Method for detecting anti-AAV particle antibodies

Abstract

Herein is reported a method for determining anti-AAV particle antibodies in a mammalian sample, the method comprising the steps of: diluting an aliquot of the sample, preferably to a final serum concentration of between 5% (v / v) and 0.5% (v / v); adding an AAV particle (AAVp) specifically bound by the anti-AAV particle antibody (AAV-ADA) to the diluted sample to produce a labeled sample; pre-incubating the labeled sample to allow the formation of an AAV-ADA-AAVp complex; and detecting the AAV-ADA-AAVp complex, thereby determining the anti-AAV antibody in the mammalian sample.

Description

[0001] The present invention belongs to the field of immunoassays, in particular the field of anti-drug antibody (ADA) assessment. In many cases, ADA assessment is challenging, especially in studies involving the administration of adeno-associated virus-based gene therapies. In this context, due to the limited availability of therapeutic AAV as assay reagents, ADA assays with low reagent requirements are needed. The present invention is based at least in part on the discovery that a complex comprising AAV-ADA can be generated for detection by pre-incubating a sample (in which anti-AAV particle antibodies (AAV-ADA) must be measured) with corresponding therapeutic AAV particles. Compared to conventional ADA assays that require labeling of therapeutic AAV particles, with this approach, the demand for therapeutic AAV particles is significantly reduced. Background Art

[0002] Assessing the immunogenicity of candidate therapeutics is an essential part of the drug development process. In the event of an immune response, immunogenicity data must be properly interpreted so that they can be correlated with clinical outcomes. Bioanalytical methods for immunogenicity testing provide this information by detecting and characterizing anti-drug antibodies (ADAs).

[0003] Like all protein therapeutics, recombinant AAV particles must be analyzed before use. However, in contrast to other biological therapies, adeno-associated virus therapies are hampered by pre-existing humoral immunity to AAV (anti-AAV particle antibodies, AAV-ADA) due to the ubiquitous nature of AAV and the concurrent widespread distribution of AAV infection in the population prior to treatment.

[0004] For common biotherapeutics, the gold standard assay format for detecting ADA in human or monkey samples is the bridging assay. ADA is complexed with a labeled drug conjugate to form a signal-giving complex. Due to the assay setup, either differently labeled drug molecules or drug-coated surfaces are required. This requires large quantities of drug.

[0005] Gimpel, AL, et al. (Mol. Ther. Meth. Clin. Dev. 20 (2021) 740-754) summarize the shortcomings of current AAV analysis methods for analyzing recombinantly produced AAV particles:

[0006] Typically, laboratory-scale production yields only a few milliliters (mL) of purified rAAV particles—equivalent to 1E12 to 1E14 vector genomes (vg)—so the sample volume required for analysis can result in significant product loss. Furthermore, viral concentrations in samples range from many orders of magnitude, from 1E10 to 1E11 vg / mL at the time of harvest from the original culture to 1E13 to 1E14 vg / mL in the purified product, and sample matrices vary widely, with residual byproducts, host cell proteins, and DNA from initial downstream steps having mass concentrations 10- to 100-fold higher than those of rAAV.

[0007] Therefore, analytical characterization of recombinantly produced AAV particles and the accompanying development of assays requires large quantities of product and thereby reduces their availability for characterization and testing.

[0008] Gorovits et al. (The AAPS Journal 23(2021)108) reviewed important considerations related to total anti-AAV antibody detection assays. Figure 1 In this paper, different applied total anti-AAV antibody assay formats are summarized. In the commonly used antigen capture and bridging formats, viral capsids are immobilized on a solid surface as capture reagents directly or via specific binding pairs. This setup uses different detection reagents in ELISA and ECLIA formats, such as HRP-conjugated anti-species Ig antibodies and HRP-conjugated protein A or G (antigen capture formats) or labeled viral capsids (bridging formats).

[0009] Therefore, there is a need for AAV-ADA assays that reduce the need for AAV material. Summary of the Invention

[0010] Herein, a novel method for determining anti-AAV particle antibodies (AAV-ADA) in a sample is reported. Compared to established assays, the method according to the invention requires, among other things, a reduced amount of AAV material.

[0011] The present invention is based, at least in part, on the discovery that by pre-incubating a sample (in which AAV-ADA is to be assayed) with corresponding therapeutic AAV particles, complexes comprising AAV-ADA can be generated for detection. Thus, the need for (i.e., the required amount of) therapeutic AAV particles as assay reagents can be significantly reduced compared to existing AAV-ADA assays employing labeled therapeutic AAV particles.

[0012] The present invention covers at least the following embodiments:

[0013] Example 1: A method for measuring anti-AAV particle antibodies in a mammalian serum sample, wherein

[0014] The method comprises the following steps in the following order:

[0015] a) adding AAV particles (AAVp) specifically bound by the anti-AAV particle antibodies (AAV-ADA) to the sample to generate a spiked sample,

[0016] b) pre-incubating the spiked sample to allow formation of rAAV-ADA-AAVp complexes,

[0017] as well as

[0018] c) detecting the AAV-ADA-AAVp complex,

[0019] Anti-AAV antibodies are thereby measured in the serum sample of the mammal.

[0020] Embodiment 2: The method according to embodiment 1, wherein the method further comprises:

[0021] Step bc) after and before step c):

[0022] bc) adding a protein or polypeptide that specifically binds to said AAVp conjugated to the first member of the binding pair (captAb-BI) to the pre-incubated spiked sample and incubating the mixture to allow formation of a ternary AAV-ADA-AAVp-captAb-BI complex,

[0023] And step c) is:

[0024] c) detecting the ternary AAV-ADA-AAVp-captAb-BI complex.

[0025] Embodiment 3: The method according to any one of embodiments 1 to 2, wherein the method further comprises a step bbc) after step b) or after step bc) and before step c): bbc) capturing the ternary complex formed in step bc) on a solid surface.

[0026] Example 4: A method for measuring anti-AAV particle antibodies in a mammalian serum sample, wherein

[0027] The method comprises the following steps in the following order:

[0028] a) diluting at least an aliquot of the sample,

[0029] b) adding AAV particles (AAVp) specifically bound by the anti-AAV particle antibodies (AAV-ADA) to the diluted sample to generate a spiked sample,

[0030] c) pre-incubating the spiked sample to allow formation of AAV-ADA-AAVp complexes,

[0031] as well as

[0032] d) detecting the AAV-ADA-AAVp complex,

[0033] The anti-AAV antibodies are thereby measured in a mammalian sample.

[0034] Embodiment 5: The method according to embodiment 4, wherein the method further comprises in step c)

[0035] Step cd) after and before step d):

[0036] cd) adding a protein or polypeptide that specifically binds to said AAVp conjugated to the first member of the binding pair (captAb-BI) to the pre-incubated spiked sample and incubating the mixture to allow formation of a ternary AAV-ADA-AAVp-captAb-BI complex,

[0037] And step d) is:

[0038] d) detecting the ternary AAV-ADA-AAVp-captAb-BI complex.

[0039] Embodiment 6: The method according to any one of embodiments 4 to 5, wherein the method further comprises a step ccd) after step c) or after step cd) and before step d): ccd) capturing the complex formed in step c) on a solid surface.

[0040] Embodiment 7: The method according to any one of embodiments 4 to 6, wherein the dilution is to a final mammalian serum concentration of less than 50% (v / v) and 0.5% (v / v) or more.

[0041] Embodiment 8: The method according to any one of embodiments 4 to 7, wherein the dilution is to a final mammalian serum concentration of less than 20% (v / v) and 0.5% (v / v) or more.

[0042] Embodiment 9: The method according to any one of embodiments 4 to 8, wherein the dilution is to a final mammalian serum concentration of less than 5% (v / v) and 0.5% (v / v) or more.

[0043] Embodiment 10: The method according to any one of embodiments 1 to 9, wherein the measuring is an assay.

[0044] Embodiment 11: The method according to any one of embodiments 1 to 9, wherein the measuring is quantitative.

[0045] Embodiment 12: The method according to any one of embodiments 1 to 11, wherein the pre-incubation lasts for 5 to 90 minutes.

[0046] Embodiment 13: The method according to any one of embodiments 1 to 12, wherein the pre-incubation lasts for 15 to 60 minutes.

[0047] Embodiment 14: The method of any one of embodiments 4 to 13, wherein the preincubation lasts for 5 to 25 minutes and the final mammalian serum concentration is about 2% (v / v).

[0048] Embodiment 15: The method of any one of embodiments 4 to 13, wherein the preincubation lasts 26 to 90 minutes and the final mammalian serum concentration is about 1% (v / v).

[0049] Embodiment 16: The method according to any one of embodiments 1 to 15, wherein the final concentration of the AAVp in the spiked sample is in the range of 0.5E9 to 1E10 viral particles per mL of spiked sample volume.

[0050] Embodiment 17: The method according to any one of embodiments 1 to 16, wherein the final concentration of the AAVp in the spiked sample is in the range of 1E9 to 7E9 viral particles per mL of spiked sample volume.

[0051] Embodiment 18: The method according to any one of embodiments 1 to 17, wherein the final concentration of the AAVp in the spiked sample is in the range of 1.25E9 to 5E9 viral particles per mL of spiked sample volume.

[0052] Embodiment 19: The method according to any one of embodiments 1 to 18, wherein the final concentration of the AAVp in the spiked sample is in the range of 1.5E9 to 3.5E9 viral particles per mL of spiked sample volume.

[0053] Embodiment 20: The method of any one of embodiments 1 to 19, wherein the volume of the sample or the aliquot of the sample is 100 μL or less.

[0054] Embodiment 21: The method of any one of embodiments 1 to 20, wherein the volume of the sample or the volume of the aliquot of the sample is 50 μL or less.

[0055] Embodiment 22: The method of any one of embodiments 1 to 21, wherein the volume of the sample or the volume of the aliquot of the sample is 10 μL or less.

[0056] Embodiment 23: The method of any one of embodiments 1 to 22, wherein the volume of the sample or the volume of the aliquot of the sample is 5 μL or less.

[0057] Embodiment 24: The method of any one of embodiments 1 to 23, wherein the volume of the sample or the volume of the aliquot of the sample is 1.5 μL or less.

[0058] Embodiment 25: The method of any one of embodiments 1 to 24, wherein the volume of the sample or the aliquot of the sample is about 1 μL.

[0059] Embodiment 26: The method according to 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 variants thereof.

[0060] Embodiment 27: The method according to any one of embodiments 1 to 26, wherein the AAV-ADA is an anti-recombinant AAV particle antibody (rAAV-ADA).

[0061] Embodiment 28: The method according to any one of embodiments 1 to 27, wherein the AAV-ADA is an anti-therapeutic AAV particle antibody (tAAV-ADA).

[0062] Embodiment 29: The method according to any one of embodiments 1 to 28, wherein the AAV-ADA is an anti-recombinant therapeutic AAV particle antibody (rtAAV-ADA).

[0063] 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.

[0064] Embodiment 31: The method of any one of embodiments 2 to 3 and 5 to 30, wherein the captAb-BI is selected from the group consisting of an anti-AAVp antibody, an AAVp-binding anticalin, and an AAVp-binding fragment thereof.

[0065] Embodiment 32: The method of any one of embodiments 2 to 3 and 5 to 31, wherein the captAb-BI is an anti-AAVp antibody or an AAVp-binding fragment thereof.

[0066] Embodiment 33: The method of any one of embodiments 2 to 3 and 5 to 32, wherein the captAb-BI is added to a final concentration of 0.1 to 0.5 μg / mL.

[0067] Embodiment 34: The method of any one of embodiments 2 to 3 and 5 to 33, wherein the captAb-BI is added to a final concentration of 0.15 to 0.35 μg / mL.

[0068] Embodiment 35: The method of any one of embodiments 2 to 3 and 5 to 34, wherein the captAb-BI is added to a final concentration of about 0.25 μg / mL.

[0069] Embodiment 36: The method according to any one of embodiments 2 to 3 and 5 to 35, wherein in step

[0070] After adding the captAb-BI in step bc) or step cd), the mixture is incubated for 5 to 90 minutes to allow the formation of the ternary AAV-ADA-AAVp-captAb-BI complex.

[0071] Embodiment 37: The method according to any one of embodiments 2 to 3 and 5 to 36, wherein in step

[0072] After adding the captAb-BI in step bc) or step cd), the mixture is incubated for 15 to 60 minutes to allow the formation of the ternary AAV-ADA-AAVp-captAb-BI complex.

[0073] Embodiment 38: The method according to any one of embodiments 2 to 3 and 5 to 37, wherein in step

[0074] After adding the captAb-BI in step bc) or step cd), the mixture is incubated for about 30 minutes to allow the formation of the ternary AAV-ADA-AAVp-captAb-BI complex.

[0075] Embodiment 39: The method according to any one of embodiments 2 to 3 and 5 to 38, wherein the

[0076] The first member of the pair is biotin, streptavidin, digoxygenin, fluorescein or theophylline.

[0077] 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 the solid surface.

[0078] Embodiment 41: The method of any one of embodiments 3 and 6 to 40, wherein if the

[0079] If the first member is biotin, the second member of the binding pair is streptavidin; if the first member is streptavidin, the second member of the binding pair is biotin or an anti-biotin antibody; if the first member is digoxigenin, the second member of the binding pair is an anti-digoxigenin antibody; if the first member is fluorescein, the second member of the binding pair is an anti-fluorescein antibody; or if the first member is theophylline, the second member of the binding pair is an anti-theophylline antibody.

[0080] Embodiment 42: The method according to any one of embodiments 3 and 6 to 41, wherein step bbc) or

[0081] The capturing in step ccd) is performed by incubating the mixture obtained in step bc) or step cd) with the solid surface for 1 to 60 minutes.

[0082] Embodiment 43: The method according to any one of embodiments 3 and 6 to 42, wherein step bbc) or

[0083] The capturing in step ccd) is performed by incubating the mixture obtained in step bc) or step cd) with the solid surface for 5 to 45 minutes.

[0084] Embodiment 44: The method according to any one of embodiments 3 and 6 to 43, wherein step bbc) or

[0085] The capturing in step ccd) is performed by incubating the mixture obtained in step bc) or step cd) with the solid surface for 10 to 25 minutes.

[0086] Embodiment 45: The method according to any one of embodiments 3 and 6 to 44, wherein step bbc) or

[0087] The capturing in step ccd) is performed by incubating the mixture obtained in step bc) or step cd) with the solid surface for about 15 minutes.

[0088] Embodiment 46: The method according to any one of embodiments 1 to 45, wherein the detection in step c) or step d) is performed 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.

[0089] 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.

[0090] In addition to the various embodiments depicted and claimed herein, the disclosed subject matter also relates to other embodiments having other combinations of features disclosed and claimed herein. Thus, the specific features presented herein, particularly those presented as individual embodiments, can be combined with one another in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of the embodiments and features disclosed herein. For purposes of illustration and description, descriptions of specific embodiments of the disclosed subject matter have been presented. They are not intended to be exhaustive or to limit the disclosed subject matter to those disclosed embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 A preferred embodiment of the method according to the present invention.

[0092] Figure 2 Protocols for antigen capture ELISA are known in the art.

[0093] Figure 3 Results of the method according to the invention obtained with eight commercially available cynomolgus macaque sera; Bar 1: detection with polyclonal anti-human IgG antibodies - no AAV2 particles added; Bar 2:

[0094] Detection with polyclonal anti-human IgG antibody - with AAV2 particles added; 3rd bar: detection with monoclonal anti-cynomolgus macaque IgG antibody - without AAV2 particles added; 4th bar: detection with monoclonal anti-cynomolgus macaque IgG antibody - with AAV2 particles added.

[0095] Figure 4 Results of the method according to the invention obtained with sixteen commercially available cynomolgus monkey sera; left bar: +AAV2, right bar: without addition of AAV2.

[0096] Figure 5 The results show that there is no epitope masking of the capture antibody in the method according to the invention.

[0097] Figure 6 Exemplary determination of the optimal concentration of the AAV2 capsid variant 7m8 as AAVp in the method according to the invention.

[0098] Figure 7 The data show that the signal obtained in the method according to the invention is correlated with the amount of AAVp in the sample during the pre-incubation step; Bar 1: amount of spiked AAV 2 (vp / mL) 132E8;

[0099] Bar 2: Amount of spiked AAV 2 (vp / mL) 66E8; Bar 3: Amount of spiked AAV 2 (vp / mL) 33E8; Bar 4: Amount of spiked AAV 2 (vp / mL) 16.5E8;

[0100] Bar 5: amount of spiked AAV 2 (vp / mL) 8.25E8; Bar 6: amount of spiked AAV 2 (vp / mL) 0E8.

[0101] Figure 8 Effect of preincubation time and serum content in the final sample; signal-to-noise ratio - average of 8 cynomolgus monkey serum samples.

[0102] Figure 9 The data show the general applicability of the method according to the invention: AAVp = AAV2 capsid variant 7m8.

[0103] Figure 10 The data show the general applicability of the method according to the invention: AAVp = empty wild-type AAV2. DETAILED DESCRIPTION

[0104] The present invention is based, at least in part, on the discovery that by pre-incubating a sample (in which AAV-ADA is to be assayed) with AAV particles of the same serotype as the therapeutic AAV particles, complexes containing AAV-ADA can be generated for subsequent detection, thereby significantly reducing the need for therapeutic AAV particles as assay reagents compared to conventional AAV-ADA assays employing labeled therapeutic AAV particles.

[0105] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0106] Methods and techniques that can be used to practice the present invention are described in, for example, Ausubel, FM (ed.), Current Protocols in Molecular Biology, Vols. I to III (1997); Glover, ND and Hames, BD (eds.), DNA Cloning: A Practical Approach, Vols. I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture—a practical approach, IRL Press Limited (1986); Watson, JD et al., Recombinant DNA, 2nd ed., CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J. (ed.), Cell Biology, 2nd ed., Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, 2nd ed., Alan R. Liss, Inc., NY (1987), the contents of which are incorporated herein by reference.

[0107] Derivatives of nucleic acids can be generated using recombinant DNA technology. Such derivatives can be modified, for example, by substitution, alteration, exchange, deletion or insertion at one or more nucleotide positions. Modification or derivatization can be performed, for example, by means of site-directed mutagenesis. Such modifications can be easily performed by those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, BD and Higgins, SG, Nucleic acid hybridization-practical approach (1985) IRL Press, Oxford, England).

[0108] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, 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," "one," and "at least one" are used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" are used interchangeably.

[0109] The term "about" refers to a range of + / - 20% of the value that follows. In certain embodiments, the term "about" refers to a range of + / - 10% of the value that follows. In certain embodiments, the term "about" refers to a range of + / - 5% of the value that follows.

[0110] The term "comprising" also encompasses the term "including".

[0111] As used herein, the term "antibody" is used in the broadest sense and encompasses various 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).

[0112] A complete antibody typically comprises two so-called light chain polypeptides (light chains) and two so-called heavy chain polypeptides (heavy chains). Each of the heavy and light chain polypeptides contains a variable domain (usually the amino-terminal portion of the polypeptide chain) and a constant region (usually the carboxyl-terminal portion).

[0113] The constant region of the heavy chain mediates the binding of the antibody i) to cells bearing Fcγ receptors (FcγRs), such as phagocytes, or ii) to cells bearing the neonatal Fc receptor (FcRn), also known as the Brambell receptor. It also mediates binding to factors including those of the classical complement system, such as component (C1q). The constant domain of an antibody heavy chain comprises the CH1, CH2, and CH3 domains, while the light chain comprises only one constant domain, CL, which can be of either the kappa or lambda isotype.

[0114] The variable domains of immunoglobulin light or heavy chains mediate the binding of antibodies to their cognate antigens. Each variable domain is composed of distinct segments, namely four framework regions (FRs) and three hypervariable regions (HVRs).

[0115] "Antibody fragment" refers to a molecule other than an intact or complete antibody that comprises a portion of the intact or complete antibody and binds to the same antigen to which the intact or complete antibody binds. 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 (dAb); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005), which is incorporated herein by reference.

[0116] The term "capture antibody" refers to an antibody used in a sandwich ELISA format to bind (i.e., capture) a target substance present in a sample for detection. A second antibody (i.e., a detection antibody) then binds to the captured target substance at a different epitope and allows detection of the capture antibody-target-complex (i.e., forming a "sandwich").

[0117] "Conjugate" is a recombinant or chemical fusion comprising at least one protein portion. The corresponding other portion can be a second protein portion or a non-protein portion. Since the portions of the conjugate are permanently connected to each other, it is a covalent conjugate. Covalent conjugation can be accomplished directly or via a linker. In certain embodiments, direct recombinant conjugation is accomplished by constructing a polypeptide fusion (i.e., by genetic fusion of two genes (e.g., encoding an antibody and a detectable marker or a member of a binding pair) and expressing them as a single polypeptide (chain)). In certain embodiments, direct chemical conjugation is accomplished by forming a covalent bond between, for example, a reactive group on an antibody and a corresponding group or receptor on a detectable marker or a member of a binding pair. In certain embodiments, direct chemical conjugation is accomplished by modifying (i.e., genetically modifying) one of the two portions to be conjugated to include a reactive group (as a non-limiting example, the reactive group is a sulfhydryl group or a carboxyl group), which forms a covalent bond with the other portion to be conjugated under appropriate conditions. As a non-limiting example, a molecule (i.e., an amino acid) having a desired reactive group (i.e., a cysteine ​​residue) can be introduced into, for example, an antibody.

[0118] Methods for covalent conjugation of nucleic acids to proteins are also known in the art (ie photocrosslinking, see for example Zatsepin et al. Russ. Chem. Rev. 74 (2005) 77-95).

[0119] In certain embodiments, the linker is a recombinant linker.

[0120] Conjugation via a recombinant linker can be performed using a single-chain peptide linker. In certain embodiments, a single-chain peptide linker comprises one to twenty amino acid residues connected by a peptide bond. In certain embodiments, amino acid is selected from twenty naturally occurring amino acids. In certain other embodiments, one or more amino acids in amino acid are selected from glycine, alanine, proline, asparagine, glutamine and lysine. In certain embodiments, the linker is a single-chain peptide linker, and the length of its amino acid sequence is at least 25 amino acid residues, and in a preferred embodiment, the length is 32 to 50 amino acid residues. In certain embodiments, the peptide linker is a (GxS)n linker, wherein G = glycine, S = serine, (x = 3, n = 8, 9 or 10) or (x = 4 and n = 6, 7 or 8), in certain embodiments, wherein x = 4, n = 6 or 7, in a preferred embodiment, wherein x = 4, n = 7.

[0121] In certain embodiments, the linker is a chemical linker.

[0122] Chemical conjugation via a linker can be performed using a variety of chemical linkers. In certain embodiments, the antibody and detectable label or member of a 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 iminoesters (such as dimethyl diimidoadipate hydrochloride), 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 (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0123] The term "detection antibody" refers to an antibody that carries a visualization or quantitative means. Such means are typically enzymes that catalyze the formation of a colored or fluorescent reaction product after adding a suitable substrate (detection reagent), such as horseradish peroxidase, urease, alkaline phosphatase, glucoamylase, or beta-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, digoxin, fluorescein, theophylline, a fluorescent marker, or a radioactive isotope. The detectable label is used to detect and / or quantitatively detect the antibody.

[0124] The term "detection reagent" refers to a reagent that allows detection and / or quantification of antibodies bound to an antigen. In certain embodiments, the detection reagent is a colorimetric substrate for an enzyme that has been conjugated to an antibody. Addition of a suitable substrate to the antibody-enzyme conjugate results in the generation of a colorimetric or fluorescent signal (e.g., after the conjugated antibody binds to the target antigen).

[0125] The term "ELISA" means enzyme-linked immunosorbent assay. Different 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, Chapter 11, John Wiley & Sons, Inc., New York (1994)). These are incorporated herein by reference.

[0126] A specific ELISA format is the so-called "direct ELISA". In this ELISA format, a target, such as an antibody, present in the sample is detected. In a direct ELISA, a sample containing the target is contacted with a solid phase, such as, for example, a stationary or fixed support (e.g., a microtiter plate well). If the target is present in the sample, the target is fixed to the solid phase and then directly detected using an enzyme-conjugated detection molecule. If the target is an antigen, the detection molecule is an antibody specific for the antigen, or if the target is an antigen-specific antibody, the detection molecule is an enzyme-conjugated antibody specific for the antigen.

[0127] Another specific ELISA format is the so-called "indirect ELISA." In this ELISA format, the antigen (or antibody) is immobilized to a solid phase (e.g., a microtiter plate well). Subsequently, an antigen-specific antibody (or antigen) is added, followed by a detection antibody specific for the antibody that specifically binds to the antigen. This detection antibody can be a "species-specific" antibody (e.g., a goat anti-rabbit antibody).

[0128] Another specific ELISA format is the so-called "sandwich ELISA." In this format, the antigen is immobilized on a solid phase (e.g., a microtiter plate well) via capture with 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 and then washed. If the target antigen is present in the sample, it is bound to the solid phase by the capture antibody.

[0129] The term "framework region" or "FR" refers to the variable domain residues other than the variable domain residues of the hypervariable region (HVR). Antibody variable domains typically contain four FRs: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following sequence in VH (or VL): FR1-HVR-H1 (HVR-L1)-FR2-HVR-H2 (HVR-L2)-FR3-HVR-H3 (HVR-L3)-FR4.

[0130] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure.

[0131] The term "in vitro" refers to the artificial environment itself or a process or reaction performed in such an artificial environment.

[0132] The term "in vivo" refers to the natural environment of a compound (eg, an animal or cell), or a process or reaction that occurs within its natural environment.

[0133] The term "immunoassay" refers to any technique that utilizes specific binding molecules (such as antibodies) to capture and / or detect a specific target in a sample for qualitative or quantitative analysis. Typically, an immunoassay is characterized by the following steps: 1) immobilizing or capturing the analyte and 2) detecting and measuring the analyte. The analyte can be captured, i.e., bound, to any solid surface, such as a membrane, plastic plate, or some other solid surface.

[0134] The term "linker" refers to a chemical linker or a single-chain peptide linker that covalently links the different entities in a conjugate. For example, a linker connects an antibody to a detectable label or one partner of a binding pair.

[0135] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (for example, containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, such variants are generally present in small amounts), each antibody comprising the population has identity. In contrast to the polyclonal antibody preparations typically comprising different antibodies for different determinants (epitopes), each antibody molecule in the monoclonal antibody preparation is directed to a single determinant on the antigen. Therefore, the modifier "monoclonal" represents that the characteristic of an antibody is obtained from a substantially homogeneous antibody population, and should not be construed as requiring antibody production by any ad hoc method. For example, monoclonal antibodies can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals comprising all or part of human immunoglobulin loci.

[0136] The term "variable domain" refers to the portion of an antibody heavy or light chain that is involved in binding an antibody to its cognate antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using the VH or VL domains from antibodies that bind to that antigen, respectively, to screen for libraries of complementary VL or VH domains.

[0137] As used herein, the term "hypervariable region" or "HVR" refers to the regions of an antibody variable domain that are highly variable in sequence and determine antigen binding specificity. These regions form the paratope or binding site of the binding region.

[0138] Typically, antibodies include six antigen-binding specificity determining regions: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary antigen-binding specificity determining regions herein include:

[0139] (a) encompasses amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3),

[0140] Hypervariable loops according to Chothia (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)) at 26-32 (H1), 53-55 (H2), and 96-101 (H3);

[0141] (b) encompasses amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3),

[0142] Hypervariable regions according to Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD) at 31-35b (H1), 50-65 (H2), and 95-102 (H3). (1991));

[0144] (a+b) encompasses amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3),

[0145] Hypervariable loops associated with the hypervariable regions at 26-35 (H1), 50-65 (H2), and 95-102 (H3) (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));

[0146] as well as

[0147] (c) encompasses amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1),

[0148] Antigen contact residues 47-58 (H2) and 93-101 (H3) (MacCallum et al. J. Mol.

[0149] Biol. 262: 732-745 (1996)).

[0150] Unless otherwise indicated, HVRs are herein identified according to the method described by Kabat et al. (supra). Those skilled in the art will appreciate that antigen-binding specificity determining region names may also be identified according to the method described by Chothia (supra), McCallum (supra), or any other scientifically accepted nomenclature system.

[0151] As used herein, the term "signal" encompasses any detectable physical change that can be used to indicate that a reaction has occurred, such as the binding of an antibody to its antigen. Signals in the form of fluorescent or colorimetric products / reagents are contemplated as specific forms of signals and can be used in the methods according to the present invention. In certain embodiments, the signal is assessed quantitatively.

[0152] The term "solid phase" refers to a non-fluid substance and includes: particles (including microparticles and beads) made of materials such as polymers, metals (paramagnetic particles, ferromagnetic particles), glass, and ceramics; gel substances such as silica gel, alumina gel, and polymer gels; capillaries, which can be made of polymers, metals, glass, and / or ceramics; zeolites and other porous substances; electrodes; microtiter plates; solid strips; and cuvettes, tubes, or other spectrometer sample containers. A solid phase component is distinguished from an inert solid surface in that a "solid phase" contains at least one portion on its surface that is intended to interact with substances in the sample directly or via compounds bound to it. The solid phase can be a fixed component, such as a tube, strip, cuvette, or microtiter plate, or it can be a non-fixed component, such as beads and microparticles. A variety of microparticles that allow non-covalent or covalent attachment of proteins and other substances can be used. Such particles include polymer particles, such as polystyrene and poly(methyl methacrylate); gold particles, such as gold nanoparticles and gold colloids; and ceramic particles, such as silica particles, glass particles, 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.

[0153] The terms "therapeutic adeno-associated virus particle" and "drug" are used interchangeably herein. These terms are used in the broadest sense and encompass a variety of structures, so long as they exhibit the desired activity.

[0154] As used herein, the term "sample" refers to any biological matrix from which the presence or amount of rAAV-ADA can be determined. Exemplary, but non-limiting, samples are serum, plasma, aqueous humor, vitreous humor, retinal tissue lysate, and tumor tissue. In a preferred embodiment, the sample is serum.

[0155] As used herein, the term "anti-AAV antibodies" refers to antibodies produced by the immune system of a recipient of therapeutic AAV particles against the therapeutic AAV particles following administration of the therapeutic AAV particles.

[0156] As used herein, the term "antibody that specifically binds to AAV particles" and its grammatical equivalents refers 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 experimental animals or by phage display methods.

[0157] As used herein, the term "immunogenicity" refers to the potential of AAV particles to induce an immune response in humans or animals. During drug development, immunogenicity is primarily assessed by measuring binding and neutralizing anti-AAV antibodies in samples.

[0158] The term "empty particle" refers to an AAV particle that has an AAV protein capsid but completely or partially lacks the recombinant AAV vector (i.e., a nucleic acid encoding a protein or transcribed into a target transcript flanked by two AAV ITRs). Thus, an empty particle is an AAV particle that does not have a functional or complete transgene (nucleic acid payload) encapsidated. Therefore, an empty capsid cannot function to transfer a nucleic acid encoding a protein or transcribed into a target transcript into a host cell.

[0159] A "recombinant AAV vector" is a nucleic acid derived from the wild-type genome of a virus (such as AAV) using molecular biological methods, in which the structural genes of the virus (e.g., AAV) are removed and replaced with a non-native nucleic acid (such as a nucleic acid that is transcribed into a therapeutic transcript or encodes a therapeutic protein). Typically, for therapeutic recombinant AAV vectors, both inverted terminal repeat (ITR) sequences of the wild-type AAV genome are retained.

[0160] A "recombinant" AAV vector is distinguished from a wild-type viral AAV genome in that all or part of the viral genome has been replaced by 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."

[0161] Recombinant AAV vectors (rAAV vectors) can be packaged into protein (capsid) shells - referred to herein as "particles" - for subsequent cell infection (transduction) in vitro, in vitro or in vivo. When the recombinant vector sequence is encapsulated or packaged into an AAV particle, the particle may also be referred to as a "rAAV particle". The capsid of the particle includes 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.

[0162] As used herein, the term "serotype" is a serologically different distinction based on the AAV capsid protein. Serological uniqueness is determined based on the lack of cross-reactivity between antibodies to one AAV compared to antibodies to another AAV. Such cross-reactivity differences are generally 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 traditional definition, a new serotype means that the target virus has been tested for serum that has specificity for neutralizing activity against all existing and characterized serotypes, and no antibodies to the target virus have been found. As more naturally occurring virus isolates are discovered and / or capsid mutants are generated, serological differences may or may not exist with any of the currently existing serotypes. Therefore, when a new virus (e.g., AAV) does not have serological differences, the new virus (e.g., AAV) will be a subgroup or variant of the corresponding serotype. Thus, for convenience and to avoid repetition, the term "serotype" refers broadly to both serologically distinct viruses (eg, AAV) as well as serologically non-distinct viruses (eg, AAV) that may be within subgroups or variants of a given serotype.

[0163] The term "transgene" is used herein for convenience to refer to a nucleic acid that is intended to be (e.g., in a recombinant AAV vector) or has been introduced into a cell or organism. A transgene includes any nucleic acid, such as a gene, that is transcribed into a transcript or encodes a polypeptide or protein.

[0164] "Vector" refers to a portion of the sequence of a recombinant plasmid that is ultimately packaged or encapsulated directly or in the form of a single strand or RNA to form a virus (e.g., AAV) particle. In the case where a recombinant plasmid is used to construct or manufacture recombinant virus particles, the virus particles do not include a "plasmid" portion that does not correspond to the vector sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called a "plasmid backbone," which is important for cloning and amplification of the plasmid, a process required for propagation and recombinant virus production, but it itself is not packaged or encapsulated into a virus (e.g., AAV) particle. Therefore, "vector" refers to the nucleic acid portion of a plasmid that is packaged or encapsulated by a virus particle (e.g., AAV).

[0165] "Plasmid" is a form of nucleic acid or polynucleotide that typically has additional elements for plasmid expression (e.g., transcription, replication, etc.) or propagation (replication). Plasmid as used herein can also be used to refer to such nucleic acid or polynucleotide sequences.

[0166] recombinant cells

[0167] In general, for efficient and large-scale production of a target protein compound, such as, for example, rAAV particles or therapeutic polypeptides, cells that express and, if possible, also secrete the protein compound are necessary. Such cells are referred to as "recombinant cells" or "recombinant production cells."

[0168] To generate "recombinant production cells," suitable mammalian cells are transfected with the desired nucleic acid sequence encoding the protein compound of interest. Depending on the polypeptide (eg, AAV), additional helper polypeptides may need to be transfected.

[0169] In order to generate stable recombinant production cells, a second step is then performed in which single cells stably expressing the protein compound of interest are selected. This can be accomplished, for example, based on the co-expression of a selection marker that has been co-transfected with the nucleic acid sequence encoding the protein compound of interest, or by expression of the protein compound itself.

[0170] For expressing coding sequence by recombinant cell, i.e. open reading frame, need extra regulatory elements, such as promoter and polyadenylation signal (sequence).Therefore, open reading frame is operably connected to described extra regulatory elements to transcribe.This can be realized by being integrated into so-called expression cassette.The minimum regulatory elements required for expression cassette to work in mammalian cell is the promoter that works in described mammalian cell, this promoter is positioned at the upstream of open reading frame, i.e. 5 ', and the polyadenylation signal (sequence) that works in described mammalian cell, this polyadenylation signal is positioned at the downstream of open reading frame, i.e. 3 '.In addition, terminator sequence may be present in 3 ' of polyadenylation signal (sequence).For expression, promoter, open reading frame / coding region and polyadenylation signal sequence must be arranged in the form that can be operably connected.

[0171] If the target protein compound is an AAV particle composed of different (monomeric) capsid polypeptides and single-stranded DNA molecules, and additional adenoviral helper functions are required for production and packaging, multiple expression cassettes are required, which differ in the desired open reading frame / coding sequence. In this case, at least an expression cassette is required for each of the transgene, the different polypeptides that form the capsid of the recombinant AAV particle, the desired helper functions, and the VA RNA. Thus, separate expression cassettes are required for each of the helper E1A, E1B, E2A, E4orf6, VA RNA, and the rep and cap genes.

[0172] As outlined in the preceding paragraph, respectively, the more complex or additionally required auxiliary polypeptide and / or RNA quantity of the target protein compound is, the higher the quantity of the required different expression cassettes is. Intrinsically, along with the increase in the number of expression cassettes, the total size of the required nucleic acid also increases. However, there is a practical upper limit to the size of transferable nucleic acids, which is in the range of about 15kbps (kilobase pairs). Exceeding this limitation, processing and processing efficiency will significantly decline. This problem can be solved by using two or more independent plasmids. Thus, different expression cassettes are assigned to different plasmids, and each plasmid only comprises some in the expression cassette thus.

[0173] For the development of stable cell lines, random integration (RI) of one or more nucleic acids carrying an expression cassette for the protein compound of interest can be used. Generally, by using RI, the nucleic acid or fragment thereof is randomly integrated into the genome of the host cell.

[0174] Alternatively to RI, targeted integration (TI) can also be used for CLD. In TI CLD, one or more nucleic acids comprising different expression cassettes are introduced / integrated into the genome of the host cell at a predetermined locus.

[0175] In TI, homologous recombination or recombinase-mediated cassette exchange reaction (RMCE) can be used to integrate the nucleic acid containing the corresponding expression cassette into a specific locus in the genome of the TI host cell.

[0176] Adeno-associated virus

[0177] For a general review of AAV and adenovirus or herpes helper functions, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The genome of AAV is described in Srivastava et al., J. Virol., 45 (1983) 555-564. Design considerations for constructing recombinant AAV vectors are described in US 4,797,368 (see also WO 93 / 24641). Additional 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 the following documents: US 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 these documents are incorporated herein by reference.

[0178] Adeno-associated virus (AAV) is a replication-defective parvovirus. It can only replicate in cells where certain viral functions are provided by a co-infecting helper virus, such as adenovirus, herpes virus, and in some cases, poxvirus, such as vaccinia. However, once the appropriate helper virus functions are present, AAV can replicate in almost any cell line of human, simian, or rodent origin.

[0179] If helper viral genes are not present, AAV establishes latency in its host cells. Its genome integrates into a specific site in chromosome 19 [(Chr)19(q13.4)], known as adeno-associated virus integration site 1 (AAVS1). For specific serotypes, such as AAV-2, other integration sites have been found, such as, for example, on chromosome 5 [(Chr)5(p13.3)], known as AAVS2, and on chromosome 3 [(Chr)3(p24.3)], known as AAVS3.

[0180] AAV is divided into different serotypes. These are assigned based on parameters such as hemagglutination, tumorigenicity, and DNA sequence homology. To date, more than 12 different serotypes and over a hundred sequences corresponding to different branches of AAV have been identified.

[0181] The capsid protein type and symmetry determine the tissue tropism of the corresponding 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.

[0182] Pseudotyping refers to the process of cross-packaging of AAV genomes or recombinant AAV vectors between different serotypes, i.e., the genome is packaged within capsid proteins from different serotypes. The serotype of the genome is determined by the serotype of its ITRs (inverted terminal repeats).

[0183] The wild-type AAV genome is approximately 4.7 kb in size. The AAV genome further comprises 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 open reading frame encoding the Rep protein provides four proteins of different sizes, which are called Rep78, Rep68, Rep52 and Rep40. These are involved in the replication, rescue and integration of AAV. The open reading frame encoding the Cap protein provides four proteins, called VP1, VP2, VP3 and AAP. VP1, VP2 and VP3 are part of the protein capsid of the AAV particle. The combined rep and cap open reading frames are flanked by so-called inverted terminal repeats (ITRs) at their 5' and 3' ends. 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.

[0184] For example, in the case of AAV serotype 2 (AAV-2), each ITR has a length of 145 nucleotides and is flanked by a coding sequence region of approximately 4470 nucleotides. Of the 145 nucleotides of 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 represented as D sequences.

[0185] The AAV genome contains three transcriptional promoters, P5, P19, and P40 (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571) for the expression of the rep and cap genes.

[0186] ITR sequence must be present in the coding region in cis.ITR provides a functional replication origin (ori), is integrated into the target cell genome required signal, and effectively excised and rescued from the host cell chromosome or recombinant plasmid.ITR further comprises a replication origin-like element, such as Rep protein binding site (RBS) and terminal resolution site (TRS).It has been found that ITR itself can have the function of transcription promoter in AAV vector (Flotte et al., J.Biol.Chem.268 (1993) 3781-3790; Flotte et al., Proc.Natl.Acad.Sci.USA 93 (1993) 10163-10167).

[0187] The rep and cap genes must be present in trans for replication and encapsidation of the viral single-stranded DNA genome or recombinant AAV vector, respectively.

[0188] The rep locus comprises two internal promoters, referred to as P5 and P19. Promoter P5 is operably linked to a nucleic acid sequence that provides a non-spliced ​​4.2 kb mRNA encoding Rep protein Rep78 (a chromatin nickase that blocks the cell cycle) and a spliced ​​3.9 kb mRNA encoding Rep protein Rep68 (a site-specific endonuclease). Promoter P19 is operably linked to a nucleic acid sequence that provides a non-spliced ​​mRNA encoding Rep protein Rep52 and a spliced ​​3.3 kb mRNA encoding Rep protein Rep40 (a DNA helicase for accumulation and packaging).

[0189] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV duplex 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).

[0190] The larger Rep proteins Rep68 and Rep78 can specifically bind to the hairpin conformation of AAV ITR. They exhibit defined enzymatic activities that are required for resolving replication at the AAV termini. Expression of Rep78 or Rep68 may be sufficient for the formation of infectious particles (Holscher, C. et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0191] All Rep proteins, primarily Rep78 and Rep68, are thought to exhibit regulatory activities such as induction and repression of AAV genes and inhibition of cell growth (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).

[0192] Recombinant overexpression of Rep78 results in a phenotype of reduced cell growth caused by induction of DNA damage, thereby arresting host cells in S phase, thereby promoting latent viral infection (Berthet, C. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0193] Tratschin et al. 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, it has been reported that after stable integration of AAV, expression in certain cell lines is only very low (see, for example, Mendelson et al., Virol. 166 (1988) 154-165).

[0194] The cap locus contains a promoter, called P40. The promoter P40 is operably linked to a nucleic acid sequence that provides a 2.6 kb mRNA that encodes the Cap proteins VP1 (87 kDa, non-spliced ​​mRNA transcript), VP2 (72 kDa, spliced ​​mRNA transcript), and VP3 (61 kDa, from an alternative start codon) by selective splicing and the use of alternative start codons. VP1 to VP3 constitute the components of the viral capsid. The capsid has the function of binding to cell surface receptors and allowing intracellular transport of the virus. VP3 accounts for approximately 90% of the total protein in the viral particle. However, all three proteins are essential for efficient capsid production.

[0195] Inactivation of all three capsid proteins, VP1 to VP3, reportedly prevents the accumulation of single-stranded progeny AAV DNA. Mutations at the amino terminus of VP1 (“Lip negative” or “Inf negative”) still allow single-stranded DNA to assemble into viral particles, thereby greatly reducing infectious titers.

[0196] The AAP open reading frame encodes the assembly activating protein (AAP). It is approximately 22 kDa in size and transports native VP protein to the nucleolus for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

[0197] A single AAV particle contains only one single-stranded DNA molecule. This can be either the "plus" or "minus" strand. AAV viral particles containing this DNA molecule are infectious. Within infected cells, the infectious single strand is converted to a double strand and subsequently amplified. This amplification produces numerous double-stranded DNA molecules, which displace the single strand and are packaged into capsids.

[0198] Adeno-associated virus (AAV) vectors can transduce both dividing and quiescent cells. It can be assumed that transgenes introduced into target cells using AAV vectors will be expressed long-term.

[0199] One disadvantage of using AAV vectors is that the size of the transgene that can be introduced into cells is limited.

[0200] Viral vectors such as parvovirus particles, including AAV serotypes and variants thereof, provide a way to deliver nucleic acids to protein-encoding cells in vitro, in vitro, and in vivo, so that the cells express the encoded protein. AAV is a virus that can be used 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 in the cells. In addition, these viruses can introduce nucleic acids / genetic material into specific sites. Since AAV is not related to human pathogenic diseases, AAV vectors are able to deliver endogenous and exogenous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial pathogenesis or disease.

[0201] Viral vectors that can be used include, but are not limited to, adeno-associated virus (AAV) particles of various serotypes (eg, AAV-1 to AAV-12, etc.) and hybrid / chimeric AAV particles.

[0202] AAV particles can be used as vehicles for efficient gene delivery. These particles possess many desirable characteristics for such applications, including tropism for both dividing and non-dividing cells. Early clinical experience with these vectors also suggests a lack of persistent toxicity. AAV is known to infect a variety of cell types in vivo and in vitro via receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting the retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells.

[0203] Recombinant AAV particles generally do not include viral genes associated with pathogenesis. Such vectors generally have one or more of the wild-type AAV genes, such as the rep and / or cap genes, deleted in whole or in part, but retain at least one functional flanking ITR sequence, which is necessary for the rescue, replication, and packaging of the recombinant vector into AAV particles. For example, only the essential parts of the vector (e.g., ITR and LTR elements, respectively) are included in the recombinant AAV vector. Thus, the recombinant AAV vector genome includes sequences required for cis replication and packaging (e.g., functional ITR sequences).

[0204] Recombinant AAV vectors and methods and uses thereof include any viral strain or serotype. As a non-limiting example, the recombinant AAV vector can be based on any AAV genome, such as, for example, AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, AAV-2i8, AAV rh74, or AAV 7m8. Such vectors can be based on the same strain or serotype (or subgroup or variant), or different from each other. As a non-limiting example, a recombinant AAV vector based on a serotype genome can be the same as one or more of the capsid proteins that package the vector. In addition, the recombinant AAV vector genome can be based on an AAV (e.g., AAV2) serotype genome that is different from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector genome can be based on AAV2, and at least one of the three capsid proteins can be AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, AAV 7m8, or a variant thereof. AAV variants include variants and chimeras of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, and AAV 7m8 capsids.

[0205] In certain embodiments of all aspects and embodiments, the adeno-associated virus (AAV) vector or particle is selected from the group consisting of serotypes including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAVrh74, and AAV 7m8, and variants thereof (e.g., capsid variants, such as amino acid insertions, additions, substitutions, and deletions). See, e.g., WO 2013 / 158879, WO 2015 / 013313, and US 2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0206] AAV and AAV variant (e.g., capsid variant) serotypes (e.g., VP1, VP2, and / or VP3 sequences) may or may not be different 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).

[0207] In certain embodiments of all aspects and embodiments, the AAV particles are related to a reference serotype and have a polypeptide or a subsequence thereof comprising or consisting of a sequence that is 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 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8 (e.g., such as ITRs, or VP1, VP2, and / or VP3 sequences).

[0208] The methods and uses of the present invention include AAV sequences (polypeptides and nucleotides) and subsequences thereof that exhibit 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, AAV rh74 or AAV 7m8), but are different 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 AAV 7m8 genes or proteins, etc.). In certain embodiments of all aspects and embodiments, the AAV polypeptide or a subsequence thereof comprises or consists of a sequence that is at least 75% or more identical, 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 a subsequence thereof, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAVrh74, or AAV 7m8 (e.g., VP1, VP2 and / or VP3 capsid or ITRs). In certain embodiments, the AAV variant has 1, 2, 3, 4, 5, 5 to 10, 10 to 15, 15 to 20, or more amino acid substitutions compared to a reference serotype.

[0209] Recombinant AAV particles, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, and AAV 7m8, as well as variants, related, hybrid, and chimeric sequences, can be constructed using recombinant techniques known to those skilled in the art to include one or more nucleic acid sequences flanked by one or more functional AAV ITR sequences (recombinant AAV vector / transgene).

[0210] Recombinant particles (e.g., rAAV particles) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are particularly useful for in vivo or ex vivo administration and delivery to a subject. Pharmaceutical compositions contain, in addition to the drug, a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition and that can be administered without excessive toxicity.

[0211] Protocols for generating adenoviral vectors have been described in US 5,998,205; US 6,228,646; US 6,093,699; US 6,100,242; WO 94 / 17810 and WO 94 / 23744.

[0212] Recombinant AAV particles

[0213] Different methods for generating rAAV particles are known in the art. For example, transfection using an AAV plasmid and an AAV helper sequence is combined with co-infection with an AAV helper virus (e.g., adenovirus, herpes virus, or vaccinia virus), or transfection using a recombinant AAV vector comprising a plasmid, an AAV helper function comprising a plasmid, and a rep / cap comprising a plasmid. Non-limiting methods for generating rAAV particles are described in, for example, US 6,001,650, US 6,004,797, WO 2017 / 096039, and WO 2018 / 226887. After recombinant rAAV particle production (i.e., generating particles in a cell culture system), rAAV particles can be obtained from host cells and cell culture supernatant and purified.

[0214] For the production of recombinant AAV particles, it is necessary to express the Rep and Cap proteins, the accessory proteins E1A, E1B, E2A and E4orf6, and the adenovirus VA RNA in a single mammalian cell. The accessory proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter, as shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), in particular the CMV IE promoter. Therefore, any promoter can be used.

[0215] In general, in order to produce recombinant AAV particles, different complementary plasmids are co-transfected into host cells. One of these plasmids contains a transgene sandwiched between two cis-acting AAV ITRs. The deleted AAV elements (i.e., open reading frames for Rep and Cap proteins) required for offspring recombinant genome replication and subsequent packaging are contained in trans on a second plasmid. Overexpression of Rep protein causes an inhibitory effect on cell growth (Li, J. et al., J. Virol. 71 (1997) 5236-5243). In addition, AAV replication requires a third plasmid containing genes for helper viruses (i.e., E1, E4orf6, E2A and VA from adenovirus).

[0216] To reduce the number of plasmids required, Rep, Cap, and adenoviral helper genes can be combined on a single plasmid.

[0217] Alternatively, the host cell may have stably expressed the E1 gene product. Such cells are HEK293 cells. The human embryonic kidney clone, denoted as 293, was generated as early as 1977 by integrating adenovirus DNA into human embryonic kidney cells (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. This covers E1A and E1B genes and adenovirus packaging signals (Louis, N. et al., Virology 233 (1997) 423-429).

[0218] When HEK293 cells are used, deleted E2A, E4orf6, and VA genes can be introduced by co-infection with adenovirus or by co-transfection with plasmids expressing E2A, E4orf6, and VA (see, 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. 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 can 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).

[0219] Therefore, cell lines in which the rep gene is integrated and expressed tend to grow slowly or express the Rep protein at very low levels.

[0220] To restrict transgene activity to specific tissues, ie, to limit integration sites, the transgene can be operably linked to an inducible or tissue-specific promoter (see, eg, Yang, Y. et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0221] 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, AAV rh.10, AAV rh.74, and AAV 7m8.

[0222] In certain embodiments of all aspects and embodiments, the rAAV particle comprises a capsid sequence that has 70% or greater sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV rh.10, AAV rh.74, or AAV 7m8 capsid sequence.

[0223] In certain embodiments of all aspects and embodiments, the rAAV particle comprises an ITR sequence having 70% or greater sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 ITR sequence.

[0224] E1A, E1B, E2, and E4

[0225] The coding sequences (open reading frames) for E1A and E1B can be derived from human adenoviruses, such as, for example, in particular human adenovirus serotype 2 or serotype 5. Exemplary sequences of human Ad5 (adenovirus serotype 5) are found in GenBank entries X02996, AC_000008, and the sequence of an exemplary human Ad2 is found in GenBank entry AC_000007. Nucleotides 505 to 3522 comprise the nucleic acid sequence encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146, as reported in EP 1 230 354, and plasmids pGS119 and pGS122, as reported in WO 2007 / 056994, can also be used as sources of E1A and E1B open reading frames.

[0226] 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 through alternative splicing. Expression of the 12S and 13S proteins triggers activation of other viral functions, including 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, cells die (apoptosis).

[0227] E1B is the second viral helper gene expressed. It is activated by proteins 12S and 13S derived from E1A. The mRNA derived from the E1B gene can be spliced ​​in two different ways, resulting in a first 55 kDa transcript and a second 19 kDa transcript. The 55 kDa E1B protein is involved in regulating the cell cycle, blocking mRNA trafficking in late infected cells, and preventing E1A-induced apoptosis. The 19 kDa E1B protein is involved in preventing E1A-induced apoptosis.

[0228] The E2 genes encode different proteins. The E2A transcript encodes the single-stranded binding protein (SSBP), which is essential for AAV replication.

[0229] In addition, the E4 gene encodes multiple proteins. The 34kDa protein (E4orf6) derived from the E4 gene, together with the 55kDa protein E1B, prevents the accumulation of cellular mRNA in the cytoplasm, but also promotes the transport of viral RNA from the nucleus to the cytoplasm.

[0230] Adenovirus VA RNA gene

[0231] Virus-associated RNA (VA RNA) is a noncoding RNA of adenovirus (Ad) that regulates translation. The adenoviral genome contains two independent copies of this RNA: VAI (VA RNA copy I) and VAII (VA RNA copy II). Both are transcribed from a type 2 polymerase III promoter by RNA polymerase III (see, e.g., Machitani, M. et al., J. Contr. Rel. 154 (2011) 285-289). For recombinant production, the adenoviral VA RNA gene can be driven by any promoter.

[0232] The structure, function and evolution of adenovirus-associated RNA were investigated using a phylogenetic approach by Ma, Y. and Mathews, MB (J. Virol. 70 (1996) 5083-5099). They provided an alignment based on 47 known human adenovirus serotypes and a consensus VA RNA sequence.

[0233] VA RNA, VAI, and VAII contain 157 to 160 nucleotides (nt).

[0234] Depending on the serotype, adenovirus contains one or two VA RNA genes. VAI is thought to play a major role in promoting viral production, while VAII can only partially compensate for the absence of VAI (Vachon, VK and Conn, GL, Virus Res. 212 (2016) 39-52).

[0235] VA RNA is not essential but plays an important role in efficient viral growth by overcoming cellular antiviral mechanisms. That is, although VA RNA is not essential for viral growth, VA RNA-deficient adenoviruses fail to grow during the initial steps of vector production, where only a few copies of the viral genome are present per cell, likely because viral genes other than VA RNA that block cellular antiviral mechanisms may not be adequately expressed (see Maekawa, A. et al. Nature Sci. Rep. 3 (2013) 1136).

[0236] Maekawa, A. et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient production of adenoviral vectors lacking genes for virus-associated RNAs that disrupt the cellular RNAi machinery, in which HEK293 cells that constitutively and highly express flippase recombinase were infected to obtain VA RNA-deficient adenoviruses through FLP recombinase-mediated excision of the VA RNA locus.

[0237] Human adenovirus 2VA RNAI corresponds to nucleotides 10586-10810 of the sequence of GenBank entry AC_000007. Human adenovirus 5VA RNAI corresponds to nucleotides 10579-10820 of the sequence of GenBank entry AC_000008.

[0238] Methods for producing rAAV particles

[0239] Carter et al. have shown 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, "Handbook of Parvoviruses", P. Tijssen ed., CRC Press, pp. 155-168 (1990)). However, the ITRs must be maintained to retain the functions of transgene replication, rescue, packaging, and integration into the target cell genome.

[0240] When cells containing the corresponding viral helper genes are transduced with AAV vectors, or vice versa, when cells containing integrated AAV provirus are transduced with a suitable helper virus, the AAV provirus is activated and enters the lytic infectious cycle (Clark, KR et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, RJ, Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0241] More generally, cells that are transfected or transduced with DNA for the recombinant production of AAV particles can be referred to as "recombinant cells". Such cells can be, for example, yeast cells, insect cells, or mammalian cells. It has been used as a recipient for nucleic acids (plasmids) encoding packaging proteins (such as AAV rep and cap), nucleic acids (plasmids) encoding accessory proteins (such as adenovirus E1A, E1B, E2A, E2B, E4orf6, and VA), and nucleic acids (plasmids) encoding proteins or transcribed into target transcripts (i.e., transgenes) placed between two AAV ITRs. The term includes the descendants of the original cells that have been transduced or transfected. It should be understood that the descendants of a single parental cell may not necessarily be completely identical to the original parent in morphology or genome or total nucleic acid complement due to natural, accidental, or deliberate mutations.

[0242] In some embodiments, the present invention provides the cell growth medium that can be used for the growth of culture medium.Many cell growth mediums that are suitable for maintaining cell viability or providing cell growth and / or propagation are commercially available.The example of this type of culture medium includes serum-free eukaryotic growth medium, such as for maintaining mammalian (for example, people) cell viability or providing the culture medium for mammalian (for example, people) cell growth.Non-limiting examples include Ham's F12 or F12K culture medium (Sigma-Aldrich), FreeStyle (FS) F17 culture medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific) and mixtures thereof.This type of culture medium can be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as the essential amino acids for mammalian (for example, people) cells.

[0243] The helper protein nucleic acid can be in the form of a plasmid, phage, transposon, or cosmid. In particular, it has been shown that helper function does not require full complement of adenoviral genes. For example, adenoviral mutants that are unable to carry out DNA replication and late gene synthesis have been shown to allow AAV replication. See, for example, Ito et al., J. Gen. Virol. 9 (1970) 243; Ishibashi et al., Virology 45 (1971) 317.

[0244] Mutants within the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions may not be involved in providing auxiliary functions (see Carter et al., Virology 126 (1983) 505). However, adenoviruses with defects in the E1 region or deletions in the E4 region are unable to support AAV replication. Therefore, for adenovirus auxiliary proteins, the E1A and E4 regions are directly or indirectly required for AAV replication (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 mutants 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, ICRC Handbook of Parvoviruses (P. Tijssen, ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)).

[0245] Studies on helper proteins provided by adenoviruses with E1B mutations have reported that the E1B 55 kDa protein is required for AAV particle production, while the E1B 19 kDa protein is not. In addition, WO 97 / 17458 and Matshushita et al. (Gene Therapy 5 (1998) 938-945) describe helper plasmids encoding various adenoviral genes. Examples of helper plasmids include the adenoviral VA RNA coding region, the adenoviral E4orf6 coding region, the adenoviral E2A 72 kDa coding region, the adenoviral E1A coding region, and the adenoviral E1B region lacking the complete E1B 55 kDa coding region (see, for example, WO 01 / 83797).

[0246] The present invention relates to a method for transferring DNA to mammalian cells. The method comprises the steps of: transferring DNA to a mammalian cell by electroporation, nuclear transfection or microinjection. In an alternative embodiment, an inorganic substance (such as calcium phosphate / DNA coprecipitation), a cationic polymer (such as polyethyleneimine, DEAE-dextran) or a cationic lipid (lipofection) can be used for nucleic acid transfer / transfection. Calcium phosphate and polyethyleneimine are the most commonly used reagents (see, for example, Baldi et al., Biotechnol. Lett. 29 (2007) 677-684) for the transfection of larger-scale nucleic acids, wherein polyethyleneimine is preferred.

[0247] Expression control elements include constitutive or regulatable control elements, such as tissue-specific expression control elements or promoters.

[0248] After producing recombinant virus (e.g., AAV) particles, if desired, the virus (e.g., rAAV) particles can be purified and / or isolated from host cells using a variety of conventional methods. Such methods include column chromatography, CsCl gradient centrifugation, iodixanol gradient centrifugation, etc.

[0249] For example, multiple column purification steps can be used, such as purification by anion exchange column, affinity column and / or cation exchange column (see, e.g., WO 02 / 12455 and US2003 / 0207439). Alternatively or in addition, iodixanol or CsCl gradients can be used (see, e.g., US2012 / 0135515; and US2013 / 0072548).

[0250] One goal of rAAV vector production and purification systems is to implement strategies to minimize / control the generation of production-related impurities, such as proteins, nucleic acids, and vector-related impurities, including wild-type / pseudo-wild-type AAV species (wtAAV) and residual DNA impurities encapsulated by AAV.

[0251] Considering that rAAV particles only account for a small part of the biomass, rAAV particles need to be purified to a purity level that can be used as a clinical human gene therapy product (see, for example, 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).

[0252] As an initial step, cultured cells producing rAAV particles are typically harvested and optionally combined with harvested cell culture supernatant (culture medium) in which cells producing rAAV particles (suspension cells or adherent cells) have been cultured. The harvested cells and optional cell culture supernatant can be used as is, as appropriate, or further lysed and / or concentrated. In addition, if infection is employed to express helper functions, the residual helper virus is inactivated. For example, adenovirus can be inactivated by heating to a temperature of about 60° C. for, for example, 20 minutes or longer, which only inactivates the helper virus because AAV is heat stable, while helper adenovirus is heat unstable.

[0253] The cells are lysed individually or in the culture supernatant of the harvest to release intracellular rAAV particles by destroying the cells (e.g., by chemical or physical means, such as detergents, microfluidization and / or homogenization). During the cell lysis period, or after the cell lysis, a nuclease (such as, for example, benzonase) is added to degrade the contaminated DNA. Typically, the resulting lysate is clarified to remove cell debris, for example, by filtering or centrifugation to obtain a clarified cell lysate. In a particular example, the lysate is filtered with a micron diameter pore filter (such as a 0.1 μm to 10.0 μm pore filter, for example, a 0.45 μm and / or pore size 0.2 μm filter) to produce a clarified lysate.

[0254] The lysate (optionally clarified) contains AAV particles (including rAAV particles with encapsidated nucleic acid and empty particles) and production / process-related impurities, such as soluble cellular components from the host cells, which may include, among other things, cellular proteins, lipids and / or nucleic acids, and cell culture medium components. The (optionally clarified) lysate is subjected to a purification step to purify the AAV particles (including the rAAV vector) from the impurities using chromatography. Prior to the first chromatography step, the clarified lysate may be diluted or concentrated with an appropriate buffer.

[0255] The first chromatography step may be a cation exchange chromatography or anion exchange chromatography. If the first chromatography step is a cation exchange chromatography, the second chromatography step may be anion exchange chromatography or size exclusion chromatography (SEC).

[0256] Alternatively, if the first chromatography step is cation exchange chromatography, the second chromatography step may be size exclusion chromatography (SEC).

[0257] Still alternatively, the first chromatography step may be affinity chromatography.If the first chromatography step is affinity chromatography, the second chromatography step may be anion exchange chromatography.

[0258] Optionally, a third chromatography step may be added to the preceding chromatography step. Typically, the optional third chromatography step follows cation exchange, anion exchange, size exclusion or affinity chromatography.

[0259] The role of cation exchange chromatography is to separate AAV particles from cells and other components present in the clarified lysate and / or column eluate from affinity chromatography or size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV particles over a wide pH range include, but are not limited to, any sulfonic acid-based resin indicated by the presence of a sulfonate functional group, 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, POROSXS, POROS SP, and POROS S (strong cation exchangers available from Thermo Fisher Scientific, Inc., Waltham, MA, USA). Additional examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), and commercial versions of the resin. and family (available from Aldrich Chemical Company, Milliwaukee, WI, USA). Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resins. Exemplary cation exchange resins include carboxymethyl (CM) resins, phosphate resins (based on phosphate functional groups), and methanesulfonate (S) resins.

[0260] The effect of anion exchange chromatography is to separate AAV particles from proteins, cells and other components present in the lysate and / or column eluate of the clarification from affinity or cation exchange or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and therefore control the amount of empty particles in the eluent. For example, the anion exchange column with the rAAV particles bound thereto can be washed with a solution of NaCl comprising a moderate concentration (e.g., about 100-125mM, such as 110-115mM), and a portion of empty particles can be eluted during the flow-through process without a large amount of eluted rAAV particles. Subsequently, the rAAV particles bonded to the anion exchange column can be eluted using a solution comprising a higher concentration of NaCl (e.g., about 130-300mM NaCl), thereby producing a column eluent of the rAAV particles comprising the rAAV vector with an empty capsid reduced or depleted and a proportionally increased amount.

[0261] 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 quaternized 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 (strong anion exchanger, available from BioRad, Hercules, CA, USA); UNO SPHERE Q (strong anion exchanger, available from BioRad, Hercules, CA, USA); POROS 50HQ (strong anion exchanger, available from Applied Biosystems, Foster City, CA, USA); POROS XQ (strong anion exchanger, available from Applied Biosystems, Foster City, CA, USA); POROS SOD (weak anion exchanger, available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (weak anion exchanger, available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE SEPHAROSE (a strong anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA); SEPHAROSE Q (a strong anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Additional exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0262] WO 2019 / 006390 reports an exemplary process for purification of recombinant AAV particles.

[0263] 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 determining the infectious titer of 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).

[0264] In order to determine the presence or amount of degraded / denatured capsids, purified rAAV particles can be subjected to SDS-polyacrylamide gel electrophoresis, which is composed of any gel (e.g., gradient gel) capable of separating the three capsid proteins, and then running the gel until the sample is separated and the gel is blotted onto a nylon or nitrocellulose membrane. Anti-AAV capsid antibodies are then used as the primary antibody that binds to the denatured capsid protein (see, e.g., Wobus et al., J. Viral. 74 (2000) 9281-9293). A secondary antibody that is bound to the primary antibody and contains a tool for detecting the primary antibody is used in the second incubation step. Semi-quantitative detection of the binding between the primary and secondary antibodies is used to determine the amount of capsids. Another method is to use an analytical HPLC or analytical ultracentrifuge using a SEC column.

[0265] Description of specific embodiments of the present invention

[0266] Herein is reported a method for measuring anti-AAV particle antibodies in a mammalian sample, comprising the steps of: diluting an aliquot of the sample, preferably to a final serum concentration of between 5% (v / v) and 0.5% (v / v); adding AAV particles (AAVp) specifically bound by the anti-AAV particle antibodies (rAAV-ADA) to the diluted sample to generate a spiked sample; pre-incubating the spiked sample to allow formation of rAAV-ADA-AAVp complexes; and detecting the rAAV-ADA-AAVp complexes, thereby measuring the anti-AAV antibodies in the mammalian sample.

[0267] Typically, in AAV production, rAAV particle titers of about 1E11 to 1E12 vg / ml and infectious titers of about 1E8 to 1E9 TU / ml can be achieved.

[0268] Typically, characterization of the immune response to administered recombinant therapeutic AAV particles (rtAAV) typically requires the rtAAV itself as the assay reagent.

[0269] An embodiment of the principle of the method according to the invention is depicted in Figure 1 This is a preferred embodiment of the method according to the present invention.

[0270] In the first step of this embodiment of the method according to the invention, a sample suspected of containing ADA (rtAAV-ADA) against recombinant therapeutic AAV particles is mixed with AAV particles (AAVp) of the same serotype as rtAAV, preferably with rtAAV itself. In this first incubation step, a binary complex (rtAAV-ADA-AAVp complex) is formed between the rtAAV-ADA present in the sample and the supplemented / spiked / added AAVp. Since the complex formation is in solution, the amount of AAV particles (e.g., rtAAV) required is very small, as the required AAV particle concentration in solution may be very low. That is, the amount of AAV particles required is significantly reduced compared to complex formation, where the AAV particles (i.e., rtAAV) are already immobilized on a solid surface, for example, in a standard ELISA in an antigen capture format. In addition, the amount of AAV particles required can be further reduced by reducing the volume of the aliquot of the sample of the application treated according to the method of the invention.

[0271] In the second step of this embodiment of the method according to the invention, an antibody (captAb-BI) that specifically binds to the capsid of AAVp conjugated to the first member of the binding pair is added to the pre-incubated sample. This captAb-BI specifically binds to the binary rtAAV-ADA-AAVp complex. Thus, a ternary rtAAV-ADA-AAVp-captAb-BI complex is formed.

[0272] It is important to note that, compared to assays using chemically modified or immobilized AAV particles, no loss of sensitivity is expected using the assay format according to the present invention. Furthermore, due to the large number of epitopes present on a single AAV particle, there is no risk of epitope saturation, masking, or competition in the methods according to the present invention. This further allows the use of lower concentrations of AAVp, i.e., the need to further reduce / lower the amount of AAVp.

[0273] In the third step of this embodiment of the method according to the invention, the ternary rtAAV-ADA-AAVp-captAb-BI-complex is immobilized or captured on a solid surface by the interaction between the first member of the binding pair of captAb-BI and the corresponding cognate second member of the binding pair conjugated to the solid surface.

[0274] In the fourth step of this embodiment of the method according to the invention, the solid-phase captured ternary complex is detected by incubation with an antibody that specifically binds to the Fc region of rtAAV-ADA, which antibody is in turn conjugated to a detectable label (dectAb). The detectable label is retained / immobilized on the solid surface only when the corresponding ternary complex is captured on the solid surface.

[0275] The principle of antigen capture assays known in the art is described in Figure 2 middle.

[0276] It can be directly seen that the most significant difference from the method according to the present invention is that the AAVp is immobilized on a 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 included in the existing method.

[0277] In addition, in existing assay formats, specific negative controls are required and must be provided, for example, for assay performance checks. Likewise, assay results can only be confirmed by adding excess AAVp or by AAV-ADA depletion.

[0278] In more detail, using the method according to the present invention,

[0279] The amount of particles required for detection and quantification of AAV-ADA can be reduced by at least an order of magnitude, i.e., to 0.1-fold or 0.05-fold or even less of the amount required for solid surface-immobilized AAVp assays;

[0280] Direct comparison of recombinant AAV material and serotypes is possible, i.e., without the need for derivatization or immobilization of AAVp; thereby preventing the risk of altering the properties of AAVp due to chemical modifications, which could result in reduced sensitivity or selectivity of the assay;

[0281] If antibodies that specifically bind to multiple or even all AAV serotypes are used as captAb-BI, a universal assay can be set up, i.e., capture and detection can be performed with the same assay reagents.

[0282] Quantification of particles of any serotype;

[0283] ● No need to modify AAV particles, i.e., no need to develop specific chemical modification methods;

[0284] • Specificity control can be performed by performing the assay according to the invention in the absence of AAV particles versus performing the assay according to the invention with added AAV particles;

[0285] ● No additional particles are required, i.e. no additional materials need to be generated and provided;

[0286] ● Existing AAV-ADA detection assays using antigen-binding formats (see Figure 2) requires a large amount of AAV particles; such AAV particles may come from a different batch than the material used for in vivo administration and generation of the sample to be analyzed; theoretically, the immunogenic epitopes of 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 invention eliminates the risk of underestimating AAV-ADA because the method according to the invention requires only a minute amount of rAAV (for spiking), which can be easily obtained from the administered material, i.e. only a fraction of 0.001 or 0.000001 of a single dose (1E9 required for the assay, a single dose in the range of 1E12 to 1E15).

[0287] The method according to the present invention is illustrated in the following example using AAV particles of the AAV2 serotype. This is presented and provided solely to illustrate the method according to the present invention and to illustrate its functionality and general applicability. This example should not be construed as limiting the scope of the present invention. The true scope of the invention is set forth in the appended claims.

[0288] To illustrate the general applicability of the method according to the invention, eight commercially available cynomolgus monkey sera have been tested (Biotrend Chemikalien GmbH, Cologne, Germany). The general method is described in Example 1 and the results are shown in Figure 3 and Table 1.

[0289] Table 1.

[0290]

[0291]

[0292] As AAVp, recombinant therapeutic AAV particles of serotype 2 with a therapeutic transgene were used at a concentration of 3.3E9 vp / mL. This corresponds to approximately 1E10 vp / plate (96-well plate). For comparison, an antigen capture assay requires approximately 5E11 vp / plate (96-well plate).

[0293] As captAb-BI, the commercially available mouse anti-AAV2 antibody A20R from Progen has been used at a concentration of 0.1 μg / mL.

[0294] As samples, eight commercially available cynomolgus monkey sera at a dilution of 1:100 (v / v) have been used.

[0295] The incubation time on the plate was 5 minutes.

[0296] As dectAb, IgG subclass-independent anti-human IgG or anti-cynomolgus monkey IgG antibodies have been used at a final concentration of 0.4 μg / mL.

[0297] For the color reaction, 50 mM anti-digoxigenin antibody conjugated to HRP was used.

[0298] As can be seen, a positive signal was obtained only when AAVp had been added to the cynomolgus monkey serum.

[0299] The applicability of the method according to the invention has been tested with an anti-human IgG antibody as dectAb (Biotrend Chemikalien GmbH, Cologne, Germany) using an otherwise commercially available cynomolgus monkey serum. Figure 4 and shown in Table 2.

[0300] Table 2.

[0301]

[0302] The absence of epitope masking by the capture antibody was also shown. To demonstrate the absence of potential anti-AAV antibody epitope masking by the capture antibody, a worst-case scenario was simulated where the anti-AAV antibodies of the study sample bound to the same epitope as the anti-AAV capture antibody. To simulate this scenario, the antibody R20A was used as the capture antibody as well as a positive control. More specifically, mouse IgG of R20A was used as the capture reagent and human IgG of R20A was used as a positive control for human anti-AAV antibodies of the study sample. Figure 5 As shown in the figures and the data in Table 3, the assay signal increases with increasing amounts of 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 bind simultaneously to a single AAV particle. Overall, there is no risk of epitope masking in the methods according to the present invention.

[0303] Table 3.

[0304]

[0305] The optimal concentration of AAV particles in the assay can be easily determined using standard titration methods. Figure 6 The AAV2 capsid variant denoted as 7m8 is shown in Figure 2. In the assay for this capsid, the optimal concentration was determined to be 6.6E9 vp / mL.

[0306] The signal obtained in the method according to the invention is related to the amount of AAVp in the sample during the pre-incubation step. Figure 3The same eight cynomolgus monkey sera from which the data are presented in Table 1. Sera that were positive in previous experiments (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. The preincubation was performed for one hour. Figure 7 and shown in Table 4.

[0307] Table 4.

[0308]

[0309] In addition, the effect of preincubation time and serum content in the final sample was analyzed. The results are shown as the average of the signal-to-noise ratios obtained with the same eight cynomolgus monkey sera as above. Figure 8 As shown in Table 5, the highest signal was obtained with a preincubation time of 5 to 15 minutes and a final serum content of 2% in the preincubation mixture. The highest signal was obtained with a preincubation time of 30 to 60 minutes and a final serum concentration of 1% in the preincubation mixture.

[0310] Table 5.

[0311]

[0312]

[0313] The general applicability of the method according to the invention is also shown. That is, comparable results with the AAV-7m8 capsid variant have been obtained with commercial empty wild-type AAV2 particles (Progene). This illustrates the serotype / capsid-independent applicability of the method according to the invention. The corresponding data are shown in Figure 9 (AAV2-7m8) and Figure 10 (AAV2 wild-type empty particles).

[0314] Calculation of the required amount of particles

[0315] In the method according to the invention, approximately 1.65E9 vp / well are required.

[0316] In the antigen capture format according to the prior art, approximately 5E10 vp / well are required.

[0317] Thus, with the method according to the invention, the amount of AAVp required can be reduced by more than an order of magnitude (7.9E9 vs. 4.8E11).

[0318] The corresponding data are shown in Table 6 below.

[0319] Table 6.

[0320]

[0321] The relationship of 3.33E7 viral particles per nanogram of AAV mass has been used to convert mass concentration (μg / mL) to particle concentration (vp / mL).

[0322] ***

[0323] All references mentioned herein are incorporated by reference in their entirety, even if not expressly stated otherwise.

[0324] ***

[0325] 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 should be understood that modifications can be made to the procedures set forth without departing from the spirit of the invention.

[0326] Example Description

[0327] Example 1

[0328] Immune complex assay for detecting anti-AAV antibodies in mammalian samples

[0329] Anti-AAV particle antibodies (AAV-ADA) were measured in serum samples of human or monkey (cynomolgus macaque) origin.

[0330] Before analysis, serum samples (1 μL) were washed with a LowCross- The sample was diluted 1 to 100 (v / v) in a 50 μL PBS (Candor Biosciences; order number 100-500) and mixed with the corresponding 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 one hour. After incubation, a biotinylated serotype-specific anti-capsid antibody (e.g., anti-AAV2 antibody (whole particles) mouse recombinant, A20R; Progen, catalog number 610298) was added to the mixture to a final concentration of 0.25 μg / mL in the mixture. The mixture of sample, AAV, and anti-capsid antibody was incubated at room temperature for an additional 30 minutes.

[0331] Afterwards, the mixture is added to a streptavidin-coated microtiter plate. The biotinylated capture antibody is captured by interacting with the streptavidin on the plate. By incubating for 15 minutes, capture is allowed. In the presence of AAV-ADA, the biotinylated anti-capsid antibody, the added AAV capsid, and the AAV-ADA complex are fixed on the plate during this step. After washing to remove unbound substances, the AAV-AAV-ADA complex added to the bound anti-capsid antibody is labeled with a horseradish peroxidase-labeled anti-human or anti-monkey IgG polyclonal antibody. The label is incubated at room temperature for one hour. After a washing step with PBS / Tween wash buffer, horseradish peroxidase (HRP) substrate 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is added to the plate (final concentration is 50 μM). The reaction of HRP and ABTS generates a colored reaction product. The signal (extinction) was measured at a wavelength of 405 nm (reference wavelength: 490 nm ([405 / 490] nm)) using an ELISA reader. The signal of each serum sample was determined in duplicate.

[0332] As a negative control, the assay was performed without the addition of AAV capsids. No complexes could form, and only nonspecific binding of the sample itself could occur. By comparing the two signals (with and without AAV addition), the signal (signal-to-noise ratio) could be corrected accordingly.

Claims

1. A method for measuring anti-AAV particle antibodies in a mammalian serum sample, the method 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 and including 50% (v / v) and 0.5% (v / v), b) adding AAV particles (AAVp) specifically bound by the anti-AAV particle antibodies (AAV-ADA) to the diluted sample to generate a spiked sample, c) pre-incubating the spiked sample to allow formation of AAV-ADA-AAVp complexes, as well as d) detecting the AAV-ADA-AAVp complex, Anti-AAV antibodies are thereby measured in the serum sample of the mammal. The method according to claim 1 , wherein the measuring is a determination. The method of claim 1 , wherein the measurement is quantitative.

4. The method according to any one of claims 1 to 3, wherein the pre-incubation lasts for 5 to 25 minutes and the final mammalian serum concentration is about 2% (v / v).

5. The method of any one of claims 1 to 3, wherein the pre-incubation lasts 26 to 90 minutes and the final mammalian serum concentration is about 1% (v / v).

6. The method according to 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 according to 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 variants thereof.

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

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

11. The method according to claim 9, wherein the method further comprises a step dc) after step cd) and before step d): dc) capturing the complex formed in step cd) on a solid surface.

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

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

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

15. The method according to any one of claims 1 to 14, wherein the detection in step d) is performed 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.

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