Methods for the detection of anti-AAV particle antibodies

Abstract

Reported herein is a method for determining anti-AAV particle antibodies in a mammalian sample, comprising the steps of diluting an aliquot of the sample to a final serum concentration, preferably between 5% (v / v) and 0.5% (v / v), adding AAV particles (AAVp) specifically bound to anti-AAV particle antibodies (AAV-5 ADA) to the diluted sample to create a spiked sample, pre-incubating the spiked sample to allow for the formation of AAV-ADA-AAVp complexes, and detecting the AAV-ADA-AAVp complexes, thereby determining anti-AAV antibodies in the mammalian sample.

Description

[Technical Field]

[0001] The present invention is in the field of immunoassays, particularly in the field of anti-drug antibody (ADA) evaluation. ADA evaluation is often challenging, especially in studies involving the administration of adeno-associated virus-based gene therapy drugs. In such cases, the limited availability of therapeutic AAV as an assay reagent makes an ADA assay requiring fewer reagents desirable. The present invention is based, at least in part, on the discovery that a sample in which anti-AAV particle antibodies (AAV-ADA) must be determined can be pre-incubated with the respective therapeutic AAV particles to generate complexes containing AAV-ADA for detection. This approach dramatically reduces the demand for therapeutic AAV particles compared to conventional ADA assays, which require labeling of the therapeutic AAV particles. [Background technology]

[0002] Background of the Invention Immunogenicity assessment of therapeutic candidates is a critical part of the drug development process. In the case of immune responses, proper interpretation of immunogenicity data is necessary to enable correlation with clinical outcomes. Bioanalytical methods used in immunogenicity testing provide the necessary information by detecting and characterizing anti-drug antibodies (ADAs).

[0003] Like all protein therapeutics, recombinant AAV particles must be analyzed before their use. However, in contrast to other biologics, adeno-associated virus therapy is hampered by pre-existing humoral immunity to AAV (anti-AAV particle antibodies, AAV-ADA), due to the ubiquitous nature of AAV and the simultaneous widespread distribution of pre-treatment AAV infections in the human population.

[0004] For typical biologics, the "gold standard" assay format for detecting ADA in human or monkey samples is a bridging assay, in which ADA is complexed with a labeled drug conjugate to form a signal-conferring complex. Depending on the assay setup, differently labeled drug molecules or drug-coated surfaces are required, which requires significant amounts of drug.

[0005] Gimpel, AL et al. (Mol. Ther. Meth. Clin. Dev. 20 (2021) 740-754) (Non-Patent Document 1) outlined the shortcomings of current AAV analysis methods used to analyze recombinantly produced AAV particles: Typically, laboratory-scale production yields only a few milliliters (mL) of purified rAAV particles, corresponding to 1E12–1E14 vector genomes (vg). Consequently, the sample volume required for analysis can cause significant product loss. At the same time, virus concentrations in samples range over several orders of magnitude, from 1E10–1E11 vg / mL at the time of raw culture harvest to 1E13–1E14 vg / mL in purified products. Furthermore, sample matrices vary significantly, with residual by-products, host cell proteins, and DNA concentrations exceeding 10–100-fold the mass concentration of rAAV in the first downstream steps.

[0006] Thus, analytical characterization of the recombinantly produced AAV particles themselves and the analyses that accompany their development requires significant quantities of the product, thereby reducing its availability for characterization and testing.

[0007] Gorovits et al. (The AAPS Journal 23 (2021) 108) (Non-Patent Document 2) outlined important considerations related to anti-AAV total antibody detection assays. Figure 1 in Gorovits summarizes different applied total anti-AAV antibody assay formats. In commonly used antigen capture and bridging formats, viral capsids are immobilized on a solid surface as capture reagents, either directly or via specific binding pairs. This setup is used in ELISA and ECLIA formats with different detection reagents, such as HRP-conjugated anti-species Ig antibodies and HRP-conjugated protein A or G (antigen capture format) or labeled viral capsids (bridging format).

[0008] Therefore, there is a need for an AAV-ADA assay with reduced AAV material demands. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Gimpel, AL et al. (Mol.Ther.Meth.Clin.Dev.20(2021)740-754) [Non-patent document 2] Gorovits et al. (The AAPS Journal 23(2021)108) Summary of the Invention

[0010] Herein, we report a novel method for the determination of anti-AAV particle antibodies (AAV-ADA) in a sample, which, among other things, requires less AAV material compared to established assays.

[0011] The present invention is based, at least in part, on the discovery that AAV-ADA-containing complexes can be generated for detection by pre-incubating a sample in which AAV-ADA must be determined with the respective therapeutic AAV particles, which can dramatically reduce the demand, i.e., the required amount, of therapeutic AAV particles as assay reagents compared to current AAV-ADA assays that use labeled therapeutic AAV particles.

[0012] The present invention encompasses at least the following embodiments: Embodiment 1: A method for measuring anti-AAV particle antibodies in a mammalian serum sample, comprising the following steps, in the above order: (a) adding AAV particles (AAVp) specifically bound by anti-AAV particle antibodies (AAV-ADA) to a sample to generate a spiked sample; (b) pre-incubating the spiked sample to allow for the formation of rAAV-ADA-AAVp complexes; (c) detecting AAV-ADA-AAVp complexes; thereby measuring anti-AAV antibodies in a mammalian serum sample. Embodiment 2: The method further comprises, after step (b) and before step (c), the step (bc): (bc) adding a protein or polypeptide that specifically binds to AAVp conjugated to a first member of the binding pair (captAb-BI) to the pre-incubated spiked sample and incubating the mixture to allow for the formation of a ternary AAV-ADA-AAVp-captAb-BI complex. and step (c) further comprises: (c) Detecting the triple AAV-ADA-AAVp-captAb-BI complex 2. The method of embodiment 1, wherein Embodiment 3: After step (b) or after step (bc) and before step (c), step (bbc): (bbc) capturing the ternary complex formed in step (bc) on a solid surface. 3. The method of any one of embodiments 1-2, further comprising: Embodiment 4: A method for measuring anti-AAV particle antibodies in a mammalian serum sample, comprising the following steps, in the above order: (a) diluting at least an aliquot of the sample; (b) adding AAV particles (AAVp) specifically bound by anti-AAV particle antibodies (AAV-ADA) to the diluted sample to generate a spiked sample; (c) pre-incubating the spiked sample to allow for the formation of AAV-ADA-AAVp complexes; (d) detecting AAV-ADA-AAVp complexes; thereby measuring anti-AAV antibodies in a mammalian sample. Embodiment 5: The method further comprises, after step (c) and before step (d), the step (cd): (cd) adding a protein or polypeptide that specifically binds to AAVp conjugated to a first member of the binding pair (captAb-BI) to the pre-incubated spiked sample and incubating the mixture to allow for the formation of a ternary AAV-ADA-AAVp-captAb-BI complex. and step (d) further comprises: (d) Detecting triple AAV-ADA-AAVp-captAb-BI complexes 5. The method of embodiment 4, wherein Embodiment 6: After step (c) or after step (cd) and before step (d), the step (ccd): (ccd) capturing the complex formed in step (c) on a solid surface. 6. The method of any one of embodiments 4 to 5, further comprising: Embodiment 7: The method of any one of embodiments 4 to 6, wherein the diluting is to a final mammalian serum concentration of less than 50% (v / v) and greater than or equal to 0.5% (v / v). Embodiment 8: The method of any one of embodiments 4 to 7, wherein the diluting is to a final mammalian serum concentration of less than 20% (v / v) and greater than or equal to 0.5% (v / v). Embodiment 9: The method of any one of embodiments 4 to 8, wherein the diluting is to a final mammalian serum concentration of less than 5% (v / v) and greater than or equal to 0.5% (v / v). Embodiment 10: The method of any one of embodiments 1 to 9, wherein the measuring is a determination. Embodiment 11: The method of any one of embodiments 1 to 9, wherein the measuring is quantitative. Embodiment 12: The method according to any one of embodiments 1 to 11, wherein the pre-incubation time is 5 to 90 minutes. Embodiment 13: The method according to any one of embodiments 1 to 12, wherein the pre-incubation time is 15 to 60 minutes. Embodiment 14: The method of any one of embodiments 4 to 13, wherein the pre-incubation is for 5 to 25 minutes and the final mammalian serum concentration is about 2% (v / v). Embodiment 15: The method of any one of embodiments 4 to 13, wherein the pre-incubation is for 26 to 90 minutes and the final mammalian serum concentration is about 1% (v / v). Embodiment 16: The method of any one of embodiments 1 to 15, wherein the final concentration of AAVp in the spiked sample is in the range of 0.5E9 to 1E10 viral particles per mL of spiked sample volume. Embodiment 17: The method of any one of embodiments 1 to 16, wherein the final concentration of AAVp in the spiked sample is in the range of 1E9 to 7E9 viral particles per mL of spiked sample volume. Embodiment 18: The method of any one of embodiments 1 to 17, wherein the final concentration of AAVp in the spiked sample is in the range of 1.25E9 to 5E9 viral particles per mL of spiked sample volume. Embodiment 19: The method of any one of embodiments 1 to 18, wherein the final concentration of AAVp in the spiked sample is in the range of 1.5E9 to 3.5E9 viral particles per mL of spiked sample volume. Embodiment 20: The method of any one of embodiments 1 to 19, wherein the sample volume or the volume of an aliquot of the sample is 100 μL or less. Embodiment 21: The method of any one of embodiments 1 to 20, wherein the sample volume or the volume of an aliquot of the sample is 50 μL or less. Embodiment 22: The method of any one of embodiments 1 to 21, wherein the sample volume or the volume of an aliquot of the sample is 10 μL or less. Embodiment 23: The method of any one of embodiments 1 to 22, wherein the sample volume or the volume of an aliquot of the sample is 5 μL or less. Embodiment 24: The method of any one of embodiments 1 to 23, wherein the sample volume or the volume of an aliquot of the sample is 1.5 μL or less. Embodiment 25: The method of any one of embodiments 1 to 24, wherein the sample volume or the volume of the aliquot of the sample is about 1 μL. Embodiment 26: The method of any one of embodiments 1 to 25, wherein the AAV-ADA specifically binds to AAVp of serotype 2 or 8 or 9 or rh74, or a variant thereof. Embodiment 27: The method of any one of embodiments 1 to 26, wherein the AAV-ADA is an anti-recombinant AAV particle antibody (rAAV-ADA). Embodiment 28: The method of any one of embodiments 1 to 27, wherein the AAV-ADA is an anti-therapeutic AAV particle antibody (tAAV-ADA). Embodiment 29: The method of any one of embodiments 1 to 28, wherein the AAV-ADA is an anti-recombinant therapeutic AAV particle antibody (rtAAV-ADA). Embodiment 30: The method of any one of embodiments 1 to 29, wherein the AAVp is conjugated to a first member of a binding pair. Embodiment 31: The method of any one of embodiments 2-3 and 5-30, wherein the captAb-BI is selected from the group consisting of anti-AAVp antibodies, AAVp-binding anticalins, and AAVp-binding fragments thereof. Embodiment 32: The method of any one of embodiments 2-3 and 5-31, wherein captAb-BI is an anti-AAVp antibody or an AAVp-binding fragment thereof. Embodiment 33: The method of any one of embodiments 2-3 and 5-32, wherein captAb-BI is added to a final concentration of 0.1-0.5 μg / mL. Embodiment 34: The method of any one of embodiments 2-3 and 5-33, wherein captAb-BI is added to a final concentration of 0.15-0.35 μg / mL. Embodiment 35: The method of any one of embodiments 2-3 and 5-34, wherein captAb-BI is added to a final concentration of about 0.25 μg / mL. Embodiment 36: The method of any one of embodiments 2 to 3 and 5 to 35, wherein after adding captAb-BI in step (bc) or step (cd), the mixture is incubated for 5 to 90 minutes to allow the formation of a triple AAV-ADA-AAVp-captAb-BI complex. Embodiment 37: The method of any one of embodiments 2 to 3 and 5 to 36, wherein after adding captAb-BI in step (bc) or step (cd), the mixture is incubated for 15 to 60 minutes to allow the formation of a triple AAV-ADA-AAVp-captAb-BI complex. Embodiment 38: The method of any one of embodiments 2 to 3 and 5 to 37, wherein after adding captAb-BI in step (bc) or step (cd), the mixture is incubated for about 30 minutes to allow the formation of a triple AAV-ADA-AAVp-captAb-BI complex. Embodiment 39: The method of any one of embodiments 2-3 and 5-38, wherein the first member of the binding pair is biotin, streptavidin, digoxigenin, fluorescein, or theophylline, respectively. Embodiment 40: The method of any one of embodiments 3 and 6 to 39, wherein the second member of the binding pair is immobilized on a solid surface. Embodiment 41: The method of any one of embodiments 3 and 6 to 40, wherein the second member of the binding pair is streptavidin when the first member is biotin, biotin or an anti-biotin antibody when the first member is streptavidin, an anti-digoxigenin antibody when the first member is digoxigenin, an anti-fluorescein antibody when the first member is fluorescein, or an anti-theophylline antibody when the first member is theophylline. Embodiment 42: The method according to any one of embodiments 3 and 6 to 41, wherein the capturing in step (bbc) or step (ccd) is performed by incubating the mixture obtained in step (bc) or step (cd) with a solid surface for 1 to 60 minutes. Embodiment 43: The method according to any one of embodiments 3 and 6 to 42, wherein the capturing in step (bbc) or step (ccd) is performed by incubating the mixture obtained in step (bc) or step (cd) with a solid surface for 5 to 45 minutes. Embodiment 44: The method according to any one of embodiments 3 and 6 to 43, wherein the capturing in step (bbc) or step (ccd) is performed by incubating the mixture obtained in step (bc) or step (cd) with a solid surface for 10 to 25 minutes. Embodiment 45: The method of any one of embodiments 3 and 6 to 44, wherein the capturing in step (bbc) or step (ccd) is performed by incubating the mixture obtained in step (bc) or step (cd) with a solid surface for about 15 minutes. Embodiment 46: The method of any one of embodiments 1 to 45, wherein detecting in step (c) or step (d) is by incubating with a detection antibody (dectAb) conjugated to a detectable label and determining the presence of the detectable label or quantifying the amount of the detectable label. Embodiment 47: The method of any one of embodiments 1 to 46, wherein the mammalian serum sample is obtained from a mammal to which the AAV particles have been administered.

[0013] In addition to the various embodiments depicted and claimed herein, the presently disclosed subject matter is directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, certain features described herein, particularly those described as individual embodiments, can be combined with each other in other ways within the scope of the disclosed subject matter, and the disclosed subject matter includes any suitable combination of the embodiments and features disclosed herein. The description of certain embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to the embodiments disclosed herein. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows a scheme of a preferred embodiment of the method according to the present invention. [Figure 2] Scheme of antigen capture ELISA known in the art. [Figure 3] Results of the method according to the invention obtained with eight commercially available cynomolgus sera. First bar: detection with polyclonal anti-human IgG antibody without added AAV2 particles; second bar: detection with polyclonal anti-human IgG antibody with added AAV2 particles; third bar: detection with monoclonal anti-cynomolgus IgG antibody without added AAV2 particles; fourth bar: detection with monoclonal anti-cynomolgus IgG antibody with added AAV2 particles. [Figure 4] The results of the method according to the present invention using 16 commercially available cynomolgus monkey sera are shown. Left bar: +AAV2, right bar: no AAV2 added. [Figure 5] Results showing the absence of epitopes with capture antibodies in a method according to the present invention. [Figure 6] 10. Exemplary determination of the optimal concentration of AAV2 capsid variant 7m8 as AAVp in a method according to the invention. [Figure 7]10 shows data showing that the signal obtained by the method according to the invention correlates with the amount of AAVp in the sample at the pre-incubation step. First bar: amount of spiked AAV2 (vp / mL) 132E8; second bar: amount of spiked AAV2 (vp / mL) 66E8; third bar: amount of spiked AAV2 (vp / mL) 33E8; fourth bar: amount of spiked AAV2 (vp / mL) 16.5E8; fifth bar: amount of spiked AAV2 (vp / mL) 8.25E8; sixth bar: amount of spiked AAV2 (vp / mL) 0E8. [Figure 8] Effect of pre-incubation time and serum content in the final sample; signal-to-noise ratio - average value of eight cynomolgus monkey serum samples. [Figure 9] Data showing the general applicability of the method according to the invention: AAVp = AAV2 capsid variant 7m8. [Figure 10] Data showing the general applicability of the method according to the invention: AAVp = empty wild type AAV2. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention The present invention is based, at least in part, on the discovery that pre-incubating a sample in which AAV-ADA must be determined with AAV particles of the same serotype as the therapeutic AAV particles can generate complexes containing AAV-ADA for subsequent detection, thereby dramatically reducing the demand for therapeutic AAV particles as assay reagents compared to conventional AAV-ADA assays in which labeled therapeutic AAV particles are used.

[0016] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0017] Methods and techniques useful for practicing the present invention are described, for example, in Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, N.D., and Hames, B.D., ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R.I. (ed.), Animal Cell Culture—a practical approach, IRL Press Limited (1986); Watson, J.D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E.L., From Genes to Clones, N.Y., VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987), the contents of which are incorporated herein by reference.

[0018] The use of recombinant DNA technology makes it possible to produce derivatives of nucleic acids. Such derivatives can be modified at individual or several nucleotide positions, for example, by substitution, alteration, replacement, deletion, or insertion. Modification or derivatization can be performed, for example, by site-directed mutagenesis. Such modifications can be easily performed by those skilled in the art (e.g., Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

[0019] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably.

[0020] The term "about" refers to a range of ±20% of the preceding numerical value. In certain embodiments, the term "about" refers to a range of ±10% of the preceding numerical value. In certain embodiments, the term "about" refers to a range of ±5% of the preceding numerical value.

[0021] The term "comprising" also encompasses the term "consisting of."

[0022] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity (AAV-binding activity).

[0023] An intact antibody generally comprises two so-called light chain polypeptides (light chains) and two so-called heavy chain polypeptides (heavy chains), each of which comprises a variable region (generally the amino terminal portion of the polypeptide chain) and a constant region (generally the carboxyl terminal portion).

[0024] The constant region of the heavy chain mediates binding of the antibody to i) cells bearing Fc gamma receptors (FcγR), such as phagocytes, or ii) cells bearing the fetal Fc receptor (FcRn), also known as the Brambell receptor. The constant region of the heavy chain also mediates binding to several factors, including factors of the classical complement system, such as the (C1q) component. The constant domain of an antibody heavy chain contains CH1, CH2, and CH3 domains, while the light chain contains a single constant domain, CL, which can be of the kappa or lambda isotype.

[0025] The variable domain of an immunoglobulin's light or heavy chain mediates the binding of the antibody to its cognate antigen. Each variable domain contains different segments: four framework regions (FR) and three hypervariable regions (HVR).

[0026] "Antibody fragment" refers to a molecule other than an intact or complete antibody that contains a portion of an intact or complete antibody and binds to the same antigen as the intact or complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005), which is incorporated herein by reference.

[0027] The term "capture antibody" refers to an antibody used in a sandwich ELISA format to bind to (i.e., capture) a target substance present in a sample to be detected. A second antibody (i.e., detection antibody) then binds to the captured target substance with a different epitope, allowing detection of the capture antibody-target complex (i.e., forming a "sandwich").

[0028] A "conjugate" is a recombinant or chemical fusion comprising at least one proteinaceous moiety. Each other moiety can be a second proteinaceous moiety or a non-proteinaceous moiety. Because the moieties of a conjugate are permanently attached to each other, it is a covalent conjugate. Covalent conjugation can be either direct or via a linker. In certain embodiments, direct recombinant conjugation is achieved by constructing a polypeptide fusion (i.e., by genetic fusion of two genes encoding, for example, an antibody and a detectable label or member of a binding pair, and expressed as a single polypeptide (chain)). In certain embodiments, direct chemical conjugation is achieved by forming a covalent bond, for example, between a reactive group on an antibody and a corresponding group or acceptor on the detectable label or member of a binding pair. In certain embodiments, direct chemical conjugation is achieved by modifying (i.e., genetically modifying) one of the two moieties to be conjugated to contain a reactive group (for example, a sulfhydryl or carboxyl group) that forms a covalent bond to one of the two moieties under appropriate conditions. As one non-limiting example, a molecule (ie, an amino acid) bearing a desired reactive group (ie, a cysteine ​​residue) can be introduced into, for example, an antibody.

[0029] Methods for covalently linking nucleic acids to proteins are also known in the art (i.e., photocrosslinking, see, eg, Zatsepin et al. Russ. Chem. Rev. 74 (2005) 77-95).

[0030] In certain embodiments, the linker is a recombinant linker.

[0031] Conjugation via a recombinant linker can be carried out using a single-stranded peptide linker. In certain embodiments, the single-stranded peptide linker comprises 1 to 20 amino acid residues linked by peptide bonds. In certain embodiments, the amino acids are selected from the 20 naturally occurring amino acids. In certain other embodiments, one or more of the amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In certain embodiments, the linker is a single-stranded peptide linker having an amino acid sequence at least 25 amino acid residues in length, and in a preferred embodiment, 32 to 50 amino acid residues in length. In certain embodiments, the peptide linker is a (GxS)n linker, where G=glycine, S=serine, and (x=3, n=8, 9, or 10) or (x=4 and n=6, 7, or 8). In certain embodiments, x=4, n=6, or 7, and in a preferred embodiment, x=4, n=7.

[0032] In certain embodiments, the linker is a chemical linker.

[0033] Linker-mediated chemical conjugation can be carried out using a variety of chemical linkers. In certain embodiments, the antibody and detectable label or member of the binding pair are conjugated using a bifunctional protein coupling agent selected from the group consisting of N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0034] The term "detection antibody" refers to an antibody that has a means for visualization or quantification. Such means are typically enzymes that catalyze the formation of a colored or fluorescent reaction product after the addition of an appropriate substrate (detection reagent), such as, for example, horseradish peroxidase, urease, alkaline phosphatase, glucoamylase, or β-galactosidase. In certain embodiments, the detection antibody is a species-specific anti-immunoglobulin antibody. In certain embodiments, the detection antibody is conjugated to a detectable label. In certain embodiments, the detectable label is selected from the group consisting of biotin, digoxigenin, fluorescein, theophylline, a fluorescent marker, or a radioisotope. The detection antibody is detected and / or quantified using this detectable label.

[0035] The term "detection reagent" refers to a reagent that allows for the detection and / or quantification of an antibody bound to an antigen. In certain embodiments, the detection reagent is a colorimetric substrate for an enzyme conjugated to the antibody. Addition of an appropriate substrate to the antibody-enzyme conjugate results in the generation of a colorimetric or fluorometric signal (e.g., after the conjugated antibody binds to the antigen of interest).

[0036] The term "ELISA" refers to enzyme-linked immunosorbent assay. Various ELISA formats and applications are known in the art (see, for example, Crowther, "Enzyme-Linked Immunosorbent Assay (ELISA)," in Molecular Biomethods Handbook, Rapley et al. [eds.], pp. 595-617, Humana Press, Inc., Totowa, NJ (1998); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988); Ausubel et al. (eds.), Current Protocols in Molecular Biology, Ch. 11, John Wiley & Sons, Inc., New York (1994)). These documents are incorporated herein by reference.

[0037] One specific ELISA format is so-called "direct ELISA". In this ELISA format, the target present in the sample, for example, antibody, is detected. In direct ELISA, the sample containing the target is brought into contact with a solid phase, for example, a fixed or immobilized support (for example, a microtiter plate well). If the target exists in the sample, it is immobilized on the solid phase, and then directly detected using a detection molecule that is conjugated with an enzyme. If the target is an antigen, the detection molecule is an antibody that is specific to the antigen, or if the target is an antibody that is specific to the antigen, the detection molecule is an antibody that is conjugated with an enzyme and that is specific to the antigen.

[0038] Another specific ELISA format is the so-called "indirect ELISA." In this ELISA format, the antigen (or antibody) is immobilized on a solid phase (e.g., a microtiter plate well). An antigen-specific antibody (or antigen) is then added, followed by a detection antibody specific to the antibody that specifically binds to the antigen. This detection antibody can be a "species-specific" antibody (e.g., a goat anti-rabbit antibody).

[0039] Another specific ELISA format is the so-called "sandwich ELISA." In this format, an antigen is immobilized on a solid phase (e.g., a microtiter plate well) via capture by an antibody that specifically binds to the antigen (i.e., a capture antibody), which is immobilized (covalently or via a specific binding pair) to the solid phase or binding pair. Typically, a sample containing the antigen is added to the solid phase, followed by washing. If the antigen of interest is present in the sample, it will be bound to the solid phase by the capture antibody.

[0040] The term "framework region" or "FR" refers to variable domain residues other than the hypervariable regions (HVRs). Antibody variable domains generally comprise four FRs: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-HVR-H1(HVR-L1)-FR2-HVR-H2(HVR-L2)-FR3-HVR-H3(HVR-L3)-FR4.

[0041] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure.

[0042] The term "in vitro" refers either to an artificial environment or to a process or reaction taking place within such an artificial environment.

[0043] The term "in vivo" refers to the natural environment of a compound (eg, an animal or a cell) or a process or reaction that takes place within that natural environment.

[0044] The term "immunoassay" refers to any technique that utilizes a specific binding molecule, such as an antibody, to capture and / or detect a specific target in a sample for qualitative or quantitative analysis. Generally, an immunoassay is characterized by the following steps: 1) immobilization or capture of the analyte; and 2) detection and measurement of the analyte. The analyte can be captured, i.e., bound, to any solid surface, such as a membrane, a plastic plate, or some other solid surface.

[0045] The term "linker" refers to a chemical or single-stranded peptide linker that covalently connects different entities in a conjugate, such as connecting an antibody to a detectable label or one partner of a binding pair.

[0046] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprising the population are identical except for variant antibodies that contain, for example, naturally occurring mutations or that may arise during the production of a monoclonal antibody preparation; such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each antibody molecule of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0047] The term "variable domain" refers to the portion of an antibody heavy or light chain that is involved in binding the antibody to its cognate antigen. The heavy and light chain variable domains (VH and VL, respectively) of native antibodies generally have a similar structure, with each domain containing four framework regions (FR) and three hypervariable regions (HVR). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain of an antibody that binds to the antigen.

[0048] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and determine antigen-binding specificity. These regions form the paratope or binding site of the binding domain.

[0049] Generally, antibodies contain six antigen-binding specificity-determining regions: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary antigen-binding specificity-determining regions herein include: (a) the hypervariable loops according to Chothia, encompassing amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the variable domain (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) hypervariable regions according to Kabat, encompassing amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) of the variable domain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (a+b) Hypervariable loops combined with hypervariable regions, including amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 26-35 (H1), 50-65 (H2), and 95-102 (H3) of the variable domain (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) + Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) antigen contact residues, which include amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0050] Unless otherwise indicated, HVRs are defined herein according to Kabat et al., supra. Those skilled in the art will understand that the designation of antigen-binding specificity-determining regions can also be defined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0051] As used herein, the term "signal" encompasses any detectable physical change that can be used to indicate that a reaction has occurred, e.g., binding of an antibody to its antigen. Signals in the form of fluorescent or colorimetric products / reagents are particular forms of signals that are contemplated as being usable in the methods of the present invention. In certain embodiments, the signal is assessed quantitatively.

[0052] "Solid phase" refers to non-fluidic substances, including particles (including microparticles and beads) made from materials such as polymers, metals (paramagnetic and ferromagnetic particles), glass, and ceramics; gel materials such as silica, alumina, and polymer gels; capillaries made from polymers, metals, glass, and / or ceramics; zeolites and other porous materials; electrodes; microtiter plates; solid strips; and cuvettes, tubes, or other spectrometer sample containers. Solid phase components are distinguished from inert solid surfaces in that the "solid phase" contains at least one moiety on its surface that is intended to interact with substances in a sample, either directly or through a compound bound to it. Solid phases can be stationary components, such as tubes, strips, cuvettes, or microtiter plates, or non-stationary components, such as beads and microparticles. A variety of microparticles can be used that allow for either non-covalent or covalent attachment of proteins and other substances. Such particles include polymeric particles, such as polystyrene and poly(methyl methacrylate); gold particles, such as gold nanoparticles and gold colloids; and ceramic particles, such as silica, glass, and metal oxide particles. See, for example, Martin, CR, et al., Analytical Chemistry - News & Features, 70 (1998) 322A-327A, or Butler, JE, Methods 22 (2000) 4-23, which are incorporated herein by reference.

[0053] The terms "therapeutic adeno-associated virus particle" and "agent" are used interchangeably herein. These terms are used in the broadest sense to encompass a variety of structures so long as they exhibit the desired activity.

[0054] As used herein, the term "sample" refers to any biological matrix in which the presence or amount of rAAV-ADA can be determined. Exemplary samples include, but are not limited to, serum, plasma, aqueous humor, vitreous humor, retinal tissue lysate, and tumor tissue. In a preferred embodiment, the sample is serum.

[0055] As used herein, the term "anti-AAV antibodies" refers to antibodies produced by the immune system of a recipient of therapeutic AAV particles against said therapeutic AAV particles following administration of said therapeutic AAV particles.

[0056] As used herein, the term "antibody that specifically binds to AAV particles" and its grammatical equivalents refer to a recombinantly produced antibody that specifically binds to AAV particles. In certain embodiments, the "antibody that specifically binds to AAV particles" has been generated by immunization of laboratory animals or by phage display methods.

[0057] As used herein, the term "immunogenicity" refers to the potential of an AAV particle to induce an immune response in a human or animal. During drug development, immunogenicity is primarily assessed by measuring the binding and neutralization of anti-AAV antibodies in samples.

[0058] The term "empty particle" refers to an AAV particle that has an AAV protein shell but lacks all or part of the recombinant AAV vector, i.e., the nucleic acid that encodes a protein or is transcribed into a transcript of interest adjacent to two AAV ITRs.Therefore, an empty particle is an AAV particle that does not have a functional or complete transgene (nucleic acid payload) encapsidated.Therefore, an empty capsid does not function to transfer the nucleic acid that encodes a protein or is transcribed into a transcript of interest into a host cell.

[0059] A "recombinant AAV vector" is a nucleic acid derived from the wild-type genome of a virus, such as AAV, by using molecular biological methods, in which the structural genes of the virus (e.g., of AAV) have been removed and replaced with a non-native nucleic acid, e.g., a nucleic acid transcribed into a therapeutic transcript or a nucleic acid encoding a therapeutic protein. Typically, therapeutic recombinant AAV vectors retain both inverted terminal repeat (ITR) sequences of the wild-type AAV genome.

[0060] A "recombinant" AAV vector is distinguished from the wild-type viral AAV genome insofar as all or part of the viral genome has been replaced with one or more non-native (i.e., heterologous) sequences. Thus, the incorporation of non-native sequences defines the viral vector as a "recombinant" vector, which in the case of AAV may be referred to as a "rAAV vector."

[0061] Recombinant AAV vectors (rAAV vectors) can be packaged into protein (capsid) shells, referred to herein as "particles," for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo. When a recombinant vector sequence is enclosed or packaged in an AAV particle, the particle can also be referred to as an "rAAV particle." The particle's capsid contains proteins that encapsulate or package the rAAV vector. Specific examples of such proteins are viral envelope proteins, and in the case of AAV, capsid proteins such as AAV VP1, VP2, and VP3.

[0062] As used herein, the term "serotype" refers to a distinction based on serologically distinct AAV capsid proteins. Serological specificity is determined based on the lack of cross-reactivity between antibodies against one AAV compared to other AAVs. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). Under the conventional definition, a new serotype means that the virus of interest has been tested against sera specific to all existing and characterized serotypes for neutralizing activity, and no antibodies that neutralize the virus of interest have been found. As more naturally occurring virus isolates are discovered and / or capsid variants are generated, serological differences may or may not emerge from any of the currently existing serotypes. Therefore, if a new virus (e.g., AAV) does not have serological differences, it is a subgroup or variant of the corresponding serotype. Thus, for convenience and to avoid repetition, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and non-serologically distinct viruses (e.g., AAV) that may be within subgroups or variants of a serotype.

[0063] The term "transgene" is used herein to conveniently refer to a nucleic acid that is contemplated, for example, in a recombinant AAV vector or introduced into a cell or organism. A transgene includes any nucleic acid, for example, a gene that is transcribed into a transcript or encodes a polypeptide or protein.

[0064] "Vector" refers to the portion of a recombinant plasmid sequence that is ultimately packaged or encapsulated, either directly or in single-stranded or RNA form, to form a viral (e.g., AAV) particle. When a recombinant plasmid is used to construct or produce a recombinant viral particle, the viral particle does not contain any portion of the "plasmid" that does not correspond to the vector sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called the "plasmid backbone," which is important for plasmid cloning and amplification, a process necessary for propagation and recombinant viral production, but is not itself packaged or encapsulated in the viral (e.g., AAV) particle. Thus, "vector" refers to the nucleic acid portion of the plasmid that is packaged or encapsulated by the viral particle (e.g., AAV).

[0065] A "plasmid" is typically a form of nucleic acid or polynucleotide that has additional elements for expression (e.g., transcription, replication, etc.) or propagation (replication) of the plasmid. As used herein, plasmid can also be used to refer to such a nucleic acid or polynucleotide sequence.

[0066] Recombinant cells Generally, efficient, large-scale production of a proteinaceous compound of interest, such as a rAAV particle or a therapeutic polypeptide, requires cells that express and possibly secrete said proteinaceous compound. Such cells are called "recombinant cells" or "recombinant production cells."

[0067] For the generation of "recombinant production cells," suitable mammalian cells are transfected with the necessary nucleic acid sequence encoding the proteinaceous compound of interest. Depending on the polypeptide, e.g., AAV, transfection of additional helper polypeptides may be necessary.

[0068] To generate stable recombinant production cells, a second step follows, in which single cells that stably express the proteinaceous compound of interest are selected. This can be done, for example, based on the co-expression of a selectable marker co-transfected with the nucleic acid sequence encoding the proteinaceous compound of interest, or by expression of the proteinaceous compound itself.

[0069] To express a coding sequence, i.e., an open reading frame, in a recombinant cell, additional regulatory elements such as a promoter and a polyadenylation signal (sequence) are required. Therefore, the open reading frame is operably linked to the additional regulatory elements for transcription. This can be achieved by incorporating it into a so-called expression cassette. The minimum control elements required for an expression cassette to be functional in mammalian cells are a promoter functional in mammalian cells located upstream, i.e., 5', of the open reading frame, and a polyadenylation signal (sequence) functional in mammalian cells located downstream, i.e., 3', of the open reading frame. In addition, a terminator sequence may be present 3' from the polyadenylation signal (sequence). For expression, the promoter, open reading frame / coding region, and polyadenylation signal sequence must be arranged in an operably linked form.

[0070] If the proteinaceous compound of interest is an AAV particle, which is composed of different (monomeric) capsid polypeptides and single-stranded DNA molecules and requires other adenoviral helper functions for production and encapsidation, multiple expression cassettes containing different open reading frames / coding sequences are required. In this case, at least one expression cassette is required for each of the transgene, the various polypeptides that form the capsid of the recombinant AAV particle, the necessary helper functions, and the VA RNA. Therefore, individual expression cassettes are required for each of the helper E1A, E1B, E2A, E4orf6, VA RNA, and rep and cap genes.

[0071] As outlined in the previous paragraph, the more complex the proteinaceous compound of interest, or the greater the number of additional helper polypeptides and / or RNAs required, the greater the number of different expression cassettes required, respectively. Essentially, the total size of the nucleic acid required increases with the number of expression cassettes. However, there is a practical upper limit to the size of the nucleic acid that can be transferred, within the range of approximately 15 kbp (kilobase pairs). Beyond this limit, handling and processing efficiency decreases significantly. This problem can be addressed by using two or more separate plasmids. This allows different expression cassettes to be assigned to different plasmids, with each plasmid containing only a portion of the expression cassette.

[0072] For the generation of stable cell lines, random integration (RI) of a nucleic acid carrying an expression cassette for a proteinaceous compound of interest can be used. Generally, by using RI, a nucleic acid or a fragment thereof is randomly integrated into the genome of a host cell.

[0073] As an alternative to RI, CLD can also use targeted integration (TI), in which one or more nucleic acids containing different expression cassettes are introduced / integrated into a predetermined locus in the genome of the host cell.

[0074] In TI, either homologous recombination or recombinase-mediated cassette exchange (RMCE) can be used to integrate nucleic acids containing respective expression cassettes into specific loci in the genome of the TI host cell.

[0075] Adeno-associated virus For a general review of AAV and adenovirus or herpesvirus helper functions, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The AAV genome is described in Srivastava et al., J. Virol., 45 (1983) 555-564. U.S. Patent No. 4,797,368 describes design considerations for constructing recombinant AAV vectors (see also WO 93 / 24641). Further references describing AAV vectors are West et al., Virol. 160 (1987) 38-47; Kotin, Hum. Gene Ther. 5 (1994) 793-801; and Muzyczka J. Clin. Invest. 94 (1994) 1351. The construction of recombinant AAV vectors is described in U.S. Patent No. 5,173,414; Lebkowski et al., Mol. Cell. Biol. 8 (1988) 3988-3996; Tratschin et al., Mol. Cell. Biol. 5 (1985) 3251-3260; Tratschin et al., Mol. Cell. Biol., 4 (1994) 2072-2081; Hermonat and Muzyczka Proc. Natl. Acad. Sci. USA 81 (1984) 6466-6470; Samulski et al. J. Virol. 63 (1989) 3822-3828, all of which are incorporated herein by reference.

[0076] Adeno-associated virus (AAV) is a replication-deficient parvovirus.It can only replicate in cells where certain viral functions are provided by co-infecting helper viruses such as adenovirus, herpesvirus, and sometimes poxviruses such as vaccinia.Nevertheless, AAV can replicate in virtually any cell line of human, monkey, or rodent origin if appropriate helper virus functions exist.

[0077] In the absence of helper virus genes, AAV establishes latency in its host cells. Its genome integrates into a specific site on chromosome 19 [(Chr)19(q13.4)], called adeno-associated virus integration site 1 (AAVS1). For certain serotypes, such as AAV-2, other integration sites have been found, such as on chromosome 5 [(Chr)5(p13.3)], called AAVS2, and on chromosome 3 [(Chr)3(p24.3)], called AAVS3.

[0078] AAVs are classified into different serotypes, which are assigned based on parameters such as hemagglutination, tumorigenicity, and DNA sequence homology. To date, more than 12 different serotypes and over 100 sequences corresponding to different clades of AAV have been identified.

[0079] The type and symmetry of the capsid protein determine the tissue tropism of each AAV. For example, AAV-2, AAV-4, and AAV-5 are specific for the retina, AAV-2, AAV-5, AAV-8, AAV-9, and AAVrh-10 are specific for the brain, AAV-1, AAV-2, AAV-6, AAV-8, and AAV-9 are specific for heart tissue, AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and AAV-10 are specific for the liver, and AAV-1, AAV-2, AAV-5, and AAV-9 are specific for the lung.

[0080] Pseudotyping refers to the process of cross-packaging of AAV genomes or recombinant AAV vectors between various serotypes, i.e., the genome is packaged within capsid proteins from different serotypes. The serotype of a genome is determined by the serotype of its ITRs (inverted terminal repeats).

[0081] The wild-type AAV genome has a size of approximately 4.7 kb. The AAV genome further contains two overlapping genes called rep and cap, which contain multiple open reading frames (see, for example, Srivastava et al., J. Viral., 45 (1983) 555-564; Hermonat et al., J. Viral., 51 (1984) 329-339; Tratschin et al., J. Virol., 51 (1984) 611-619). The Rep protein encoding open reading frame provides four proteins of different sizes called Rep78, Rep68, Rep52, and Rep40. These are involved in AAV replication, rescue, and integration. The Cap protein encoding open reading frame provides four proteins called VP1, VP2, VP3, and AAP. VP1, VP2, and VP3 are part of the proteinaceous capsid of the AAV particle. The combined rep and cap open reading frames are flanked at their 5' and 3' ends by so-called inverted terminal repeats (ITRs). For replication, AAV requires, in addition to the Rep and Cap proteins, the products of the adenoviral genes E1A, E1B, E4orf6, E2A, and VA or the corresponding factors of another helper virus.

[0082] For example, in the case of AAV serotype 2 (AAV-2), each ITR is 145 nucleotides long and flanks a coding sequence region of approximately 4,470 nucleotides. Of the 145 nucleotides in the ITR, 125 nucleotides have a palindromic sequence and can form a T-shaped hairpin structure. This structure functions as a primer during viral replication. The remaining 20 unpaired nucleotides are designated as the D sequence.

[0083] The AAV genome has three transcription promoters, P5, P19, and P40, for expression of the rep and cap genes (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571).

[0084] The ITR sequence must be present in cis with respect to the coding region. The ITR provides a functional origin of replication (ori), the signal required for integration into the genome of target cells, and efficient excision and rescue from host cell chromosomes or recombinant plasmids. The ITR also contains replication origin-like elements such as Rep protein binding sites (RBS) and terminal release sites (TRS). It has been found that the ITR itself can function as a transcription promoter in AAV vectors (Flotte et al., J. Biol. Chem. 268 (1993) 3781-3790; Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163-10167).

[0085] For replication and encapsidation of the viral single-stranded DNA genome or recombinant AAV vector, the rep and cap genes must be present in trans.

[0086] The rep locus contains two internal promoters, designated P5 and P19. Promoter P5 is operably linked to a nucleic acid sequence that provides an unspliced ​​4.2 kb mRNA encoding the Rep protein Rep78 (a chromatin nickase that arrests the cell cycle) and a spliced ​​3.9 kb mRNA encoding the Rep protein Rep68 (a site-specific endonuclease). Promoter P19 is operably linked to a nucleic acid sequence that provides an unspliced ​​mRNA encoding the Rep protein Rep52 and a spliced ​​3.3 kb mRNA encoding the Rep protein Rep40 (a DNA helicase for accumulation and packaging).

[0087] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV double-stranded DNA replication, whereas the smaller Rep proteins, Rep52 and Rep40, appear to be essential for progeny single-stranded DNA accumulation (Chejanovsky & Carter, Virology 173 (1989) 120-128).

[0088] The larger Rep proteins, Rep68 and Rep78, can specifically bind to the hairpin conformation of the AAV ITR. They exhibit the required enzymatic activity for resolving replication at the AAV termini. Expression of Rep78 or Rep68 can be sufficient for infectious particle formation (Holscher, C., et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0089] All Rep proteins, primarily Rep78 and Rep68, appear to exhibit regulatory activities such as AAV gene induction and repression and inhibitory effects on cell proliferation (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894; Labow et al., Mol. Cell. Biol., 7 (1987) 1320-1325; Khleif et al., Virology, 181 (1991) 738-741).

[0090] Recombinant overexpression of Rep78 results in a phenotype involving reduced cell proliferation due to the induction of DNA damage, which arrests host cells in S phase and thereby promotes latent infection by the virus (Berthet, C., et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0091] reported that the P5 promoter is negatively autoregulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of Rep protein expression, only very low expression has been reported for certain cell lines after stable AAV integration (see, for example, Mendelson et al., Virol. 166 (1988) 154-165).

[0092] The cap locus contains one promoter, designated P40. Through alternative splicing and the use of alternative start codons, promoter P40 is operably linked to a nucleic acid sequence that provides a 2.6-kb mRNA encoding the Cap proteins VP1 (87 kDa, unspliced ​​mRNA transcript), VP2 (72 kDa from a spliced ​​mRNA transcript), and VP3 (61 kDa from an alternative start codon). VP1 through VP3 constitute the viral capsid. The capsid functions by binding to cell surface receptors and enabling intracellular transport of the virus. VP3 accounts for approximately 90% of the total viral particle protein. Nevertheless, all three proteins are essential for efficient capsid production.

[0093] It has been reported that inactivation of all three capsid proteins, VP1-VP3, prevents the accumulation of single-stranded progeny AAV DNA. Mutations in the VP1 amino terminus ("lipid-negative" or "Inf-negative") still allow assembly of single-stranded DNA into viral particles, thereby significantly reducing infectious titers.

[0094] The AAP open reading frame encodes the assembly activating protein (AAP), which is approximately 22 kDa in size and transports native VP proteins to the nucleolus for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

[0095] Each AAV particle contains only one single-stranded DNA molecule. This can be either a "plus" or "minus" strand. AAV viral particles containing the DNA molecule are infectious. Inside the infected cell, the infectious single strand is converted to a double strand and then amplified. Amplification results in a large pool of double-stranded DNA molecules, from which the single strand is displaced and packaged into capsids.

[0096] Adeno-associated virus (AAV) vectors can transduce dividing as well as quiescent cells, and transgenes introduced into target cells using AAV vectors are likely to be expressed for a long period of time.

[0097] One drawback of using AAV vectors is the limited size of the transgene that can be introduced into a cell.

[0098] Viral vectors, such as parvovirus particles containing AAV serotypes and their variants, provide a means for delivering nucleic acids encoding proteins to cells ex vivo, in vitro, and in vivo, so that the cells express the encoded proteins. AAV is a useful virus as a gene therapy vector because it can penetrate cells and introduce nucleic acids / genetic material so that the nucleic acids / genetic material can be stably maintained within the cells. Furthermore, these viruses can introduce nucleic acids / genetic material at specific sites. Because AAV is not associated with pathogenic diseases in humans, AAV vectors can deliver endogenous and exogenous polynucleotide sequences (e.g., therapeutic proteins and drugs) to human patients without causing substantial pathogenesis or disease.

[0099] Viral vectors that can be used include, but are not limited to, adeno-associated virus (AAV) particles of multiple serotypes (eg, AAV-1 to AAV-12, etc.) and hybrid / chimeric AAV particles.

[0100] AAV particles can be used as vehicles for effective gene delivery. Such particles have several desirable characteristics for such applications, including targeting both dividing and non-dividing cells. Early clinical experience with these vectors has also shown no persistent toxicity. AAV is known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells.

[0101] Recombinant AAV particles typically do not contain viral genes related to pathogenesis.Such vectors typically have one or more of the wild-type AAV genes deleted in whole or in part, such as rep and / or cap genes, but retain at least one functional adjacent ITR sequence as necessary for the rescue, replication and packaging of recombinant vector into AAV particles.For example, only the essential parts of the vector, such as ITR and LTR elements, are included in the recombinant AAV vector.Therefore, the recombinant AAV vector genome contains the sequences required for replication and packaging in cis (for example, functional ITR sequence).

[0102] Recombinant AAV vectors and their methods and uses include any virus strain or serotype.As a non-limiting example, recombinant AAV vectors can be based on any AAV genome, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, AAV-2i8, AAVrh74 or AAV7m8.Such vectors can be based on the same strain or serotype (or subgroup or variant), or can be different from each other.As a non-limiting example, recombinant AAV vectors based on one serotype genome can be identical in one or more of the capsid proteins that package the vector.In addition, recombinant AAV vector genomes can be based on AAV (for example, AAV2) serotype genomes that are different from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector genome may be based on AAV2, but at least one of the three capsid proteins may be, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAVrh74, AAV7m8, or a variant thereof. AAV variants include variants and chimeras of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAVrh74, and AAV7m8 capsids.

[0103] In certain embodiments of all aspects and embodiments, the adeno-associated virus (AAV) vector or particle is selected from the group of serotypes including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAVrh74, and AAV7m8, and variants thereof (e.g., capsid variants, e.g., amino acid insertions, additions, substitutions, and deletions). See, e.g., WO 2013 / 158879, WO 2015 / 013313, and U.S. Patent Application Publication No. 2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0104] AAV and AAV variant (e.g., capsid variant) serotypes (e.g., VP1, VP2 and / or VP3 sequences) may or may not be distinct from other AAV serotypes, including, for example, AAV1-AAV12 (e.g., different from the VP1, VP2 and / or VP3 sequences of any of the AAV1-AAV12 serotypes).

[0105] In certain embodiments of all aspects and embodiments, AAV particles related to a reference serotype have a polypeptide or subsequence thereof that comprises or consists of a sequence at least 80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAVrh74, or AAV7m8 (e.g., ITR sequences, or VP1, VP2, and / or VP3 sequences, etc.).

[0106] The methods and uses of the invention include AAV sequences (polypeptide and nucleotide) and subsequences, genes or proteins thereof that show less than 100% sequence identity to a reference AAV serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAVrh74, or AAV7m8, but are different from and not identical to known AAV genes or proteins such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAVrh74, or AAV7m8, etc. In certain embodiments of all aspects and embodiments, the AAV polypeptide or subsequence thereof comprises or consists of a sequence that is at least 75% identical or greater, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to any reference AAV sequence or subsequence thereof, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAVrh74, or AAV7m8 (e.g., VP1, VP2 and / or VP3 capsid or ITR). In certain embodiments, the AAV variant has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to a reference serotype.

[0107] Recombinant AAV particles, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAVrh74 and AAV7m8, as well as variant, related, hybrid and chimeric sequences, can be constructed using recombinant techniques known to those skilled in the art to contain one or more nucleic acid sequences (recombinant AAV vectors / transgenes) flanked by one or more functional AAV ITR sequences.

[0108] Recombinant particles (e.g., rAAV particles) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are particularly useful for administration and delivery to subjects in vivo or ex vivo. In addition to the drug, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent that does not itself induce a harmful immune response in the individual receiving the composition and can be administered without undue toxicity.

[0109] Protocols for the production of adenoviral vectors are described in U.S. Pat. Nos. 5,998,205; 6,228,646; 6,093,699; 6,100,242; WO 94 / 17810 and WO 94 / 23744.

[0110] Recombinant AAV particles Various methods are known in the art for producing rAAV particles. For example, transfection with an AAV plasmid and an AAV helper sequence in conjunction with co-infection with an AAV helper virus (e.g., adenovirus, herpesvirus, vaccinia virus), or transfection with a recombinant AAV vector containing a plasmid, a plasmid containing an AAV helper function, or a plasmid containing rep / cap. Non-limiting methods for producing rAAV particles are described, for example, in U.S. Patent No. 6,001,650, U.S. Patent No. 6,004,797, International Publication No. 2017 / 096039, and International Publication No. 2018 / 226887. After recombinant rAAV particle production (i.e., particle production in a cell culture system), rAAV particles can be obtained and purified from host cells and cell culture supernatant.

[0111] The production of recombinant AAV particles requires the expression of Rep and Cap proteins, helper proteins E1A, E1B, E2A and E4orf6, and adenovirus VA RNA in a single mammalian cell. The helper proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter, particularly the CMV IE promoter, as described by Matsushita et al. (Gene Ther. 5 (1998) 938-945). Therefore, any promoter can be used.

[0112] Typically, to produce recombinant AAV particles, different complementary plasmids are co-transfected into host cells. One of the plasmids contains a transgene sandwiched between two cis-acting AAV ITRs. The missing AAV elements required for replication and subsequent packaging of the progeny recombinant genome, namely the open reading frames for the Rep and Cap proteins, are contained in trans on the second plasmid. Overexpression of the Rep protein results in an inhibitory effect on cell growth (Li, J., et al., J. Virol. 71 (1997) 5236-5243). In addition, a third plasmid containing helper virus genes, namely E1, E4orf6, E2A, and VA from adenovirus, is required for AAV replication.

[0113] To reduce the number of plasmids required, the Rep, Cap and adenoviral helper genes can be combined on a single plasmid.

[0114] Alternatively, the host cell may already stably express the E1 gene product. Such cells are HEK293 cells. The human embryonic kidney clone designated 293 was created in 1977 by integrating adenovirus DNA into human embryonic kidney (HEK) cells (Graham, FL, et al., J. Gen. Virol. 36 (1977) 59-74). The HEK293 cell line contains base pairs 1 to 4344 of the adenovirus serotype 5 genome, which encompasses the E1A and E1B genes and the adenovirus packaging signal (Louis, N., et al., Virology 233 (1997) 423-429).

[0115] When using HEK293 cells, the missing E2A, E4orf6, and VA genes can be introduced by co-infection with adenovirus or by co-transfection with E2A, E4orf6, and VA expression plasmids (e.g., Samulski, RJ, et al., J. Virol. 63 (1989) 3822-3828; Allen, JM, et al., J. Virol. 71 (1997) 6816-6822; Tamayose, K., et al., Hum. Gene Ther. 7 (1996) 507-513; Flotte, TR, et al., Gene Ther. 2 (1995) 29-37; Conway, JE, et al., J. Virol. 71 (1997) 8780-8789; Chiorini, JA, et al., Hum. Gene Ther. Ther.6(1995)1531-1541;Ferrari,FK,et al.,J.Virol.70(1996)3227-3234;Salvetti,A.,et al.,Hum.Gene Ther.9(1998)695-706;Xiao,X.,et al., J. Virol. 72 (1998) 2224-2232; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Zhang, X., et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors may be used (see, e.g., Conway, JE, et al., J. Virol. 71 (1997) 8780-8789; Johnston, KM, et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, AJ, et al., Gene Ther. 2 (1995) 481-485; Fisher, JK, et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, KM, et al., Hum. Gene Ther. 8 (1997) 359-370).

[0116] Thus, cell lines in which the rep gene is integrated and expressed tend to grow slowly or express very low levels of Rep protein.

[0117] To restrict transgene activity to specific tissues, i.e., to limit the integration site, the transgene can be operably linked to an inducible or tissue-specific promoter (see, e.g., Yang, Y., et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0118] In certain embodiments of all aspects and embodiments, the rAAV particles are derived from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74 and AAV7m8.

[0119] In certain embodiments of all aspects and embodiments, the rAAV particles comprise a capsid sequence having 70% or greater sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74, or AAV7m8 capsid sequence.

[0120] In certain embodiments of all aspects and embodiments, the rAAV particles comprise ITR sequences having 70% or greater sequence identity to the ITR sequences of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0121] E1A, E1B, E2, and E4 The coding sequences for E1A and E1B (open reading frames) can be derived from, for example, a human adenovirus, particularly human adenovirus serotype 2 or serotype 5. An exemplary sequence for human Ad5 (adenovirus serotype 5) is found in GenBank entry X02996, AC_000008, and an exemplary sequence for human Ad2 is found in GenBank entry AC_000007. Nucleotides 505-3522 contain the nucleic acid sequence encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146, reported in EP 1230354, and plasmids pGS119 and pGS122, reported in WO 2007 / 056994, can also be used as sources of E1A and E1B open reading frames.

[0122] E1A is the first viral helper gene expressed after adenoviral DNA enters the cell nucleus. The E1A gene encodes the 12S and 13S proteins, which are derived from the same E1A mRNA by alternative splicing. Expression of the 12S and 13S proteins activates other viral functions, E1B, E2, E3, and E4. Furthermore, expression of the 12S and 13S proteins forces cells into the S phase of the cell cycle. If only E1A-derived proteins are expressed, the cells will die (apoptosis).

[0123] E1B is the second viral helper gene expressed. It is activated by the E1A-derived proteins 12S and 13S. The E1B gene-derived mRNA can be spliced ​​in two different ways, resulting in a 55-kDa transcript and a 19-kDa transcript. The E1B 55-kDa protein is involved in regulating the cell cycle, preventing the transport of cellular mRNA during the late stages of infection, and preventing E1A-induced apoptosis. The E1B 19-kDa protein is involved in preventing E1A-induced apoptosis of cells.

[0124] The E2 gene encodes various proteins: The E2A transcript encodes a single-stranded binding protein (SSBP) that is essential for AAV replication.

[0125] The E4 gene also encodes several proteins: the 34 kDa protein derived from the E4 gene (E4orf6), which, together with the 55 kDa E1B protein, prevents the accumulation of cellular mRNA in the cytoplasm, but also promotes the transport of viral RNA from the cell nucleus to the cytoplasm.

[0126] Adenovirus VA RNA gene Virus-associated RNA (VA RNA) is a non-coding RNA of adenovirus (Ad) that regulates translation. The adenovirus genome contains two independent copies of this RNA: VAI (VA RNA copy I) and VAII (VA RNA copy II). Both are transcribed by RNA polymerase III from a type 2 polymerase III promoter (see, for example, Machitani, M., et al., J. Contr. Rel. 154 (2011) 285-289). For recombinant production, the adenovirus VA RNA gene can be driven by any promoter.

[0127] Ma, Y. and Mathews, M.B. (J. Virol. 70 (1996) 5083-5099) used a phylogenetic approach to study the structure, function, and evolution of adenovirus-associated RNAs. They provided an alignment and consensus VA RNA sequence based on 47 known human adenovirus serotypes.

[0128] The VA RNAs, VAI and VAII, contain 157 to 160 nucleotides (nt).

[0129] Depending on the serotype, adenoviruses contain one or two VA RNA genes. VAI is thought to play a dominant proviral role, while VAII can only partially compensate for the absence of VAI (Vachon, VK and Conn, GL, Virus Res. 212 (2016) 39-52).

[0130] Although VA RNA is not essential, it plays an important role in efficient viral propagation by overcoming cellular antiviral mechanisms. That is, although VA RNA is not essential for viral propagation, VA RNA-deficient adenoviruses cannot propagate during the early stages of vector production when only a few copies of the viral genome are present per cell, possibly because viral genes other than VA RNA cannot be sufficiently expressed, which blocks cellular antiviral mechanisms (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[0131] Maekawa, A. et al. (Nature Sci. Rep. 3 (2013) 1136) reported efficient production of adenovirus vectors lacking genes for virus-associated RNAs that disrupt the cellular RNAi machinery. Here, HEK293 cells that constitutively and highly express flippase recombinase were infected to obtain VA RNA-deleted adenoviruses by FLP recombinase-mediated excision of the VA RNA locus.

[0132] Human adenovirus 2 VA RNAI corresponds to nucleotides 10586 to 10810 of the sequence of GenBank entry AC_000007. Human adenovirus 5 VA RNAI corresponds to nucleotides 10579 to 10820 of the sequence of GenBank entry AC_000008.

[0133] Methods for producing rAAV particles Carter et al. showed that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with a transgene (Carter, BJ, in "Handbook of Parvoviruses," ed. by P. Tijssen, CRC Press, pp. 155-168 (1990)). However, the ITRs must be maintained to preserve the functions of replication, rescue, packaging, and integration of the transgene into the genome of the target cell.

[0134] When cells containing the respective viral helper genes are transduced by an AAV vector, or vice versa, when cells containing an integrated AAV provirus are transduced by the appropriate helper virus, the AAV provirus is activated and enters a lytic infection cycle (see Clark, KR et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, RJ, Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0135] More generally, cells transfected or transduced with DNA for recombinant production of AAV particles can be referred to as "recombinant cells." Such cells can be, for example, yeast, insect, or mammalian cells. They have been used as recipients of nucleic acids (plasmids) encoding packaging proteins such as AAV rep and cap, adenovirus helper proteins such as E1A, E1B, E2A, E2B, E4orf6, and VA, and nucleic acids (plasmids) encoding proteins or transcribed into a desired transcription product, i.e., a transgene located between two AAV ITRs. This term includes the progeny of the original transduced or transfected cell. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or genome or total nucleic acid complement to the original parent due to natural, accidental, or deliberate mutations.

[0136] Numerous cell growth media suitable for maintaining cell viability or providing cell growth and / or proliferation are commercially available. Examples of such media include serum-free eukaryotic growth media, such as media for maintaining viability or media for providing mammalian (e.g., human) cell growth. Non-limiting examples include Ham's F12 or F12K medium (Sigma-Aldrich), FreeStyle (FS) F17 medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific), and mixtures thereof. Such media can be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as essential amino acids for mammalian (e.g., human) cells.

[0137] Helper protein nucleic acid can be in the form of plasmid, phage, transposon or cosmid.In particular, it has been demonstrated that the full complement of adenovirus genes is not required for helper function.For example, it has been shown that adenovirus mutants that are unable to DNA replicate and synthesize late genes can allow AAV replication.See, for example, Ito et al., J.Gen.Virol.9(1970)243 and Ishibashi et al., Virology 45(1971)317.

[0138] Mutations in the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing helper function (see Carter et al., Virology 126 (1983) 505). However, adenoviruses defective in the E1 region or lacking the E4 region cannot support AAV replication. Thus, in the case of adenovirus helper proteins, the E1A and E4 regions are required for AAV replication either directly or indirectly (see, for example, Laughlin et al., J. Virol. 41 (1982) 868; Janik et al., Proc. Natl. Acad. Sci. USA 78 (1981) 1925; Carter et al., Virology 126 (1983) 505). Other characterized adenovirus variants include E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., Virology 104 (1980) 502); E2A (Handa et al., J. Gen. Virol. 29 (1975) 239; Strauss et al., J. Virol. 17 (1976) 140; Myers et al., J. Virol. 35 (1980) 665; Jay et al., Proc. Natl. Acad. Sci. USA 78 (1981) 2927; Myers et al., J. Biol. Chem. 256 (1981) 567); E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)).

[0139] Studies of helper proteins provided by adenoviruses with mutations in E1B have reported that the E1B 55kDa protein is required for AAV particle production, but the E1B 19kDa protein is not. Furthermore, WO 97 / 17458 and Matshushita et al. (Gene Therapy 5 (1998) 938-945) described helper function plasmids encoding various adenovirus genes. Examples of helper plasmids include the adenovirus VA RNA coding region, the adenovirus E4orf6 coding region, the adenovirus E2A 72kDa coding region, the adenovirus E1A coding region, and an adenovirus E1B region lacking an intact E1B 55kDa coding region (see, for example, WO 01 / 83797).

[0140] Various methods for DNA transfer into mammalian cells have been reported in the art. All of these are useful in the methods of the present invention. For example, electroporation, nucleofection, or microinjection for nucleic acid transfer / transfection can be used. Alternatively, inorganic substances (e.g., calcium phosphate / DNA co-precipitation, etc.), cationic polymers (e.g., polyethyleneimine, DEAE-dextran, etc.), or cationic lipids (lipofection) can be used for nucleic acid transfer / transfection. Calcium phosphate and polyethyleneimine are the most commonly used reagents for larger-scale nucleic acid transfection (e.g., Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), with polyethyleneimine being preferred.

[0141] Expression control elements include constitutive or regulatable control elements, such as tissue-specific expression control elements or promoters.

[0142] After production of recombinant viral (e.g., AAV) particles, if desired, the viral (e.g., rAAV) particles can be purified and / or isolated from the host cells using a variety of conventional methods, including column chromatography, CsCl gradient centrifugation, iodixanol gradient centrifugation, and the like.

[0143] For example, multiple column purification steps may be used, such as purification with anion exchange columns, affinity columns, and / or cation exchange columns (see, e.g., WO 02 / 12455 and U.S. Patent Application Publication No. 2003 / 0207439). Alternatively or additionally, iodixanol or CsCl gradients may be used (see, e.g., U.S. Patent Application Publication No. 2012 / 0135515 and U.S. Patent Application Publication No. 2013 / 0072548).

[0144] The goal of the rAAV vector production and purification system is to implement strategies to minimize / control the generation of production-related impurities such as protein, nucleic acid, and vector-related impurities, including wild-type / pseudo-wild-type AAV species (wtAAV) and AAV-encapsulated residual DNA impurities.

[0145] Given that rAAV particles represent only a small portion of the biomass, they must be purified to a purity that allows them to be used as clinical human gene therapy products (e.g., Smith PH, et al., Mo. Therapy 7 (2003) 8348; Chadeuf G., et al., Mo. Therapy 12 (2005) 744; report from the CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0146] As a first step, typically, cultured cells that produced rAAV particles are harvested, optionally in combination with the harvested cell culture supernatant (medium) in which the rAAV particle-producing cells (in suspension or adherent state) were cultured. The harvested cells and, optionally, the cell culture supernatant can be used as is, or additionally dissolved and / or concentrated. Furthermore, if infection is used to express helper function, residual helper virus is inactivated. For example, adenovirus can be inactivated by heating to a temperature of approximately 60°C for, for example, 20 minutes or more; this inactivates only the helper virus, since AAV is thermostable while the helper adenovirus is thermolabile.

[0147] Cells alone or in the harvested culture supernatant are lysed by disrupting the cells, e.g., by chemical or physical means such as detergents, microfluidization, and / or homogenization, to release intracellular rAAV particles. During or after cell lysis, a nuclease, e.g., benzonase, is added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cellular debris, e.g., by filtration or centrifugation, to yield a clarified cell lysate. In certain instances, the lysate is filtered through a micron-diameter pore size filter (e.g., a 0.1-10.0 μm pore size filter, e.g., a 0.45 μm and / or 0.2 μm pore size filter) to generate a clarified lysate.

[0148] The lysate (optionally clarified) contains AAV particles (including nucleic acid-encapsulated rAAV particles and empty particles) and production / process-related impurities, such as soluble cellular components from the host cells, which may include, inter alia, cellular proteins, lipids, and / or nucleic acids, and cell culture medium components. The clarified lysate is then optionally subjected to a purification step to purify the AAV particles (including the rAAV vector) from impurities using chromatography. The clarified lysate can be diluted or concentrated with an appropriate buffer before the first chromatography step.

[0149] The first chromatography step can be cation exchange chromatography or anion exchange chromatography. If the first chromatography step is cation exchange chromatography, the second chromatography step can be anion exchange chromatography or size exclusion chromatography (SEC).

[0150] Alternatively, if the first chromatographic step is cation exchange chromatography, the second chromatographic step can be size exclusion chromatography (SEC).

[0151] Further alternatively, the first chromatographic step can be affinity chromatography. If the first chromatographic step is affinity chromatography, the second chromatographic step can be anion exchange chromatography.

[0152] Optionally, a third chromatography step may be added to the preceding chromatography steps. Typically, the optional third chromatography step follows cation exchange, anion exchange, size exclusion or affinity chromatography.

[0153] Cation exchange chromatography functions to separate AAV particles from cellular and other components present in the clarified lysate and / or column eluate from affinity or size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV particles over a wide pH range include any sulfonic acid-based resin indicated by the presence of sulfonate functional groups, including aryl- and alkyl-substituted sulfonates such as sulfopropyl or sulfoethyl resins. Representative matrices include, but are not limited to, POROS HS, POROS HS 50, POROS XS, POROS SP, and POROS S (strong cation exchangers available from Thermo Fisher Scientific, Inc., Waltham, MA, USA). Further examples include Capto S, Capto S ImpAct, and Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), as well as the commercially available DOWEX®, AMBERLITE®, and AMBERLYST® resin families available from Aldrich Chemical Company (Milliwaukee, WI, USA). Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resin. Exemplary cation exchange resins include carboxymethyl (CM), phospho (based on phosphate functionality), and methylsulfonate (S) resins.

[0154] Anion exchange chromatography functions to separate AAV particles from proteins, cellular components, and other components present in the clarified lysate and / or column eluate from affinity chromatography, cation exchange chromatography, or size exclusion chromatography. Anion exchange chromatography can be used to reduce and thereby control the amount of empty particles in the eluate. For example, an anion exchange column to which rAAV particles are bound can be washed with a solution containing a moderate concentration of NaCl (e.g., about 100-125 mM, e.g., 110-115 mM), resulting in the elution of a portion of the empty particles in the flow-through without substantially eluting the rAAV particles. Subsequently, the rAAV particles bound to the anion exchange column can be eluted with a solution containing a higher concentration of NaCl (e.g., about 130-300 mM NaCl), producing a column eluate with a reduced or depleted amount of empty capsids and a proportionally increased amount of rAAV vector-containing rAAV particles.

[0155] Exemplary anion exchange resins include, but are not limited to, those based on polyamine resins and other resins. Examples of strong anion exchange resins include those generally based on quaternary nitrogen atoms, including, but not limited to, quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography materials include, but are not limited to, MACRO PREP Q (a strong anion exchanger available from BioRad, Hercules, CA, USA); UNO SPHERE Q (a strong anion exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (a strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (a strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (a weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (a weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (GE DEAE Sepharose (a strong anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA); DEAE Sepharose (a weak anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA); Q Sepharose (a strong anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Further exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0156] An exemplary process for the purification of recombinant AAV particles is reported in WO 2019 / 006390.

[0157] Methods for determining the infectious titer of rAAV particles containing a transgene are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for assaying for empty particles and rAAV particles with packaged transgenes are known in the art (see, e.g., Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[0158] To determine the presence or amount of disassembled / denatured capsids, purified rAAV particles are subjected to SDS-polyacrylamide gel electrophoresis using any gel capable of separating the three capsid proteins, such as a gradient gel. The gel is then run until the sample is separated, and the gel can be blotted onto a nylon or nitrocellulose membrane. An anti-AAV capsid antibody is then used as the primary antibody to bind to the denatured capsid protein (see, for example, Wobus et al., J. Viral. 74 (2000) 9281-9293). A secondary antibody, which binds to the primary antibody and includes a means for detecting it, is used in the second incubation step. The binding between the primary and secondary antibodies is detected semiquantitatively to determine the amount of capsid. Another method is analytical HPLC using an SEC column or analytical ultracentrifuge.

[0159] Description of Specific Embodiments of the Invention Reported herein is a method for measuring anti-AAV particle antibodies in a mammalian sample, comprising the steps of diluting an aliquot of the sample to a final serum concentration of preferably 5% (v / v) to 0.5% (v / v), adding AAV particles (AAVp) specifically bound by anti-AAV particle antibodies (rAAV-ADA) to the diluted sample to generate a spiked sample, pre-incubating the spiked sample to allow for the formation of rAAV-ADA-AAVp complexes, and detecting the rAAV-ADA-AAVp complexes, thereby measuring anti-AAV antibodies in the mammalian serum sample.

[0160] Typically, in AAV production, the titer of rAAV particles can be about 1E11 to 1E12 vg / ml, and the infectious titer can be about 1E8 to 1E9 TU / ml.

[0161] In general, characterization of the immune response to administered recombinant therapeutic AAV particles (rtAAV) generally requires the rtAAV itself as an assay reagent.

[0162] One embodiment of the principle of the method according to the invention is illustrated in Figure 1. This is one preferred embodiment of the method according to the invention.

[0163] In the first step of this embodiment of the method according to the present invention, a sample suspected of containing ADA against recombinant therapeutic AAV particles (rtAAV-ADA) is mixed with AAV particles (AAVp) of the same serotype as rtAAV, preferably rtAAV itself. During this first incubation step, a binary complex is formed between the rtAAV-ADA present in the sample and the supplemented / spiked / added AAVp (rtAAV-ADA-AAVp complex). Because complex formation occurs in solution, the required concentration of AAV particles in solution can be very low, which means that the amount of AAV particles, e.g., rtAAV, required is significantly reduced. This dramatically reduces the amount of AAV particles required compared to complex formation in which the AAV particles, i.e., rtAAV, are immobilized on a solid surface, as in standard ELISAs, e.g., antigen capture formats. Furthermore, the amount of AAV particles required can be further reduced by reducing the volume of the applied sample aliquot treated by the method according to the present invention.

[0164] In the second step of this embodiment of the method according to the invention, an antibody that specifically binds to the capsid of AAVp conjugated to a first member of a binding pair (captAb-BI) is added to the pre-incubated sample. This captAb-BI specifically binds to the binary rtAAV-ADA-AAVp complex, thereby forming a ternary rtAAV-ADA-AAVp-captAb-BI complex.

[0165] It should be noted that compared with the assay using chemically modified or immobilized AAV particles, the assay format according to the present invention is not expected to reduce sensitivity.In addition, due to the large number of epitopes present on a single AAV particle, the method according to the present invention does not have the risk of epitope saturation, masking, or competition.This allows the use of even lower concentrations of AAVp, i.e., even reduced / less amount of AAVp is required.

[0166] In the third step of this embodiment of the method according to the invention, the triple rtAAV-ADA-AAVp-captAb-BI complex is immobilized or captured on a solid surface by interaction between the first member of the captAb-BI binding pair and the cognate second member of each of the binding pairs, which are conjugated on the solid surface.

[0167] In the fourth step of this embodiment of the method according to the invention, the solid-phase captured ternary complexes are detected by incubation with an antibody that specifically binds to the Fc region of rtAAV-ADA, which is then conjugated to a detectable label (dectAb), which is retained / immobilized on the solid surface only if the respective ternary complex is captured on the solid surface.

[0168] The principle of an antigen capture assay known in the art is illustrated in FIG.

[0169] The most striking difference with the method according to the present invention can be seen directly in the immobilization of AAVp on the solid surface: since the entire surface must be covered by AAVp, a large amount of AAVp is required, i.e., a high AAVp consumption is involved in the method according to the state of the art.

[0170] Furthermore, in state-of-the-art assay formats, specific negative controls are required and must be provided, e.g., to check assay performance. Similarly, confirmation of assay results is only possible by addition of excess AAVp or AAV-ADA depletion.

[0171] More specifically, the method according to the invention The amount of particles required to detect and quantitate AAV-ADA can be reduced by at least one order of magnitude, i.e., to 0.1-fold or 0.05-fold or even less than that required for assays using solid surface-immobilized AAVp; • Direct comparison of recombinant AAV material and serotypes is possible, i.e., there is no need for derivatization or immobilization of AAVp; thereby preventing the risk of altering the properties of AAVp due to chemical modifications, which may result in a decrease in the sensitivity or selectivity of the assay; • If antibodies that specifically bind to multiple or even all AAV serotypes are used as captAb-BIs, a generic assay can be set up, i.e., particles of any serotype can be captured and detected / quantified with the same assay reagents; No need for AAV particle modification, i.e., no need to develop specific chemical modification methods; • A specificity control may be performed by performing an assay according to the invention without AAV particles versus performing an assay according to the invention with added AAV particles; • No additional particles are required, i.e. no further material needs to be produced and provided; Current state-of-the-art AAV-ADA detection assays using antigen-binding formats (see Figure 2) require significant quantities of AAV particles; such AAV particles may be from a different batch than the material used for in vivo administration and generation of the sample being analyzed; theoretically, immunogenic epitopes on the administered AAV particles may not be contained / present in AAV particles from a different batch used in the assay; the method according to the present invention eliminates such risks of underestimation of AAV-ADA, because the method according to the present invention requires only trace amounts of rAAV (for spiking) that can be easily obtained from the administered material, i.e., only 0.001 or 0.000001 of a single dose (a single dose in the range of 1E9, 1E12 to 1E15 required for the assay).

[0172] The method of the present invention is exemplified below using a sample using AAV particles of the AAV2 serotype. This is presented and provided only to illustrate the method of the present invention and to demonstrate the functionality and general applicability of the method of the present invention. This example should not be construed as limiting the scope of the present invention. The true scope of the present invention is set forth in the appended claims.

[0173] To demonstrate the general applicability of the method according to the invention, eight commercially available cynomolgus monkey sera (Biotrend Chemikalien GmbH, Cologne, Germany) were tested. The general method is described in Example 1 and the results are shown in Figure 3 and Table 1.

[0174] [Table 1]

[0175] For AAVp, serotype 2 recombinant therapeutic AAV particles carrying a therapeutic transgene were used at a concentration of 3.3E9 vp / mL, which corresponds to approximately 1E10 vp / plate (96-well plate). For comparison, an antigen capture assay would require approximately 5E11 vp / plate (96-well plate).

[0176] As captAb-BI, the mouse anti-AAV2 antibody A20R commercially available from Progen was used at a concentration of 0.1 μg / mL.

[0177] Eight commercially available cynomolgus monkey sera were used as samples at a dilution of 1:100 (v / v).

[0178] The incubation time on the plate was 5 minutes.

[0179] As dectAb, either an IgG subclass independent anti-human IgG or anti-cynomolgus IgG antibody is used at a final concentration of 0.4 μg / mL.

[0180] For the color reaction, 50 mM anti-digoxigenin antibody conjugated to HRP was used.

[0181] It can be seen that a positive signal is obtained only when AAVp is added to cynomolgus serum.

[0182] The applicability of the method according to the invention has been tested with additional commercially available cynomolgus monkey serum using anti-human IgG antibody as dectAb (Biotrend Chemikalien GmbH, Cologne, Germany). The results are shown in Figure 4 and Table 2.

[0183] [Table 2]

[0184] The absence of epitope masking by the capture antibody was also demonstrated. To demonstrate the absence of potential anti-AAV antibody epitope masking by the capture antibody, a worst-case scenario was simulated—the anti-AAV antibody in the test sample binds to the same epitope as the anti-AAV capture antibody. To mimic this situation, antibody R20A was used as the capture antibody and positive control. More precisely, for the human anti-AAV antibody in the study sample, mouse IgG from R20A was applied as the capture reagent, and human IgG from R20A was applied as the positive control. As shown in the graph in Figure 5 and the data in Table 3, the assay signal increases with increasing amounts of the positive control. This means that there is virtually no competition for binding sites: one or more capture antibodies and one or more positive control antibodies can simultaneously bind to a single AAV particle. Overall, there is no risk of epitope masking in the method according to the present invention.

[0185] [Table 3]

[0186] The optimal concentration of AAV particles in the assay can be readily determined using standard titration approaches. This is shown for the AAV2 capsid variant designated 7m8 in Figure 6. In assays with this capsid, the optimal concentration was determined to be 6.6E9 vp / mL.

[0187] The signal obtained by the method according to the present invention correlates with the amount of AAVp in the sample at the preincubation step. The same eight cynomolgus monkey sera used to generate the data shown in Figure 3 and Table 1 were used. The sera that were positive in the previous experiment (see Figure 3 and Table 1) were also positive in this experiment. It can be seen that the optimal AAVp concentration is in the range of 1.65E9 to 3.3E9. Preincubation was performed for 1 hour. The results are shown in Figure 7 and Table 4.

[0188] [Table 4]

[0189] Furthermore, the effects of preincubation time and serum content in the final sample were analyzed. The results are shown in Figure 8 and Table 5 as the average signal-to-noise ratios obtained with the same eight cynomolgus monkey sera as above. It can be seen that the highest signal was obtained with a final serum content of 2% in the preincubation mixture for preincubation times of 5 to 15 minutes. For preincubation times of 30 to 60 minutes, the highest signal was obtained with a final serum concentration of 1% in the preincubation mixture.

[0190] [Table 5]

[0191] The general applicability of the method according to the present invention has also been demonstrated. Using commercially available empty wild-type AAV2 particles (Progene), comparable results were obtained as compared with the AAV-7m8 capsid variant. This demonstrates the serotype / capsid-independent applicability of the method according to the present invention. The respective data are shown in Figure 9 (AAV2-7m8) and Figure 10 (AAV2 wild-type empty particles).

[0192] Calculating the required particle amount In the method according to the invention, approximately 1.65E9 vp / well is required.

[0193] Prior art antigen capture formats require approximately 5E10 vp / well.

[0194] Thus, the method according to the invention can reduce the amount of AAVp required by more than an order of magnitude (7.9E9 vs. 4.8E11).

[0195] The respective data are shown in Table 6 below.

[0196] [Table 6]

[0197] To convert mass concentration (μg / mL) to particle concentration (vp / mL), the relationship of 3.33E7 viral particles per nanogram of AAV mass is used.

[0198] All references mentioned herein are incorporated by reference in their entirety, even if not expressly stated otherwise.

[0199] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit or scope of the invention. [Example]

[0200] Description of the Examples Example 1 Immune complex assay for the detection of anti-AAV antibodies in mammalian samples Anti-AAV particle antibodies (AAV-ADA) were determined in serum samples of human or monkey (cynomolgus) origin.

[0201] Prior to analysis, serum samples (1 μL) were diluted 1 to 100 (v / v) with LowCross-Buffer® (Candor Biosciences; order number 100-500) and mixed with the respective AAV capsid (e.g., AAV2, Roche Diagnostics GmbH, Mannheim, Germany) to a concentration of 1.65E9 capsids / mL. The total volume of the mixture was 120 μL. The mixture was incubated at room temperature for 1 hour. After incubation, a biotinylated serotype-specific anti-capsid antibody (e.g., anti-AAV2 antibody (intact particle) mouse recombinant A20R; Progen, catalog number 610298) was added to the mixture at a final concentration of 0.25 μg / mL. The mixture of sample, AAV, and anti-capsid antibody was incubated at room temperature for an additional 30 minutes.

[0202] The mixture was then added to a streptavidin-coated microtiter plate. The biotinylated capture antibody was captured by interaction with streptavidin on the plate. A 15-minute incubation allowed for capture. In the presence of AAV-ADA, the biotinylated anti-capsid antibody, added AAV capsid, and AAV-ADA complexes were immobilized on the plate during this step. After washing to remove unbound material, the bound anti-capsid antibody-loaded AAV-AAV-ADA complexes were labeled with horseradish peroxidase-conjugated anti-human or anti-monkey IgG polyclonal antibodies. This labeling occurred by incubation at room temperature for 1 hour. After a wash step with PBS / Tween wash buffer, the horseradish peroxidase (HRP) substrate 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was added to the plate (final concentration 50 μM). The reaction of HRP with ABTS produced a colored reaction product. The signal (quenching) was measured using an ELISA reader at a wavelength of 405 nm (reference wavelength: 490 nm ([405 / 490] nm)). The signal for each serum sample was determined in duplicate.

[0203] As a negative control, perform the assay without adding AAV capsid. No complexes can form; only nonspecific binding of the sample itself can occur. By comparing the two signals (with and without AAV addition), the signal can be corrected accordingly (signal-to-noise ratio).

Claims

1. 1. A method for measuring anti-AAV particle antibodies in a mammalian serum sample, comprising the following steps in the following order: (a) diluting at least an aliquot of said sample to a final mammalian serum concentration of between 50% (v / v) and 0.5% (v / v), inclusive; (b) adding AAV particles (AAVp) specifically bound by said anti-AAV particle antibodies (AAV-ADA) to the diluted sample to generate a spiked sample; (c) pre-incubating the spiked sample to allow for the formation of AAV-ADA-AAVp complexes; (d) detecting the AAV-ADA-AAVp complex; and thereby measuring the anti-AAV antibodies in the mammalian serum sample.

2. The method of claim 1 , wherein the measurement is a determination.

3. The method of claim 1 , wherein the measurement is quantitative.

4. 4. The method of claim 1, wherein the pre-incubation is for 5 to 25 minutes and the final mammalian serum concentration is about 2% (v / v).

5. 4. The method of claim 1, wherein the pre-incubation is for 26 to 90 minutes and the final mammalian serum concentration is about 1% (v / v).

6. 6. The method of any one of claims 1 to 5, wherein the final concentration of the AAVp in the spiked sample is in the range of 1.5E9 to 3.5E9 particles per mL of spiked sample volume.

7. The method of any one of claims 1 to 6, wherein the AAV-ADA specifically binds to AAVp of serotype 2 or 8 or 9 or rh74, or a variant thereof.

8. 8. The method of any one of claims 1 to 7, wherein the AAVp is conjugated to a first member of a binding pair.

9. After step (c) and before step (d), step (cd): (cd) adding to the pre-incubated spiked sample a polypeptide or protein that specifically binds to the AAVp conjugated to a first member of a binding pair (captAb-BI) and incubating the mixture to allow formation of an AAV-ADA-AAVp-captAb-BI complex. and step (d) further comprises: (d) detecting the AAV-ADA-AAVp-captAb-BI complex. The method according to any one of claims 1 to 8, wherein

10. After step (c) and before step (d), step (dc): (dc) capturing the complex formed in step (c) on a solid surface. The method of any one of claims 1 to 8, further comprising:

11. After step (cd) and before step (d), step (dc): (dc) capturing the complex formed in step (cd) on a solid surface.

10. The method of claim 9, further comprising:

12. The method of any one of claims 10 to 11, wherein the second member of the binding pair is immobilized on the solid surface.

13. 13. The method of any one of claims 8 to 12, wherein the first member of a binding pair is selected from the group comprising biotin, streptavidin, digoxigenin, fluorescein, or theophylline, and the second member of the binding pair is selected from the group comprising streptavidin, biotin, an anti-digoxigenin antibody, and an anti-fluorescein antibody, or an anti-theophylline antibody.

14. The method of any one of claims 9 to 13, wherein the polypeptide or protein that specifically binds to AAVp is an anti-AAVp antibody or an AAVp-binding fragment thereof.

15. 15. The method of any one of claims 1 to 14, wherein said detecting in step (d) is by incubating with a detection antibody (dectAb) conjugated to a detectable label and determining the presence of said detectable label or quantifying the amount of said detectable label.

Images