: a. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 33, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 34, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 35, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 36, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 37, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 38; b. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO:39, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:40, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:41, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:42, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:43, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:44; c. A VH CDR1 having the amino acid sequence set forth in SEQ ID NO:46, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:47, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:48, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:49, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:50, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:51; d. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO:52, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:53, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:54, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:55, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:56, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:57; or e. a VH CDR1 having the amino acid sequence set forth in SEQ ID NO:58, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:59, a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:60, a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:61, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:62, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:63; A polynucleotide comprising a nucleic acid sequence encoding

17. the polynucleotide comprises a nucleic acid sequence encoding a heavy chain variable region and a nucleic acid sequence encoding a light chain variable region; a. the nucleic acid encoding the heavy chain variable region comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:11, and SEQ ID NO:19; and b) The polynucleotide of claim 16, wherein the nucleic acid encoding the light chain variable region comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:12, and SEQ ID NO:20.

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