Anti-AAV2.7m8 antibodies and methods of use thereof

Abstract

The present disclosure relates to antibodies that specifically bind to AAV2.7m8 capsid protein as antigens, and the use of these antibodies in assays for detecting and / or quantifying the presence of neutralizing antibodies during treatment with AAV2.7m8 gene therapy, and / or detecting and / or quantifying AAV2.7m8 capsid protein. The present disclosure also relates to a kit comprising the anti-AAV2.7m8 antibody of the present disclosure.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 591,622, filed October 19, 2023, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Invention Field

[0003] This disclosure relates to antibodies that specifically bind to the AAV2.7m8 capsid protein as an antigen, and the use of these antibodies in assays for detecting and / or quantifying the presence of neutralizing antibodies and / or detecting and / or quantifying the AAV2.7m8 capsid protein during AAV2.7m8 gene therapy treatment. This disclosure also relates to kits comprising the anti-AAV2.7m8 antibodies of this disclosure.

[0004] References to sequence lists

[0005] An official copy of this sequence list is submitted together with this specification as an ST26 format XML file named "17234-044WO1.xml", created on October 16, 2024, and is 22,160 bytes in size. This sequence list, submitted together, is an integral part of this specification and is incorporated herein by reference in its entirety. Background Technology

[0006] Recombinant viruses have shown great promise and practicality as delivery vehicles for therapeutic nucleic acids used in gene therapy applications. A variety of different recombinant viruses are used in these gene therapy applications based on many factors, including the size of the nucleic acid to be delivered, the target cells or tissues to which the nucleic acid is delivered, the need for short-term or long-term expression of the therapeutic nucleic acid, and the integration of the therapeutic nucleic acid into the recipient genome. Examples of viruses used in gene therapy applications include adeno-associated virus (AAV), adenovirus, lentivirus, and herpes simplex virus (HSV).

[0007] Subretinal (SR) administration of AAV-mediated gene therapy has proven effective in treating several ocular diseases. Available data suggest that neutralizing antibodies (nAbs) do not block AAV vector transduction after SR delivery due to ocular immune privilege. Intravitreal (IVT) administration is less invasive than SR and may be more beneficial for diseased, fragile retinas. IVT delivery of AAV vectors may also be advantageous for broader retinal gene expression. However, pre-existing nAbs may negatively impact transduction and gene expression after IVT delivery. Detection and quantification of nAbs before or after IVT administration are crucial for successful gene therapy treatment.

[0008] Therefore, there is a need in this field to accurately detect and quantify nAbs associated with AAV gene therapy. Summary of the Invention

[0009] This disclosure generally relates to the field of gene therapy using AAV vectors. Specifically, this disclosure relates to antibodies that specifically bind to the AAV2.7m8 capsid protein used in gene therapy. This disclosure also relates to the use of these anti-AAV2.7m8 antibodies for the detection and / or quantification of the AAV2.7m8 capsid protein. Furthermore, this disclosure relates to the use of these anti-AAV2.7m8 antibodies in assays (and kits) for the detection and / or quantification of neutralizing antibodies (nAbs) that may be present in subjects receiving gene therapy treatment containing the AAV2.7m8 capsid protein or as candidates for receiving said treatment.

[0010] This invention is intended to introduce the subject matter of this disclosure, but does not cover all and every embodiment, combination, or variation contemplated and described within this disclosure. Other embodiments are contemplated and described through the detailed description, drawings, and claims.

[0011] In at least one embodiment, this disclosure provides an antibody that specifically binds to the AAV2.7m8 capsid, wherein the antibody comprises the first light chain complementarity-determining region (CDR-L1) of SEQ ID NO: 5, the second light chain complementarity-determining region (CDR-L2) of SEQ ID NO: 6, the third light chain complementarity-determining region (CDR-L3) of SEQ ID NO: 7, the first heavy chain complementarity-determining region (CDR-H1) of SEQ ID NO: 13, the second heavy chain complementarity-determining region (CDR-H2) of SEQ ID NO: 14, and the third heavy chain complementarity-determining region (CDR-H3) of SEQ ID NO: 15.

[0012] In at least one embodiment of the antibody disclosed herein, the antibody comprises a light chain variable domain (VL) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 4; and / or a heavy chain variable domain (VH) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 12.

[0013] In at least one embodiment of the antibody disclosed herein, the antibody comprises: a light chain (LC) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 20; and / or a heavy chain (HC) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 21.

[0014] In at least one embodiment of the antibody disclosed herein, the antibody is: (i) a monoclonal antibody; (ii) a chimeric antibody; (iii) a humanized antibody; (iv) a full-length IgG antibody, optionally wherein the IgG antibody has an isotype selected from IgG1, IgG2, IgG3 and IgG4; (v) an antibody fragment, optionally selected from the group consisting of F(ab')2, Fab', Fab, Fv, single-domain antibody (VHH) and scFv; (vi) the antibody is a multispecific antibody, optionally a bispecific antibody; and / or (vii) the antibody is a synthetic antibody, wherein the HVR is grafted onto a scaffold or framework other than an immunoglobulin scaffold or framework; optionally, a scaffold selected from alternative protein scaffolds and artificial polymer scaffolds.

[0015] In at least one embodiment of the antibody disclosed herein, the antibody is an immunoconjugate, optionally wherein the immunoconjugate comprises a detectable portion. In at least one embodiment, the immunoconjugate comprises a detectable portion selected from radiolabeled, fluorescently labeled, and indirectly labeled portions.

[0016] In at least one embodiment of the antibody disclosed herein, the antibody is characterized by one or more of the following properties: (a) at a concentration of 1 x 10 -8 M or smaller, 1 x 10 -9 M or smaller, 1 x 10 -10 M or smaller or 1 x 10 -11 A binding affinity of M or less is required to bind to the AAV2.7m8 capsid protein; optionally, said binding affinity is determined by the equilibrium dissociation constant (K0) of the AAV2.7m8 capsid polypeptide. D (a) Measurement; or (b) Binding to the AAV2.7m8 capsid peptide of SEQ ID NO: 2 with a binding affinity at least 2, 3, 5, or 10 times greater than that to the AAV2 capsid protein; optionally wherein the relative binding affinity is measured by the equilibrium dissociation constant (K0) of the AAV2.7m8 capsid peptide of SEQ ID NO: 2 and the AAV2 capsid peptide of SEQ ID NO: 1. D )Measurement.

[0017] In at least one embodiment, this disclosure provides an antibody that specifically binds to the same AAV2.7m8 capsid protein epitope as the antibody of this disclosure; optionally, said epitope comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 22) located at positions 588-597 of SEQ ID NO: 2.

[0018] In at least one embodiment, this disclosure provides an antibody that competitively binds to an epitope of the AAV2.7m8 capsid protein and has a 1 x 10-1 -9 M or smaller, 1 x 10 -10 M or smaller or 1 x 10 -11 M or less binding affinity; optionally, said binding affinity is determined by the equilibrium dissociation constant (K0) of the AAV2.7m8 capsid peptide of SEQ ID NO: 2. D Measurement. In at least one embodiment, the epitope comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 22) located at positions 588-597 of SEQ ID NO: 2.

[0019] In other embodiments, this disclosure also provides an isolated polynucleotide or vector encoding the anti-AAV2.7m8 antibody of this disclosure.

[0020] In another embodiment, this disclosure provides an isolated host cell containing a polynucleotide or vector encoding the anti-AAV2.7m8 antibody of this disclosure.

[0021] In another embodiment, this disclosure also provides a method for generating the anti-AAV2.7m8 antibody of this disclosure, comprising culturing a host cell containing a polynucleotide or vector encoding the anti-AAV2.7m8 antibody to generate the antibody.

[0022] In at least one embodiment, this disclosure also provides a kit comprising: an assay reagent; and the anti-AAV2.7m8 antibody of this disclosure. In at least one embodiment, the assay reagent in the kit comprises: (a) an AAV2.7m8 vector encoding a luciferase gene; (b) a luciferase substrate; and / or (c) cells from a transducible cell line; optionally, said cells are from a 293T cell line.

[0023] In at least one embodiment, this disclosure also provides a method for detecting the activity of a neutralizing antibody against AAV2.7m8 in a human serum sample, the method comprising: (a) providing: (i) a human serum sample; (ii) an assay solution containing cells; (iii) a carrier solution containing an AAV2.7m8 vector encoding a luciferase gene; (iv) a luciferase substrate solution; and (v) an AAV2.7m8 antibody solution containing an antibody that specifically binds to the AAV2.7m8 capsid; (b) combining aliquots of the human serum sample, the assay solution, the carrier solution, and the luciferase substrate solution to form a sample solution; (c) combining aliquots of the AAV2.7m8 antibody solution, the assay solution, the carrier solution, and the luciferase substrate solution to form a positive control solution; and (d) detecting a luminescent signal from the sample solution and the positive control solution; thereby, the relative luminescent signal level indicates the presence and / or amount of neutralizing antibody activity in the human serum sample.

[0024] In at least one embodiment of the method, the antibody that specifically binds to the AAV2.7m8 capsid is the anti-AAV2.7m8 antibody of this disclosure.

[0025] In at least one embodiment of a method for detecting the activity of neutralizing antibodies against AAV2.7m8 in human serum samples, step (c) comprises combining at least 2, 3, 4, 5, 6, 7, 8, 9 or more different aliquots with different dilutions of the AAV2.7m8 antibody solution to form at least 2, 3, 4, 5, 6, 7, 8, 9 or more positive control solutions with different concentrations of AAV2.7m8 antibody for use in preparing reaction curves.

[0026] In at least one embodiment of a method for detecting the activity of a neutralizing antibody against AAV2.7m8 in a human serum sample, the method further includes the steps of: combining an aliquot of the assay solution and the luciferase substrate solution to form a negative control solution; and detecting a luminescent signal from the negative control solution.

[0027] In at least one embodiment of the method for detecting the activity of neutralizing antibodies against AAV2.7m8 in human serum samples, cells pre-exposed to an AAV2.7m8 vector encoding a luciferase gene are derived from a transducible cell line; optionally, said cell line is 293T.

[0028] In at least one embodiment, this disclosure also provides a method for detecting AAV2.7m8 capsid protein, the method comprising: contacting the protein with an anti-AAV2.7m8 antibody of this disclosure, and detecting the antibody bound to the protein. In at least one embodiment, the AAV2.7m8 capsid protein is immobilized. In at least one embodiment, the AAV2.7m8 capsid protein is in solution. In at least one embodiment, the antibody comprises a detectable label. In at least one embodiment, the detectable label is selected from radioactive labels, fluorescent labels, and indirect labeling portions.

[0029] Brief description of the attached figures

[0030] A better understanding of the novel features and advantages of this disclosure will be obtained by referring to the following detailed description and accompanying drawings (also referred to herein as "Figure" and "FIG."), which illustrate illustrative embodiments utilizing the principles of this disclosure, in which:

[0031] Figure 1A , 1B Octet BLI plots (1C, 1D, 1E, 1F, 1G, 1H, and 1I) depict nine rabbit anti-AAV2.7m8 antibodies (clones 1-8 and 11) prepared as described in Example 1 and for which AAV2.7m8 affinity was determined. The curve labeled "Antibody 1" corresponds to the rabbit anti-AAV2.7m8 antibody from "clone 1," and so on. "Antibody 6" corresponds to "clone 6," the anti-AAV2.7m8 antibody with the highest affinity, which was sequenced and used for the nAb determination in Example 2. Detailed Implementation

[0032] In the description of this document and the appended claims, unless the context clearly indicates otherwise, the singular form “a / an” includes plural references. Thus, for example, a reference to “protein” includes more than one protein, and a reference to “compound” refers to more than one compound. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a basis for using exclusive terms such as “only” or “just” in conjunction with the elements of the claim, or for using “negative” limitations. The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” is interchangeable and not intended to be restrictive. It should also be understood that when the term “comprising” is used in the description of various embodiments, those skilled in the art will appreciate that in certain specific cases, the language “consistently of” or “comprises of” may be used instead to describe the embodiments.

[0033] When providing numerical ranges, unless the context explicitly indicates otherwise, it should be understood that each intermediate integer of the value and each tenth of each intermediate integer of the value, unless the context explicitly indicates otherwise, is encompassed within the scope of this invention, including the upper and lower limits of the range and any other specified values ​​or intermediate values ​​within the stated range. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered by this invention, but are subject to any specific exclusions within the stated range.

[0034] Numerical ranges include the numbers that define the range. In this document, the term "approximately" is used to mean plus or minus ten percent (10%) of a value. For example, "approximately 100" refers to any value between 90 and 110. The term "approximately" for a range means that range minus 10% of its lowest value and plus 10% of its highest value. References to "approximately" for a value or parameter include (and describe) implementations for that value or parameter itself.

[0035] In this disclosure, the scientific format with exponent notation can be used, where a portion of the numerical value is replaced by E+n, where E (the exponent) multiplies the aforementioned number by 10 to the power of n. For example, the scientific format with two decimal places displays 12345678901 as 1.23E10, which is 1.23 multiplied by 10 to the power of 10, and can also be written alternatively as 1.23×10 10 Similarly, 1.23E-10 can also be written as 1.23×10. -10 .

[0036] Generally, the nomenclature used in this article and the techniques and procedures described herein include those techniques and methods that are well understood and commonly used by those skilled in the art, such as those commonly described in the following literature: Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Volumes 1–3, ColdSpring Harbor Laboratory, Cold Spring Harbor, NY, 2012 (hereinafter referred to as “Sambrook”); and Current Protocols in Molecular Biology, edited by FM Ausubel et al., originally published as a book by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. in 1987, and regularly supplemented until 2011, now available online in journal format as Current Protocols in Molecular Biology, Volumes 00–130 (1987–2020), published by Wiley & Sons, Inc. on Wiley Online Library (hereinafter referred to as “Ausubel”).

[0037] Unless otherwise stated, nucleic acids are written from left to right in the 5' to 3' orientation; amino acid sequences are written from left to right in the amino to carboxyl orientation.

[0038] 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 invention pertains. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive. For the purposes of interpreting this disclosure, the following description of terminology will apply, and where appropriate, terms used in the singular will also include plural forms, and vice versa.

[0039] The headings provided herein are not intended to limit the various aspects or embodiments of the invention, and are incorporated herein by reference in their entirety. Therefore, the terms directly defined below are incorporated herein by reference for a more complete definition.

[0040] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a / an” and “the” are intended to include the plural forms as used herein. Furthermore, the extent to which the terms “including,” “includes,” “having,” “has,” “with,” or variations thereof are used in the detailed description and / or claims is intended to be inclusive in a manner similar to the term “comprising.” As used herein, the term “comprising” is synonymous with “including” or “containing” and has an inclusive or open-ended meaning.

[0041] The terms “subject,” “patient,” or “individual” refer to mammals, including but not limited to primates, such as human and non-human primates (“NHPs”), such as African green monkeys and rhesus monkeys, mammalian competitive animals, mammalian farm animals, mammalian pets, and rodents. In some implementations, the subject is a human.

[0042] Biological samples that can be used in the methods of this disclosure include any sample obtained from a subject and containing a biomolecule (e.g., RNA or DNA) relevant to a method for measuring the expression level of a gene signature used to identify the risk of an inflammatory response. Therefore, it is contemplated that biological samples may include blood, vitreous fluid, cerebrospinal fluid, peritoneal fluid, pleural fluid, bronchoalveolar lavage fluid, sputum, nasal brush samples, throat swabs, urine, amniotic fluid, plasma, serum, saliva, semen, bone marrow, tissue or fine-needle biopsy samples, feces, skin, or cells of their origin. In another embodiment, the biological sample includes blood. Biological samples may be obtained from the subject at any point in time relevant to a particular method, such as before any rAAV treatment (e.g., screening potential candidates) or at any time during or after rAAV treatment (e.g., monitoring a patient). It is also contemplated that biological samples may include tissue sections, such as frozen sections for immunohistochemical analysis.

[0043] As used herein, the terms “treat,” “treating,” “treatment,” and other grammatical equivalents mean to alleviate, reduce, or improve a disease or condition or its symptoms; to prevent additional symptoms of said disease or condition; to improve or prevent the underlying cause of symptoms; to suppress a disease or condition, such as curbing its development; to alleviate a disease or condition; to cause the remission of a disease or condition; or to stop the symptoms of a disease or condition, and are intended to include prevention and avoidance. The terms also include achieving therapeutic and / or preventive benefits, such as benefits achieved by eradicating or improving one or more physiological symptoms associated with the disease or condition, such that improvement is observed in the subject, although the subject may still have the disease or condition.

[0044] As used herein, the terms “administer,” “administering,” “administration,” etc., can refer to methods for delivering a therapeutic agent or pharmaceutical composition to a desired biological site of action. These methods include intravitreal or subretinal injection into the eye.

[0045] As used herein, the term "pharmaceutical composition" or simply "composition" may refer to a bioactive compound optionally mixed with at least one pharmaceutically acceptable chemical component, such as, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, etc.

[0046] As used herein, the terms “effective amount,” “therapeutic effective amount,” or “pharmaceutical effective amount” can refer to an adequate amount of at least one pharmaceutical composition or compound that will, to a certain extent, alleviate one or more signs or symptoms of the treated eye disease, eye condition, or eye disorder. In some embodiments, an effective amount of the pharmaceutical composition may be administered in a unit dose to a subject in need (as described in further detail elsewhere herein).

[0047] As used herein, "vector" refers to a macromolecule or macromolecule associated with a polynucleotide and that can be used to mediate the delivery of polynucleotides into cells. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery mediators.

[0048] "Neutralizing antibody" or "nAb" refers to an antibody that recognizes a specific antigen and inhibits the effect of the antigen in a host (e.g., a human). As used herein, an antibody may be a single antibody or multiple antibodies. In some embodiments, a neutralizing antibody may inhibit the effect of antigens from at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or more different AAV viral strains. For example, a neutralizing antibody may inhibit the infectivity of AAV (e.g., cell entry) or AAV-mediated gene expression. Neutralizing antibodies that bind to a virus (e.g., AAV) may exert a materially adverse effect, for example, on the infectivity of the virus, viral transduction, virus-mediated gene expression, or another characteristic or activity of the virus.

[0049] As used in this article, “biological species” is any molecule capable of forming specific interactions with any other biomolecule.

[0050] As used in this article, “nucleic acid” can be DNA, RNA, single-stranded or double-stranded or certain aptamers and any variant thereof.

[0051] As used in this article, a "protein" is any molecule whose units are linked together by peptide bonds, typically amino acids linked together to form a protein.

[0052] As used herein, a "vector" is any molecule capable of delivering therapeutic or diagnostic molecules into a biological system, such as in a gene delivery system.

[0053] As used herein, a "payload" molecule refers to a therapeutic or diagnostic active molecule that is administered to a mammal (preferably a human) and delivered to the mammal when carried within a carrier molecule and subsequently released in the body.

[0054] AAV particles, variants, and AAV2.7m8 capsid protein

[0055] As used herein, “AAV” refers to adeno-associated virus and can be used to refer to the virus itself or its derivatives. Unless otherwise stated, the term covers all subtypes as well as naturally occurring and recombinant forms. The term “AAV” includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV. “Primate AAV” refers to AAV that infects primates, “non-primate AAV” refers to AAV that infects non-primates, and “bovine AAV” refers to AAV that infects bovine mammals, etc.

[0056] The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeat (TR), Rep protein, and capsid subunit, are well known in the art. For example, such sequences are available in the literature or in public databases such as GenBank. See, for example, GenBank accessions NC_002077 (AAV-1), AF063497 (AAV-1), NC_001401 (AAV-2), AF043303 (AAV-2), NC_001729 (AAV-3), NC-001829 (AAV-4), U89790 (AAV-4), NC_006152 (AAV-5), AF028704 and AAB95450 (AAV-6), AF513851 (AAV-7), AF513852 (AAV-8) and NC_006261 (AAV-8), the disclosures of which are incorporated herein by reference to teach AAV nucleic acid and amino acid sequences. See also, for example, Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; International Patent Publications WO00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303.

[0057] The polynucleotide sequence encoding any AAV capsid protein can be readily generated based on the amino acid sequence and the known genetic code (including codon-optimized sequences).

[0058] As used herein, “AAV variant” or “AAV mutant” refers to a viral particle consisting of: a) a variant AAV capsid protein, wherein the variant AAV capsid protein contains at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to the corresponding parental AAV capsid protein, wherein the AAV capsid protein does not correspond to the amino acid sequence of naturally occurring AAV capsid proteins; and optionally b) a heterologous nucleic acid containing a nucleotide sequence encoding a heterologous gene product, wherein the variant AAV capsid protein confers increased binding to heparin or heparan sulfate proteoglycans compared to binding to an AAV viral particle containing the corresponding parental AAV capsid protein. In some embodiments, the variant capsid protein confers: a) increased infectivity to retinal cells compared to the infectivity of AAV viral particles containing the corresponding parental AAV capsid protein; b) altered cytotropy compared to the tropism of AAV viral particles containing the corresponding parental AAV capsid protein; and / or c) increased ability to bind to and / or cross the ILM compared to AAV viral particles containing the corresponding parental AAV capsid protein.

[0059] In some implementations, AAV variants may have variant AAV capsid proteins, for example, the capsid protein of AAV may include an insertion within the capsid protein. Exemplary insertions may include VP1 of AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10), AAV rh.10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, bovine AAV, AAV2 / 7m8, AAVShH10, AAV2.5T, AAV2.5T / 7m8, AAV9 / 7m8, and AAV5 / 7m8.

[0060] The AAV2.5T variant capsid protein and viral particles are described in U.S. Patent No. 9,233,131 (wherein the VP1-encoded amino acid sequence of AAV2.5T is provided as SEQ ID NO:42) and Figures 10A to 10B of U.S. Patent No. 9,233,131.

[0061] The AAV2.7m8 capsid protein is described in U.S. Patent No. 9,193,956.

[0062] The polypeptide sequences of the wild-type AAV2 capsid protein and the variant AAV2.7m8 capsid protein are provided in Table 1 (hereinafter) and the attached sequence listing.

[0063] Table 1: AAV2 and AAV2.7m8 capsid polypeptide sequences

[0064] Name AA Sequence SEQ ID NO: Wild-type AAV2 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL 1 AAV2.7m8 <![CDATA[MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LALGETTRPA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL]]> 2 191205-18-PAC (Loop) Polypeptide CRGNLALGETTRPAR 3

[0065] As shown in bold underline in SEQ ID NO: 2, the AAV2.7m8 variant capsid protein contains a 10-amino acid "7m8" insertion in LALGETTRPA (SEQ ID NO: 22) between amino acids 587 and 588 of the wild-type AAV2 genome.

[0066] Anti-AAV neutralizing antibody

[0067] As described elsewhere in this article, AAV-mediated gene therapy is effective in treating eye diseases. Pre-existing antibodies capable of binding to and neutralizing the AAV vectors used in such gene therapies can adversely affect transduction and gene expression after IVT delivery. Detection and quantification of nAbs before or after IVT administration are crucial for successful gene therapy.

[0068] Anti-AAV2.7m8 antibody

[0069] Antibodies that specifically bind to the therapeutic AAV capsid protein can be used in assays to detect AAV neutralizing antibodies (nAbs). Specifically, these anti-AAV antibodies can be used as positive controls in assays for detecting and quantifying pre-existing nAb levels in patient samples. In at least one embodiment, this disclosure relates to anti-AAV2.7m8 antibodies that bind to variant AAV2.7m8 capsid protein. Various antibody production techniques known in the art can be used to generate and screen such antibodies that specifically and with high affinity bind to the AAV2.7m8 capsid protein. This disclosure provides an exemplary anti-AAV2.7m8 antibody (“clone 6”) generated in rabbits using the 191205-18-PAC (loop) polypeptide of SEQ ID NO: 3, which is derived from the AAV2.7m8 polypeptide sequence of SEQ ID NO: 2. The generation and screening of this anti-AAV2.7m8 antibody are described in Example 1, and the relevant amino acid sequences of the antibody CDR, framework, VH domain, and VL domain are provided in Table 2 and the appended sequence listing.

[0070] Table 2: Exemplary "Clon 6" anti-AAV2.7m8 antibody sequence

[0071] Region AA Sequence SEQ ID NO: VL <![CDATA[MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAALGGTVTINC QSSQSVAR DNRLA WFQQKPGQPPKLLIY RASTLAS GVPSRFKGSGSGTQFTLTISGVQCDDAATYYC QGGHSAGDGRA FGGGTEVVVKG]]> 4 CDR-L1 QSSQSVARDNRLA 5 CDR-L2 RASTLAS 6 CDR-L3 QGGHSAGDGRA 7 FR-L1 AQVLTQTPSPVSAALGGTVTINC 8 FR-L2 WFQQKPGQPPKLLIY 9 FR-L3 GVPSRFKGSGSGTQFTLTISGVQCDDAATYYC 10 FR-L4 FGGGTEVVVKG 11 VH <![CDATA[METGLRWLLLVAVLKGVQCQSVEESRGGLIKPTDTLTLTCTVSGFSLS DYGVN WVRQAPGNGLEWIG IISNGGRTYYASWAKS RATITRNTNENTVTLKMTSLTAAADTATYFCAR EDLYVASDI WGPGTLVTVSS]]> 12 CDR-H1 DYGVN 13 CDR-H2 IISNGGRTYYASWAKS 14 CDR-H3 EDLYVASDI 15 FR-H1 SVEESRGGLIKPTDTLTLTCTVSGFSLS 16 FR-H2 WVRQAPGNGLEWIG 17 FR-H3 RATITRNTNENTVTLKMTSLTAADTATYFCAR 18 FR-H4 WGPGTLVTVSS 19 LC MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAALGGTVTINCQSSQSVARDNRLAWFQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGGHSAGDGRAFGGGTEVVVKG 20 HC METGLRWLLLVAVLKGVQCQSVEESRGGLIKPTDTLTLTCTVSGFSLSDYGVNWVRQAPGNGLEWIGIISNGGRTYYASWAKSRATITRNTNENTVTLKMTSLTAADTATYFCAREDLYVASDIWGPGTLVTVSS 21

[0072] As demonstrated in the examples, the "clone 6" anti-AAV2.7m8 antibody exhibits high binding specificity and affinity for the AAV2.7m8 capsid protein of SEQ ID NO: 2. As further described below, this high specificity and affinity make the antibody particularly suitable for use in many assays involving the detection and quantification of the AAV2.7m8 capsid protein.

[0073] Typically, the CDR sequence of an antibody defines the binding and affinity characteristics of the antibody to its specific antigen. This disclosure envisions a series of anti-AAV2.7m8 antibodies comprising the six CDR sequences shown in Table 2. Therefore, this disclosure envisions a series of antibodies that specifically bind to the AAV2.7m8 capsid with the affinity and specificity of "clone 6," wherein said antibodies comprise the first light chain complementarity-determining region (CDR-L1) of SEQ ID NO: 5, the second light chain complementarity-determining region (CDR-L2) of SEQ ID NO: 6, the third light chain complementarity-determining region (CDR-L3) of SEQ ID NO: 7, the first heavy chain complementarity-determining region (CDR-H1) of SEQ ID NO: 13, the second heavy chain complementarity-determining region (CDR-H2) of SEQ ID NO: 14, and the third heavy chain complementarity-determining region (CDR-H3) of SEQ ID NO: 15.

[0074] In addition, this disclosure envisions an anti-AAV2.7m8 antibody, wherein the antibody comprises a light chain variable domain (VL) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 4; and / or a heavy chain variable domain (VH) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 12.

[0075] In another embodiment, this disclosure envisions an anti-AAV2.7m8 antibody comprising a light chain (LC) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO:20; and / or a heavy chain (HC) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO:21.

[0076] The exemplary "clone 6" anti-AAV2.7m8 antibody was generated in rabbits and contains a rabbit framework and immunoglobulin fragments; however, those skilled in the art will recognize that equivalent functional anti-AAV2.7m8 antibodies can be prepared in other antibody frameworks and formats. Therefore, in at least one embodiment, this disclosure also contemplates that the anti-AAV2.7m8 antibody may be in any of the following antibody formats: (i) a monoclonal antibody; (ii) a chimeric antibody; (iii) a humanized antibody; (iv) a full-length IgG antibody, optionally wherein the IgG antibody has an isotype selected from IgG1, IgG2, IgG3 and IgG4; (v) an antibody fragment, optionally selected from the group consisting of F(ab')2, Fab', Fab, Fv, single-domain antibody (VHH) and scFv; (vi) the antibody is a multispecific antibody, optionally a bispecific antibody; and / or (vii) the antibody is a synthetic antibody, wherein the HVR is grafted onto a scaffold or framework other than an immunoglobulin scaffold or framework; optionally, a scaffold selected from alternative protein scaffolds and artificial polymer scaffolds.

[0077] Similarly, those skilled in the art will recognize that the exemplary "clone 6" anti-AAV2.7m8 antibody of this disclosure can be modified using standard antibody and / or peptide conjugation techniques well known in the art. Therefore, in at least one embodiment, this disclosure contemplates that the anti-AAV2.7m8 antibody can be an immunoconjugate, such as a conjugate with a detectable moiety (e.g., a radiolabeled, fluorescently labeled, or indirectly labeled moiety). Modification with a detectable moiety can allow the anti-AAV2.7m8 antibody to be used in a range of assays for the detection and quantification of the AAV2.7m8 capsid protein.

[0078] The exemplary “clone 6” anti-AAV2.7m8 antibody in Table 2 has been defined according to its CDR structure. However, since it is generated and screened to specifically bind the 7m8 sequence of SEQ ID NO: 2, this disclosure contemplates that other anti-AAV2.7m8 antibodies that specifically bind to the same epitope can be prepared and used in the determination of specific detection and quantification of the AAV2.7m8 capsid protein. Therefore, in at least one embodiment, this disclosure provides an antibody that can competitively bind to the human AAV2.7m8 capsid polypeptide of SEQ ID NO: 2 (e.g., having 1 x 10^6 ppm) against the “clone 6” anti-AAV2.7m8 antibody. -9 M or smaller, 1 x 10 -10 M or smaller or 1 x 10 -11 M or less binding affinity), or more specifically, binding to an epitope containing the amino acid sequence LALGETTRPA (SEQ ID NO: 22) located at positions 588-597 of SEQ ID NO: 2.

[0079] In the embodiments, the exemplary "Clon 6" anti-AAV2.7m8 antibody is characterized by a high affinity for the AAV2.7m8 capsid protein of SEQ ID NO: 2. This disclosure contemplates a series of anti-AAV2.7m8 antibodies having functional characteristics similar to those exhibited by the "Clon 6" antibody. For example, an anti-AAV2.7m8 antibody characterized by one or more of the following properties: (a) at 1 x 10 -8 M or smaller, 1 x 10 -9 M or smaller, 1 x 10 -10 M or smaller or 1 x 10 -11 A binding affinity of M or less is required to bind to the AAV2.7m8 capsid protein; optionally, said binding affinity is determined by the equilibrium dissociation constant (K0) of the AAV2.7m8 capsid protein. D (a) Measurement; or (b) Binding to the AAV2.7m8 capsid of SEQ ID NO: 2 with a binding affinity at least 2, 3, 5, or 10 times greater than the binding affinity to the AAV2 capsid protein; optionally wherein the relative binding affinity is measured by the equilibrium dissociation constant (K0) of the AAV2.7m8 capsid peptide of SEQ ID NO: 2 and the AAV2 capsid peptide of SEQ ID NO: 1. D )Measurement.

[0080] Uses of anti-AAV2.7m8 antibody

[0081] As described elsewhere herein, the antibodies of this disclosure that bind specifically to the AAV2.7m8 capsid protein with high affinity can be used in assays to detect and / or quantify the presence of AAV2.7m8 neutralizing antibodies in a sample. For example, anti-AAV2.7m8 antibodies can be used as a positive control in methods for detecting the activity of neutralizing antibody (nAb) against AAV2.7m8 in human serum samples. Typically, such nAb assays include the following steps: (a) providing: (i) a human serum sample; (ii) an assay solution containing cells; (iii) a vector solution containing an AAV2.7m8 vector encoding a luciferase gene; (iv) a luciferase substrate solution; and (v) an antibody solution containing an anti-AAV2.7m8 antibody that specifically binds to the AAV2.7m8 capsid; (b) combining aliquots of the human serum sample, the assay solution, the vector solution, and the luciferase substrate solution to form a sample solution; (c) combining aliquots of the AAV2.7m8 antibody solution, the assay solution, the vector solution, and the luciferase substrate solution to form a positive control solution; and (d) detecting luminescent signals from the sample solution and the positive control solution; thereby, the relative luminescent signal levels indicate the presence and / or amount of neutralizing antibody activity in the human serum sample. It is envisioned that step (c) may include combining at least 2, 3, 4, 5, 6, 7, 8, 9 or more different aliquots of the sample with different dilutions of the AAV2.7m8 antibody solution to form at least 2, 3, 4, 5, 6, 7, 8, 9 or more positive control solutions with different concentrations of AAV2.7m8 antibody, for use in preparing reaction curves. It is also envisioned that the assay method may include other steps. For example, combining aliquots of the assay solution with a luciferase substrate solution to form a negative control solution; and detecting the luminescent signal from the negative control solution.

[0082] Additionally, it is envisioned that, in the assay method, cells may be pre-exposed to the AAV2.7m8 vector encoding the luciferase gene, derived from a transducible cell line; optionally, said cell line is 293T.

[0083] In addition, it is envisioned that the anti-AAV2.7m8 antibody could be used for other assays and diagnostic purposes to detect and quantify the AAV2.7m8 capsid protein.

[0084] In at least one embodiment, this disclosure also provides a method for detecting AAV2.7m8 capsid protein, the method comprising: contacting the protein with an anti-AAV2.7m8 antibody of this disclosure, and detecting the antibody bound to the protein. In at least one embodiment, the AAV2.7m8 capsid protein is immobilized. In at least one embodiment, the AAV2.7m8 capsid protein is in solution. In at least one embodiment, the antibody comprises a detectable label. In at least one embodiment, the detectable label is selected from radioactive labels, fluorescent labels, and indirect labeling portions.

[0085] Furthermore, this disclosure relates to the use of these anti-AAV2.7m8 antibodies in assays (and kits) for detecting and / or quantifying neutralizing antibodies (nAbs) that may be present in subjects receiving gene therapy treatment containing the AAV2.7m8 capsid protein or as candidates for receiving said treatment. It is envisioned that anti-AAV2.7m8 antibodies as disclosed herein may be included in kits having reagents for assays performed with the antibodies to detect and / or quantify the AAV2.7m8 capsid protein in samples, such as blood samples from patients. For example, the assay reagents included in a kit containing the antibody may include, but are not limited to: (a) an AAV2.7m8 vector encoding a luciferase gene; (b) a luciferase substrate; and / or (c) cells from a permissible transduction cell line; optionally, said cells are from the 293T cell line. The kit may also include instructions on how to use the kit to determine the relative amounts of payload and vector in a biological sample.

[0086] The scope of this invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims.

[0087] Example

[0088] The following representative embodiments illustrate various features and implementations of this disclosure and are intended to be illustrative rather than restrictive. Those skilled in the art will readily understand that the specific embodiments are merely illustrative of the invention, as more fully described in the appended claims. Each embodiment and feature described in this application should be understood to be interchangeable and combined with each embodiment contained therein.

[0089] Example 1: Preparation of anti-AAV2.7m8 antibody

[0090] This embodiment illustrates the preparation of rabbit anti-AAV2.7m8 antibodies of this disclosure and a comparative BLI affinity study of the binding of these antibodies to the target antigen AAV2.7m8 capsid protein relative to their affinity for wild-type AAV2 capsid protein.

[0091] Materials and methods

[0092] The polypeptide sequences of AAV2 and AAV2.7m8 used for antibody generation and screening are provided in Table 1 (above) and the attached sequence listing.

[0093] A. Antibody generation, screening, and cloning

[0094] A 14 aa cyclic polypeptide specific to AAV2.7m8 was modified with N-terminus Cys to provide the 191205-18-PAC (cyclic) polypeptide CRGNLALGETTRPAR (SEQ ID NO: 3). This polypeptide was conjugated to a keyhole cyanobacterial hemocyanin (KLH) carrier protein. Rabbits were immunized with this KLH-polypeptide conjugate at least four times, and blood was collected to provide 20-25 mL of serum per animal. The collected serum was treated with an affinity column modified with the specific AAV2.7m8 cyclic polypeptide of SEQ ID NO: 3 to enrich B cells that specifically bind to AAV2.7m8.

[0095] ELISA was performed on affinity-purified rabbit serum samples to confirm the presence of antibodies exhibiting single-specific binding affinity for AAV2.7m8 (SEQ ID NO: 2). Paired heavy and light chains from enriched B cells identified in the ELISA were cloned and used to transfect HEK293 cells. A total of nine rabbit antibodies (clones 1-8 and 11) with high affinity for AAV2.7m8 were identified using a screening assay from the supernatant of transfected HEK293 cells.

[0096] B. BLI Affinity Assay for Anti-AAV2.7m8 Antibody

[0097] The binding affinity of the anti-AAV2.7m8 antibody was determined using standard Octet biolayer interferometry (BLI). In short, (1) nine rabbit anti-AAV2.7m8 antibodies (clones 1-8 and 11) were immobilized onto a protein A biosensor; (2) the tip of each immobilized antibody biosensor was immersed in an analyte well containing buffer and either AAV.7m8-luciferase carrier (34.4 nM) or AAV2-CAG-luciferase carrier (17.2 nM); and (3) the tip of a control biosensor without immobilized antibody was also immersed in an analyte well to test for nonspecific binding.

[0098] BLI signals generated by the association and dissociation of analytes with immobilized antibodies were analyzed using standard Octet data software and analysis. First, all recorded curves were reference-subtracted using control wells (biosensors that underwent the same steps as the experimental wells but without the analyte in the association step), and then all traces were aligned with the start of the association step. A 1:1 binding model of nine rabbit anti-AAV2.7m8 antibodies was fitted using the entire association and dissociation step.

[0099] The AAV.7m8 target analyte did not exhibit nonspecific binding to the tip of the control biosensor in the absence of a fixed antibody. The commercially available “AAV2-CAG-luciferase” vector target analyte exhibited nonspecific binding to the tip of the control biosensor, which may be due to undisclosed impurities in the commercial formulation.

[0100] result

[0101] The AAV2.7m8-luciferase vector target analyte bound to six of the nine rabbit anti-AAV2.7m8 antibodies tested. None of the nine rabbit anti-AAV2.7m8 antibodies bound to the AAV2-CAG-luciferase vector target analyte. Figures 1A to 1I As shown in the BLI signal trace, clone 1 ( Figure 1A ), Clones 6 ( Figure 1H ) and clone 7 ( Figure 1C The anti-AAV2.7m8 antibody showed the highest specific binding affinity to the AAV.7m8 target analyte.

[0102] Based on its high affinity for the AAV2.7m8 target analyte measured in BLI, the "Clon 6" anti-AAV2.7m8 antibody was selected for sequencing. The CDR, FR, and VL and VH domain sequences of "Clon 6" are provided in Table 2.

[0103] Example 2: Assay for anti-AAV2.7m8 neutralizing antibody (nAb)

[0104] This embodiment illustrates the assay method for the exemplary "clone 6" anti-AAV2.7m8 antibody using Example 1, and for comparison, the level of AAV2 capsid neutralizing antibody in biological samples is detected and / or quantified using an "IVIG" antibody pool that binds to wild-type AAV2 capsid protein antigen.

[0105] In summary, in the assay, a solution of transducible 293T cells is exposed to the AAV2.7m8 vector encoding the firefly luciferase gene. Successful transduction of the virus-encoded luciferase gene results in an increased luminescence signal when the luciferase substrate is added to the solution. Adding a human serum sample containing a neutralizing antibody (nAb) capable of neutralizing the AAV2.7m8 vector to this solution leads to reduced AAV2.7m8 transduction and reduced luminescence. Serial dilutions of an exemplary antibody (“clone 6” or “IVIG”) added to a solution of 293T cells exposed to the AAV2.7m8 virus similarly reduce luminescence and thus serve as a positive control. The presence and amount of nAb are determined by monitoring a decrease in the luminescence signal level relative to the signal level obtained from cells exposed to AAV.7m8-luciferase virus pre-incubated with non-neutralizing serum.

[0106] Materials and methods

[0107] The "clone 6" anti-AAV2.7m8 antibody was serially diluted and then combined with the luciferase vector (AAV.7m8-luciferase). The antibody-vector mixture was incubated at 37°C and 5% CO2 for 1 hour, and then added to pre-seeded HEK 293 cells at an MOI of 2.5E5-5E5 vg / cell. Plates of transduced cells were incubated at 37°C and 5% CO2 for 18 to 24 hours. After incubation, the plates were cooled to room temperature, and 100 μL of One-Glo (Promega) reagent was added to each well. The luminescence signal was read using a SpectraMax M3 instrument (Molecular Devices), and the inhibition percentage was calculated and plotted using 4-PL fitting on SoftMax Pro software (version 7.1.2).

[0108] The detailed list of materials and the description of the testing procedures are as follows:

[0109] A. Reagents and Materials

[0110] "Clon6" anti-AAV2.7m8 antibody positive alternative control.

[0111] 293T complete growth medium: cIMDM (IMDM w / GlutaMax + 10% hi FBS)

[0112] 293 Transduction Medium: TM (OptiMEM w / GlutaMax + 1% FBS)

[0113] AAV2.7m8.CMV.Luc vector (batch number 18-293-005).

[0114] AAV2-CBA-luciferase (Vector Biolabs, batch number 109128-200807).

[0115] Sterile PBS, Ca++, Mg++ free, Gibco: 10010-23, batch number 2085516.

[0116] Hi Fetal Bovine Serum (FBS), Gibco: 10438-026. Batch No. 2086963.

[0117] Trypsin-EDTA 0.05% 1X, Gibco: 25300-054. Batch No. 2085644.

[0118] One-Glo, Promega, E6130, batch number 0000275700.

[0119] IMDM and GlutaMax, Gibco: 31980-030, lot number 2150863.

[0120] OptiMEM and GlutaMax, Gibco: 51985-034, lot number 2053509).

[0121] Reagent reservoir, Costar 4870.

[0122] 96-well flat-bottomed tissue culture plate, white microwells, Thermo Scientific: 136102.

[0123] 96-well U-bottom polypropylene plate, Greiner Bio-one REF 650261.

[0124] Molecular Devices SpectraMax M3.

[0125] SoftMax Pro software.

[0126] B. Measurement Protocol

[0127] After thawing, the 293T cells were allowed to grow for at least four generations.

[0128] Day 1 Laminated cells

[0129] 1. Warm fresh culture medium, trypsin, and transduction medium (TM).

[0130] 2. Remove the culture medium from the flask.

[0131] 3. Rinse the cells with 10 mL of PBS and aspirate from the flask.

[0132] 4. Add 2.0 mL of 0.5% trypsin-EDTA and gently tap the flask for 3–5 minutes periodically until the cells no longer adhere to the flask.

[0133] 5. When the cells no longer adhere to the culture medium, add 8.0 mL of fresh culture medium, mix well and transfer to a 15 mL centrifuge tube.

[0134] 6. Centrifuge at 130 xg for 10 minutes at room temperature.

[0135] 7. Carefully aspirate the supernatant.

[0136] 8. Use a serum pipette to pipette up and down at least six times to resuspend the precipitate in 5.0 mL of fresh culture medium.

[0137] 9. Add 0.5 mL of cells to 19.5 mL of fresh culture medium in a new flask and incubate for the next passage.

[0138] 10. Add slightly more than 100 µL of resuspended cells to an Eppendorf centrifuge tube.

[0139] 11. Add exactly 100 µL from the first Eppendorf centrifuge tube to the second Eppendorf centrifuge tube.

[0140] 12. Add exactly 100 µL of trypan blue to the second Eppendorf centrifuge tube, mix thoroughly without causing cell lysis.

[0141] 13. Fill the two chambers of a disposable hematology counter with stained cells.

[0142] 14. Count cells on Countess II FL according to the following analytical and quantitative templates:

[0143] viable cell count: Chamber A ____ x 10 6 + Room B_____X10 6 .

[0144] Mean viable cell count = _____ x 10 6 cells / mL;

[0145] Survival rate: Live A = ____% Dead A = ____%; Live B = ____% Dead B = ____%

[0146] Dilution (5 plates);

[0147] (i) Expected concentration: 1.875E5 cells / mL ;

[0148] (ii) Total volume required: ___(number of plates) x 96 = ___(number of wells) x 0.080mL = ____mL + 20% =_____mL;

[0149] (iii) Volume of living cells: ____ mL x 1.875 x 10 5 Cells / mL = ________ cells ÷ mean viable cell count = ___ mL

[0150] (iv) The volume of TM: ____mL (total volume required) - ___mL (volume of living cells) = _______mL.

[0151] 15. Add 80 µL of diluted cells to all wells of a 96-well white tissue culture plate and incubate overnight. Cell count per well: 1.5E 4 cells / well.

[0152] Day 2 Preparation of standards and AAV vectors; and transduction of 293T cells.

[0153] 1. For row AD, prepare “clone 6” mAb 3-12; (ii) add 135 μL TM to well A2; (iii) mix 15 μL “clone 6” mAb into well A2; (iv) add 150 μL 7m8 supernatant to wells B2-D2; (v) take 75 μL from wells A2-A11, B2-B11, C2-C11 and D2-D11 for serial dilution.

[0154] 2. Prepare 2.5 mL of diluted vector AAV2.7m8-CMV-LUC, batch number ADVM19-293-012, 9.53E12 vg / mL (MOI is 2.5E5 vg / cell) as follows: (i) 1.5E4 cells / well X 2.5E5 vg / cell = 3.75E9 vg / well = 3.75E9 vg / 40 μL = 9.38E10 vg / mL; (ii) Dilute the stock vector: 9.38E10 vg / mL / 9.53E12 vg / mL = 9.84 µL stock solution + 2.490 mL TM.

[0155] 3. Prepare 2.5 mL of diluted vector AAV2-CBA-LUC, batch number #, 1.0E13 vg / mL (MOI is 5.33E5 vg / cell) as follows: (i) 1.5E4 cells / well X 5.33E5 vg / cell = 8E9 vg / well = 8E9 vg / 40 µL = 2E11 vg / mL; (ii) Dilute the stock vector: 2E11 vg / mL / 1.0E13 vg / mL = 20 µL stock solution + 2.480 mL TM.

[0156] 4. Pre-bind “clone 6” mAb as follows: (i) Add 30 μL of diluted vector to all wells of a new U-shaped bottom pre-binding plate; (ii) Add 30 μL of diluted mAb and the sample from the dilution plate to the pre-binding plate; (iii) Add a sterile cap and incubate at 37°C and 5% CO2 for 1 hour.

[0157] 5. Transduction was performed as follows: (i) 20 μL of the pre-conjugated plate was added in duplicate to the cell plate; (ii) the plate was sealed with a multi-well sealing film and incubated at 37°C and 5% CO2 for 24 hours.

[0158] Day 3 Reading and data analysis

[0159] 1. Thaw One-Glo at room temperature. Keep away from light.

[0160] 2. Remove the plate from the incubator and allow it to equilibrate to room temperature for 30 minutes.

[0161] 3. Add 100 μL to each well in columns 1-12.

[0162] 4. Shake the plate at 200 RPM for 2.5 minutes.

[0163] 5. Use the SoftMax Pro reading board on SpectraMax within 30 minutes.

[0164] 6. Perform analysis using SoftMax Pro software.

[0165] result

[0166] The comparison results of “clone 6” and “IVIG” obtained in the neutralizing antibody assay are shown in Table 3 below.

[0167] Table 3

[0168]

[0169] As these results show, the luciferase inhibition exhibited by the "clone 6" antibody was significantly greater than that exhibited by the "IVIG" antibody. The EC50 value for "clone 6" was 9.9 x 10⁻⁶. -4 This indicates that, relative to the EC50 value measured for the "IVIG" antibody (2.0 x 10⁻⁶), -1 This assay demonstrates a 200-fold increase in binding specificity. This increased binding specificity reflects the fact that the "clone 6" antibody is generated to specifically bind to the AAV2.7m8 capsid protein rather than the AAV2 capsid protein. Therefore, the "clone 6" antibody can provide a more sensitive (e.g., 200-fold) and accurate assay for detecting antibodies in patient samples that can neutralize the AAV2.7m8 vector.

[0170] Despite the appended claims, the disclosure set forth herein is also limited by the following embodiments, which may be advantageous individually or in combination with one or more other embodiments disclosed herein. Without limiting the foregoing description, certain non-limiting embodiments of this disclosure are provided with the following numbered options, wherein each individually numbered embodiment may be used or combined with any of the foregoing or following embodiments. Therefore, this is intended to support all such combinations and is not necessarily limited to the specific combinations explicitly provided below:

[0171] Implementation Scheme 1: An antibody that specifically binds to the AAV2.7m8 capsid, wherein the antibody comprises the first light chain complementarity-determining region (CDR-L1) of SEQ ID NO: 5, the second light chain complementarity-determining region (CDR-L2) of SEQ ID NO: 6, the third light chain complementarity-determining region (CDR-L3) of SEQ ID NO: 7, the first heavy chain complementarity-determining region (CDR-H1) of SEQ ID NO: 13, the second heavy chain complementarity-determining region (CDR-H2) of SEQ ID NO: 14, and the third heavy chain complementarity-determining region (CDR-H3) of SEQ ID NO: 15.

[0172] Implementation Scheme 2: The antibody as described in Implementation Scheme 1, wherein the antibody comprises a light chain variable domain (VL) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 4; and / or a heavy chain variable domain (VH) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 12.

[0173] Implementation Scheme 3: An antibody as described in any one of Implementation Schemes 1 to 2, wherein the antibody comprises: a light chain (LC) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 20; and / or a heavy chain (HC) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 21.

[0174] Implementation Scheme 4: An antibody as described in any one of Implementation Schemes 1 to 3, wherein the antibody is: (i) a monoclonal antibody; (ii) a chimeric antibody; (iii) a humanized antibody; (iv) a full-length IgG antibody, optionally wherein the IgG antibody has an isotype selected from IgG1, IgG2, IgG3 and IgG4; (v) an antibody fragment, optionally selected from the group consisting of F(ab')2, Fab', Fab, Fv, single-domain antibody (VHH) and scFv; (vi) the antibody is a multispecific antibody, optionally a bispecific antibody; and / or (vii) the antibody is a synthetic antibody, wherein the HVR is grafted onto a scaffold or framework other than an immunoglobulin scaffold or framework; optionally, a scaffold selected from alternative protein scaffolds and artificial polymer scaffolds.

[0175] Implementation Scheme 5: An antibody as described in any one of Implementation Schemes 1 to 4, wherein the antibody is an immunoconjugate, and optionally the immunoconjugate contains a detectable label.

[0176] Implementation Scheme 6: The antibody as described in Implementation Scheme 5, wherein the immunoconjugate comprises a detectable label selected from radiolabeled, fluorescently labeled and indirectly labeled portions.

[0177] Implementation Scheme 7: An antibody as described in any one of Implementation Schemes 1 to 6, wherein the antibody is characterized by one or more of the following properties:

[0178] With 1 x 10 -8 M or smaller, 1 x 10 -9 M or smaller, 1 x 10 -10 M or smaller or 1 x 10 -11 M or less binding affinity binds to the AAV2.7m8 capsid; optionally, said binding affinity is determined by the equilibrium dissociation constant (K0) of the AAV2.7m8 capsid peptide of SEQ ID NO: 2. D ) measurement; and

[0179] Binding to the AAV2.7m8 capsid with a binding affinity at least 2, 3, 5, or 10 times greater than the binding affinity to the AAV2 capsid; optionally, wherein the relative binding affinity is determined by the equilibrium dissociation constant (K0) of the AAV2.7m8 capsid peptide of SEQ ID NO: 2 and the AAV2 capsid peptide of SEQ ID NO: 1. D To measure.

[0180] Implementation Scheme 8: An antibody that specifically binds to an epitope of the same AAV2.7m8 capsid as any one of the antibodies described in Implementation Schemes 1 to 7; optionally, said epitope comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 22) located at positions 588-597 of SEQ ID NO: 2.

[0181] Implementation Scheme 9: An antibody that competitively binds to the epitope of the AAV2.7m8 capsid as described in any one of Implementation Schemes 1 to 8, and has a 1 x 10 -9 M or smaller, 1 x 10 -10 M or smaller or 1 x 10 -11 M or less binding affinity; optionally, said binding affinity is determined by the equilibrium dissociation constant (K0) of the AAV2.7m8 capsid peptide of SEQ ID NO: 2. D )Measurement.

[0182] Implementation Scheme 10: The antibody as described in Implementation Scheme 9, wherein the epitope comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 22) located at positions 588-597 of SEQ ID NO: 2.

[0183] Implementation Scheme 11: An isolated polynucleotide or vector encoding an antibody as described in any one of Implementation Schemes 1 to 10.

[0184] Implementation Scheme 12: An isolated host cell comprising oligonucleotides or vectors as described in Implementation Scheme 11.

[0185] Implementation Scheme 13: A method for generating antibodies, the method comprising culturing host cells as described in Implementation Scheme 12 to generate antibodies.

[0186] Implementation Scheme 14: A kit comprising: an assay reagent; and an antibody as described in any one of Implementation Schemes 1 to 10.

[0187] Implementation Scheme 15: The kit as described in Implementation Scheme 14, wherein the assay reagent comprises: (a) an AAV2.7m8 vector encoding a luciferase gene; (b) a luciferase substrate; and / or (c) cells from a transducible cell line; optionally wherein the cells are from the 293T cell line.

[0188] Implementation Scheme 16: A method for detecting the activity of a neutralizing antibody against AAV2.7m8 in a human serum sample, the method comprising: (a) providing: (i) a human serum sample; (ii) an assay solution containing cells; (iii) a carrier solution containing an AAV2.7m8 vector encoding a luciferase gene; (iv) a luciferase substrate solution; and (v) an AAV2.7m8 antibody solution containing an antibody that specifically binds to the AAV2.7m8 capsid; (b) combining aliquots of the human serum sample, the assay solution, the carrier solution, and the luciferase substrate solution to form a sample solution; (c) combining aliquots of the AAV2.7m8 antibody solution, the assay solution, the carrier solution, and the luciferase substrate solution to form a positive control solution; and (d) detecting a luminescent signal from the sample solution and the positive control solution; thereby, the relative luminescent signal level indicates the presence and / or amount of neutralizing antibody activity in the human serum sample.

[0189] Implementation Scheme 17: The method as described in Implementation Scheme 16, wherein the antibody that specifically binds to the AAV2.7m8 capsid is an antibody as described in any one of Implementation Schemes 1 to 10.

[0190] Implementation Scheme 18: The method of any one of Implementation Schemes 16 to 17, wherein step (c) comprises combining at least 2, 3, 4, 5, 6, 7, 8, 9 or more different aliquots of the sample with different dilutions of the AAV2.7m8 antibody solution to form at least 2, 3, 4, 5, 6, 7, 8, 9 or more positive control solutions with different concentrations of the AAV2.7m8 antibody for use in preparing reaction curves.

[0191] Implementation Scheme 19: The method of any one of Implementation Schemes 16 to 18, wherein the method further comprises the steps of: combining an aliquot of the assay solution and the luciferase substrate solution to form a negative control solution; and detecting the luminescence signal from the negative control solution.

[0192] Implementation Scheme 20: The method of any one of Implementation Schemes 16 to 19, wherein the cells pre-exposed to the AAV2.7m8 vector encoding the luciferase gene are derived from a transducible cell line; optionally, wherein the cell line is 293T.

[0193] Implementation Scheme 21: A method for detecting AAV2.7m8 capsid protein, the method comprising: contacting the protein with an antibody as described in any one of Implementation Schemes 1 to 10, and detecting the antibody bound to the protein.

[0194] Implementation Scheme 22: The method as described in Implementation Scheme 21, wherein the AAV2.7m8 capsid protein is immobilized.

[0195] Implementation Scheme 23: The method as described in Implementation Scheme 21, wherein the AAV2.7m8 capsid protein is in solution.

[0196] Implementation Scheme 24: The method of any one of Implementation Schemes 21 to 23, wherein the antibody comprises a detectable marker.

[0197] Implementation Scheme 25: The method as described in Implementation Scheme 24, wherein the detectable marker is selected from radioactive markers, fluorescent markers, and indirect markers.

[0198] While the foregoing disclosure of the invention has been described in detail by way of example and illustration for clarity and understanding, this disclosure (including the embodiments, descriptions, and implementations described herein) is for illustrative purposes and is intended to be exemplary, and should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various modifications or changes can be made to the embodiments, descriptions, and implementations described herein, and that these modifications or changes are included within the spirit and scope of this disclosure and the appended claims. Furthermore, those skilled in the art will recognize that various methods and procedures are equivalent to the methods and procedures described herein. All such equivalents should be understood to be within the scope of this disclosure and covered by the appended claims.

Claims

1. An antibody that specifically binds to the AAV2.7m8 capsid, wherein the antibody comprises the first light chain complementarity-determining region (CDR-L1) of SEQ ID NO: 5, the second light chain complementarity-determining region (CDR-L2) of SEQ ID NO: 6, the third light chain complementarity-determining region (CDR-L3) of SEQ ID NO: 7, the first heavy chain complementarity-determining region (CDR-H1) of SEQ ID NO: 13, the second heavy chain complementarity-determining region (CDR-H2) of SEQ ID NO: 14, and the third heavy chain complementarity-determining region (CDR-H3) of SEQ ID NO:

15.

2. The antibody of claim 1, wherein the antibody comprises a light chain variable domain (VL) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 4; and / or a heavy chain variable domain (VH) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO:

12.

3. The antibody of claim 1, wherein the antibody comprises: a light chain (LC) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 20; and / or a heavy chain (HC) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO:

21.

4. The antibody of claim 1, wherein the antibody is: (i) a monoclonal antibody; (ii) a chimeric antibody; (iii) a humanized antibody; (iv) a full-length IgG antibody, optionally wherein the IgG antibody has an isotype selected from IgG1, IgG2, IgG3 and IgG4; (v) an antibody fragment, optionally selected from the group consisting of F(ab')2, Fab', Fab, Fv, single-domain antibody (VHH) and scFv; (vi) the antibody is a multispecific antibody, optionally a bispecific antibody; and / or (vii) the antibody is a synthetic antibody, wherein the HVR is grafted onto a scaffold or framework other than an immunoglobulin scaffold or framework; optionally, a scaffold selected from alternative protein scaffolds and artificial polymer scaffolds.

5. The antibody of claim 1, wherein the antibody is an immunoconjugate, and optionally the immunoconjugate comprises a detectable marker.

6. The antibody of claim 5, wherein the immunoconjugate comprises a detectable label selected from radiolabeled, fluorescently labeled, and indirectly labeled portions.

7. The antibody of claim 1, wherein the antibody is characterized by one or more of the following properties: (a) with 1 x 10 -8 M or smaller, 1 x 10 -9 M or smaller, 1 x 10 -10 M or smaller or 1 x 10 -11 M or less binding affinity binds to the AAV2.7m8 capsid; optionally, said binding affinity is determined by the equilibrium dissociation constant (K0) of the AAV2.7m8 capsid peptide of SEQ ID NO:

2. D ) measurement; and (b) Binding to the AAV2.7m8 capsid with a binding affinity at least 2, 3, 5, or 10 times greater than the binding affinity to the AAV2 capsid; optionally, wherein the relative binding affinity is determined by the equilibrium dissociation constant (K0) of the AAV2.7m8 capsid peptide of SEQ ID NO: 2 and the AAV2 capsid peptide of SEQ ID NO:

1. D To measure.

8. An antibody that specifically binds to an epitope of the same AAV2.7m8 capsid as the antibody of claim 1; optionally, said epitope comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 22) located at positions 588-597 of SEQ ID NO:

2.

9. An antibody that competitively binds to an epitope of the AAV2.7m8 capsid with the antibody of claim 1, and has a 1 x 10⁻⁶ ppm concentration. -9 M or smaller, 1 x 10 -10 M or smaller or 1 x 10 -11 M or less binding affinity; optionally, said binding affinity is determined by the equilibrium dissociation constant (K0) of the AAV2.7m8 capsid peptide of SEQ ID NO:

2. D )Measurement.

10. The antibody of claim 9, wherein the epitope comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 22) located at positions 588-597 of SEQ ID NO:

2.

11. An isolated polynucleotide or vector encoding the antibody as claimed in claim 1.

12. An isolated host cell comprising the oligonucleotide or vector as described in claim 11.

13. A method for producing antibodies, the method comprising culturing a host cell as described in claim 12 to produce antibodies.

14. A kit comprising: an assay reagent; and the antibody as claimed in claim 1.

15. The kit of claim 14, wherein the assay reagent comprises: (a) an AAV2.7m8 vector encoding a luciferase gene; (b) a luciferase substrate; and / or (c) cells from a transducible cell line; optionally wherein the cells are from a 293T cell line.

16. A method for detecting the activity of a neutralizing antibody against AAV2.7m8 in a human serum sample, the method comprising: (a) Provide: (i) human serum samples; (ii) Assay solutions containing cells; (iii) a vector solution containing an AAV2.7m8 vector encoding a luciferase gene; (iv) a luciferase substrate solution; and (v) an AAV2.7m8 antibody solution containing an antibody that specifically binds to the AAV2.7m8 capsid; (b) combining an aliquot of the human serum sample, the assay solution, the vector solution, and the luciferase substrate solution to form a sample solution; (c) Combine the aliquots of the AAV2.7m8 antibody solution, the assay solution, the carrier solution, and the luciferase substrate solution to form a positive control solution; (d) Detecting the luminescence signals from the sample solution and the positive control solution; thereby, the relative luminescence signal level indicates the presence and / or amount of neutralizing antibody activity in the human serum sample.

17. The method of claim 16, wherein the antibody that specifically binds to the AAV2.7m8 capsid is the antibody of claim 1.

18. The method of claim 16, wherein step (c) comprises combining at least 2, 3, 4, 5, 6, 7, 8, 9 or more different aliquots with different dilutions of the AAV2.7m8 antibody solution to form at least 2, 3, 4, 5, 6, 7, 8, 9 or more positive control solutions with different concentrations of the AAV2.7m8 antibody for use in preparing reaction curves.

19. The method of claim 16, wherein the method further comprises the following step: The aliquots of the assay solution and the luciferase substrate solution were combined to form a negative control solution; And detect the luminescence signal from the negative control solution.

20. The method of claim 16, wherein the cells pre-exposed to the AAV2.7m8 vector encoding the luciferase gene are derived from a transducible cell line; optionally, wherein the cell line is 293T.

21. A method for detecting AAV2.7m8 capsid protein, the method comprising: The protein is contacted with the antibody as described in claim 1, and the antibody bound to the protein is detected.

22. The method of claim 21, wherein the AAV2.7m8 capsid protein is immobilized.

23. The method of claim 21, wherein the AAV2.7m8 capsid protein is in solution.

24. The method of claim 21, wherein the antibody comprises a detectable marker.

25. The method of claim 24, wherein the detectable marker is selected from radioactive markers, fluorescent markers, and indirect markers.

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