Monoclonal antibodies to chemically modified nucleic acids and uses thereof

Abstract

To provide a pan-specific antibody which is a monoclonal antibody specifically binding to a chemically modified nucleic acid molecule and binds to the chemically modified nucleic acid molecule without depending on a nucleotide sequence. Also provided are methods of using the antibodies to detect chemically modified nucleic acid molecules in a sample.SOLUTION: Provided is an isolated monoclonal antibody that specifically binds to a chemically modified nucleic acid without depending on the nucleotide sequence, wherein the monoclonal antibody comprises (i) a light chain variable region comprising complementarity-determining regions CDRL1, CDRL2 and CDRL3, and (ii) a heavy chain variable region comprising complementarity-determining regions CDRH1, CDRH2 and CDRH3, wherein the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3 each have a specific amino acid sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 993,575, filed March 23, 2020, which is incorporated herein by reference in its entirety.

[0002] Description of electronically submitted text files

[0001] This application contains a Sequence Listing which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the Sequence Listing in computer readable format, created on March 22, 2021, is titled A-2544-WO-PCT_ST25 and is 176 kilobytes in size.

[0003] The present invention relates to the fields of biological analysis and immunology. Specifically, the present invention relates to monoclonal antibodies that specifically bind to chemically modified nucleic acid molecules and methods for producing such monoclonal antibodies. The present invention also relates to methods for using the monoclonal antibodies to detect nucleic acid molecules, for example, in biological samples (e.g., samples from subjects treated with chemically modified nucleic acid molecules). [Background technology]

[0004] Nucleic acid-based drugs continue to evolve as a unique and effective therapeutic class, with numerous nucleic acid therapeutics being studied in clinical trials and others receiving regulatory approval for various human indications (Sridharan and Gogtay, British Journal of Clinical Pharmacology, Vol. 82:659-672, 2016; Stein et al., Molecular Therapy, Vol. 25:1069-1075, 2017; Adams et al., New England Journal of Medicine, Vol. 379:11-21, 2018). The development of therapeutic nucleic acid molecules requires an understanding of their pharmacokinetic parameters, metabolism, and distribution. Therefore, robust and sensitive bioanalytical assays and reagents capable of detecting nucleic acid molecules in biological fluids, cells, tissues, and tissue samples are essential. Various polymerase chain reaction (Cheng et al., Oligonucleotides, Vol. 19:203-208, 2009; Cheng et al., In: Therapeutic Oligonucleotides: Methods and Protocols (Goodchild, ed.), Humana Press, pp. 183-197, 2011), size exclusion chromatography (Shimoyama et al., Journal of Pharmaceutical and Biomedical Analysis, Vol. 136:55-65, 2017), and liquid chromatography-mass spectrometry-based methods (Basiri et al., Bioanalysis, Vol. 6:1525-1542, 2014; Ewles et al., Bioanalysis, Vol. 6:447-464, 2014) exist, but are limited by their sensitivity and time-consuming extraction steps.

[0005] Immunoassays offer high specificity, high throughput, and high sensitivity for the analysis of a wide range of analytes in biological samples (Darwish, Int. J. Biomed. Sci., Vol. 2:217-235, 2006). However, effective immunoassays require immunoassay reagents, which are novel antibodies that specifically bind to the analyte of interest. Generating antibodies against nucleic acids is challenging because of the low immunogenicity of nucleic acids (see, e.g., Hu et al., Expert Rev. Mol. Diagn., Vol. 14:895-916, 2014 and Feederle and Schepers, RNA Biology, Vol. 14:1089-1098, 2017). Even more challenging is the generation of antibodies (especially monoclonal antibodies) that recognize chemically modified nucleic acid molecules independently of the nucleotide sequence or chemical modification pattern, because most antibodies that recognize modified nucleotides target a single modified nucleotide ( Feederle and Schepers, 2017 ). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sridharan and Gogtay,British Journal of Clinical Pharmacology,Vol.82:659-672,2016 [Non-patent document 2] Stein et al.,Molecular Therapy,Vol.25:1069-1075,2017 [Non-patent document 3] Adams et al.,New England Journal of Medicine,Vol.379:11-21,2018 [Non-patent document 4] Cheng et al.,Oligonucleotides,Vol.19:203-208,2009 [Non-patent document 5] Cheng et al., In: Therapeutic Oligonucleotides: Methods and Protocols (Goodchild, ed.), Humana Press, pgs.183-197, 2011 [Non-patent document 6] Shimoyama et al.,Journal of Pharmaceutical and Biomedical Analysis,Vol.136:55-65,2017 [Non-Patent Document 7] Basiri et al.,Bioanalysis,Vol.6:1525-1542,2014 [Non-patent document 8] Ewles et al.,Bioanalysis,Vol.6:447-464,2014 [Non-Patent Document 9] Darwish,Int.J.Biomed.Sci.,Vol.2:217-235,2006 [Non-Patent Document 10] Hu et al.,Expert Rev.Mol.Diagn.,Vol.14:895-916,2014 [Non-Patent Document 11] Feederle and Schepers,RNA Biology,Vol.14:1089-1098,2017 Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there is a need in the art for novel antibodies that specifically bind to chemically modified nucleic acid molecules, and methods for making such antibodies, which would be particularly useful in the development of novel immunoassays for detecting chemically modified nucleic acid molecules in various types of biological samples. [Means for solving the problem]

[0008] The present invention provides monoclonal antibodies that specifically bind to chemically modified nucleic acid molecules, and methods for making such antibodies. The present invention also provides methods for using such antibodies to detect chemically modified nucleic acid molecules in biological samples, particularly in samples obtained from subjects to whom the chemically modified nucleic acid molecules have been administered.

[0009] In some embodiments, the present invention provides a method for producing a monoclonal antibody that specifically binds to a chemically modified nucleic acid molecule. In one embodiment, the method includes conjugating a plurality of nucleic acid molecules to beads to form an immunogen, each of which contains one or more modified nucleotides; administering the immunogen to an animal; obtaining splenocytes from the immunized animal; selecting splenocytes that are IgG-positive and bind to the chemically modified nucleic acid molecule, thereby isolating cells that produce antigen-specific antibodies; plating the cells that produce the antigen-specific antibodies in single-cell culture; and isolating monoclonal antibodies from the single-cell culture. In certain embodiments, the method further includes lysing B cells derived from the single-cell culture and sequencing antibody genes from the clonal B cells. In an alternative embodiment, the method comprises conjugating a plurality of nucleic acid molecules to beads to form an immunogen, each of which contains one or more modified nucleotides; administering the immunogen to an animal; obtaining splenocytes from the immunized animal; fusing the splenocytes with a myeloma cell line to thereby generate hybridoma cells; establishing hybridoma cell lines from the hybridoma cells; and identifying hybridoma cell lines that produce antibodies that bind to the chemically modified nucleic acid molecule of interest. The animal to which the immunogen is administered can be any immunocompetent animal (e.g., mouse, rabbit, rat, goat, or non-human primate). In a specific embodiment, the animal to which the immunogen is administered is a rabbit.

[0010] The present invention includes monoclonal antibodies or antigen-binding fragments thereof produced by any of the methods described herein. The monoclonal antibodies and antigen-binding fragments thereof find use in a variety of applications, such as the detection and isolation of chemically modified nucleic acid molecules in body fluids and tissues using immunoassays, immunoprecipitation, and immunohistochemistry techniques. In some embodiments, the present invention provides antibodies that specifically bind to chemically modified nucleic acid molecules independent of their nucleotide sequence. Antibodies with such binding specificity are referred to herein as pan-specific antibodies. In certain embodiments, the pan-specific antibodies or antigen-binding fragments thereof of the present invention comprise one or more CDRs or variable regions from any of the pan-specific antibodies described herein. For example, in some embodiments, the pan-specific antibody or antigen-binding fragment thereof comprises a CDRL1 comprising a sequence selected from SEQ ID NOs: 1-3; a CDRL2 comprising a sequence selected from SEQ ID NOs: 14-16; a CDRL3 comprising a sequence selected from SEQ ID NOs: 25-27; a CDRH1 comprising a sequence selected from SEQ ID NOs: 51-53; a CDRH2 comprising a sequence selected from SEQ ID NOs: 64-66; and a CDRH3 comprising a sequence selected from SEQ ID NOs: 77-79. The pan-specific antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 38-40. In these and other embodiments, the pan-specific antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region comprising a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 90-92. In one embodiment, the pan-specific antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a sequence selected from SEQ ID NOs: 38-40 and a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 90-92.

[0011] In other embodiments, the present invention provides antibodies that sequence-specifically bind to an RNAi construct comprising the nucleotide sequence of SEQ ID NO: 192. Such antibodies (referred to herein as 1851 RNAi construct-specific antibodies) specifically bind to the 1851 RNAi construct molecules described herein and do not significantly bind to or significantly cross-react with other chemically modified nucleic acid molecules that differ in nucleotide sequence. 1851 RNAi construct-specific antibodies or antigen-binding fragments thereof of the present invention can comprise one or more CDRs or variable regions from any of the 1851 RNAi construct-specific antibodies described herein. For example, in some embodiments, the 1851 RNAi construct-specific antibody or antigen-binding fragment thereof comprises a CDRL1 comprising a sequence selected from SEQ ID NOs: 4-8; a CDRL2 comprising a sequence selected from SEQ ID NOs: 17-20; a CDRL3 comprising a sequence selected from SEQ ID NOs: 28-32; a CDRH1 comprising a sequence selected from SEQ ID NOs: 54-58; a CDRH2 comprising a sequence selected from SEQ ID NOs: 67-71; and a CDRH3 comprising a sequence selected from SEQ ID NOs: 80-84. The 1851 RNAi construct-specific antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 41-45. In these and other embodiments, the 1851 RNAi construct-specific antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region comprising a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 93-97. In one embodiment, the 1851 RNAi construct-specific antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a sequence selected from SEQ ID NOs: 41-45, and a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 93-97.

[0012] In yet other embodiments, the present invention provides antibodies that specifically bind to N-acetyl-galactosamine (GalNAc) moieties, such as those described herein. GalNAc moiety-specific antibodies or antigen-binding fragments thereof of the invention can comprise one or more CDRs or variable regions from any of the GalNAc moiety-specific antibodies described herein. For example, in some embodiments, the GalNAc moiety-specific antibodies or antigen-binding fragments thereof include a CDRL1 comprising a sequence selected from SEQ ID NOs: 9-13; a CDRL2 comprising a sequence selected from SEQ ID NOs: 19 and 21-24; a CDRL3 comprising a sequence selected from SEQ ID NOs: 33-37; a CDRH1 comprising a sequence selected from SEQ ID NOs: 59-63; a CDRH2 comprising a sequence selected from SEQ ID NOs: 72-76; and a CDRH3 comprising a sequence selected from SEQ ID NOs: 85-89. In certain embodiments, the GalNAc moiety-specific antibodies or antigen-binding fragments thereof can comprise a light chain variable region comprising a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 46-50. In these and other embodiments, the GalNAc moiety-specific antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region comprising a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 98 to 102. In one embodiment, the GalNAc moiety-specific antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a sequence selected from SEQ ID NOs: 46 to 50, and a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 98 to 102.

[0013] The present invention also includes one or more isolated polynucleotides and expression vectors, or components thereof, encoding any of the antibodies or antigen-binding fragments described herein, as well as recombinant host cells containing the encoding polynucleotides and expression vectors. Methods of producing the antibodies or antigen-binding fragments of the present invention using recombinant methods are also contemplated. In some embodiments, such methods include culturing a host cell containing an expression vector encoding the antibody or antigen-binding fragment under conditions that allow expression of the antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment from the culture medium or host cell.

[0014] Any of the monoclonal antibodies or antigen-binding fragments thereof described herein can be conjugated to a detectable label for use in various immunoassays, such as those described herein. In some embodiments, the detectable label is a fluorophore (e.g., fluorescein, rhodamine, Alexa dye molecules, etc.), a metal nanoparticle (e.g., gold nanoparticle, silver nanoparticle, composite nanoparticle, etc.), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, etc.), a radiolabel (e.g., ribonucleotides, nucleotides, etc.), or a nucleotide sequence. 125 I, 131 I, 3 H, 35 S, etc.), or an ECL luminophore (e.g., a ruthenium complex, an iridium complex, etc.). In certain embodiments, the detectable label conjugated to the monoclonal antibody or antigen-binding fragment of the invention is an ECL luminophore, such as a ruthenium complex.

[0015] The present invention provides methods for detecting chemically modified nucleic acid molecules in a sample using the monoclonal antibodies or antigen-binding fragments of the present invention. In some embodiments, the methods include providing a surface comprising a capture antibody that specifically binds to the chemically modified nucleic acid molecule; contacting the surface with the sample under conditions that allow the chemically modified nucleic acid molecule, if present in the sample, to bind to the capture antibody on the surface; contacting the surface with a detection reagent that comprises a detectable label conjugated to a binding partner that specifically binds to the chemically modified nucleic acid molecule; and detecting a signal from the detectable label. In certain embodiments, the capture antibody is any of the pan-specific antibodies described herein, such as the 14K10 antibody.

[0016] In some embodiments of the detection methods of the present invention, the binding partner in the detection reagent can be a labeled form of one of the pan-specific antibodies described herein (e.g., a labeled 14K10 antibody) or another antibody (e.g., a polyclonal antibody) that specifically binds to the chemically modified nucleic acid molecule. The binding partner in the detection reagent can vary depending on the specific chemically modified nucleic acid molecule to be detected. For example, in some embodiments where the chemically modified nucleic acid molecule to be detected is an 1851 RNAi construct, the binding partner in the detection reagent can be any of the 1851 RNAi construct-specific antibodies described herein. In other embodiments where the chemically modified nucleic acid molecule to be detected is covalently linked to a ligand containing a GalNAc moiety, the binding partner in the detection reagent can be any of the GalNAc moiety-specific antibodies described herein (e.g., the 14D4 antibody). In still other embodiments where the chemically modified nucleic acid molecule to be detected is conjugated to an antibody, the binding partner in the detection reagent can be the antibody's target antigen, an anti-Fc region antibody, or an anti-idiotypic antibody. The detectable label conjugated to the binding partner can be any type of signal-generating entity (e.g., a fluorophore, a metal nanoparticle, an enzyme, a radiolabel, an ECL luminophore, such as those described herein).

[0017] The present invention also includes methods for detecting anti-drug antibodies against chemically modified nucleic acid molecules in a subject using a labeled form of a monoclonal antibody or antigen-binding fragment thereof (e.g., a pan-specific antibody) of the present invention in a competitive immunoassay format. In some embodiments, the method includes providing a surface comprising the chemically modified nucleic acid molecule; contacting the surface with a sample obtained from the subject to which the chemically modified nucleic acid molecule has been administered; contacting the surface with a detection reagent, wherein the detection reagent comprises a monoclonal antibody of the present invention conjugated to a detectable label; and detecting a signal from the detectable label, wherein the signal from the detectable label indicates the absence of anti-drug antibodies in the sample. In certain embodiments, the detection reagent comprises any one of the pan-specific antibodies of the present invention conjugated to a detectable label. In one particular embodiment, the detection reagent comprises a 14K10 antibody conjugated to a detectable label.

[0018] The methods of the present invention can be used to generate monoclonal antibodies against, or detect or measure in a sample, any type of chemically modified nucleic acid molecule, such as those described herein. In some embodiments, the chemically modified nucleic acid molecule comprises one or more modified nucleotides selected from 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNAs), or combinations thereof. The chemically modified nucleic acid molecule can also comprise one or more phosphorothioate internucleotide linkages. In certain embodiments, the chemically modified nucleic acid molecule used in or detected or measured according to the methods of the present invention is double-stranded. For example, in some embodiments, the chemically modified nucleic acid molecule is an RNAi construct comprising a sense strand and an antisense strand. In certain embodiments, the chemically modified nucleic acid molecule used in or detected or measured according to the methods of the present invention is covalently linked to a ligand, such as any of the ligands described herein. In some embodiments, the ligand comprises a GalNAc moiety. The GalNAc moiety can be a multivalent GalNAc moiety, such as a trivalent or tetravalent GalNAc moiety. In one embodiment, the GalNAc moiety has the structure of Structure 1. In another embodiment, the GalNAc moiety is a TL01 GalNAc moiety. In yet another embodiment, the GalNAc moiety is a TL02 GalNAc moiety. In yet another embodiment, the GalNAc moiety is a TL03 GalNAc moiety.

[0019] The methods of the present invention can be used to detect or measure chemically modified nucleic acid molecules in various sample types. In some embodiments, the sample is a biological sample, such as serum, plasma, cell lysate, subcellular fraction, or tissue (e.g., tissue homogenate). Such samples can be obtained from an animal or human subject to which a chemically modified nucleic acid molecule has been administered. The sample can be obtained from the subject before, during, or after treatment with the chemically modified nucleic acid molecule. In some embodiments, the sample is obtained from a cell culture (e.g., supernatant, subcellular fraction, or lysate) that has been exposed to the chemically modified nucleic acid molecule.

[0020] The present invention also encompasses kits for detecting chemically modified nucleic acid molecules according to the methods described herein. The kits may include various combinations of monoclonal antibodies or antigen-binding fragments thereof described herein, in unlabeled or labeled form. In some embodiments, the kits include a capture antibody immobilized on a surface (e.g., a well in a microtiter plate) that specifically binds to the chemically modified nucleic acid molecule; a detection reagent comprising a detectable label conjugated to a binding partner that specifically binds to the chemically modified nucleic acid molecule; and instructions for contacting the immobilized capture antibody and detection reagent with a sample and for detecting a signal from the detectable label. In certain embodiments, the capture antibody and the binding partner in the detection reagent are selected from the pan-specific antibodies described herein. In one embodiment, the capture antibody is a 14K10 antibody, and the detection reagent comprises a labeled form of the 14K10 antibody. In certain other embodiments, the capture antibody is one of the pan-specific antibodies described herein, and the binding partner in the detection reagent is one of the GalNAc moiety-specific antibodies described herein. In one particular embodiment, the capture antibody is the 14K10 antibody and the detection reagent comprises a labeled form of the 14D4 antibody. [Brief explanation of the drawings]

[0021] [Figure 1]Schematic showing the generation of multivalent GalNAc-siRNA-coated nanobead immunogens: siRNA molecules conjugated to a trivalent GalNAc moiety (represented by a solid rectangle) at the 5' end of the sense strand were covalently attached to biotin (represented by a solid circle) at the 5' end of the antisense strand. The biotinylated siRNA molecules were attached to streptavidin nanobeads with an average diameter of 0.1 μm. [Figure 2A] Figure 1 is a plot comparing the binding of rabbit monoclonal antibodies to 1851 RNAi constructs with and without GalNAc moieties, as measured by a colorimetric ELISA assay. Optical density (OD) values ​​at 450 nm are shown, with higher values ​​indicating higher levels of antibody binding. The black box indicates a monoclonal antibody that exhibits a higher level of binding to 1851 RNAi constructs with GalNAc moieties compared to RNAi constructs without GalNAc moieties, indicating that this antibody likely binds to the GalNAc moiety of the RNAi construct (GalNAc binder). [Figure 2B]This plot compares the binding of rabbit monoclonal antibodies to the 1851 RNAi construct containing a GalNAc moiety with the binding of the 6189 RNAi construct containing a GalNAc moiety, as measured by a colorimetric ELISA assay. The 6189 RNAi construct has the same GalNAc moiety, chemical modification pattern, and format as the 1851 RNAi construct, but differs in the nucleotide sequences in the sense and antisense strands. OD values ​​at 450 nm are shown; higher values ​​indicate higher levels of antibody binding. The black box at the top shows a monoclonal antibody that showed binding to both RNAi constructs, suggesting that this antibody either binds to the GalNAc moiety (GalNAc binder) or binds to double-stranded RNA structures independent of nucleotide sequence (pan-siRNA binder). The black box at the bottom shows a monoclonal antibody that showed a higher level of binding to the 1851 RNAi construct compared to the 6189 NRAi construct, indicating that this antibody specifically binds to the 1851 RNAi construct (1851 binder). [Figure 3A] Binding activity as measured by geometric mean in flow cytometry using fluorochrome-conjugated secondary antibodies for recombinant antibodies 5I17, 14P2, 14F4, and 14K10 to the indicated antigens is shown. These recombinant antibodies were evaluated for binding at a concentration of 1 μg / mL. [Figure 3B] Figure 1 shows the binding activity as measured by geometric mean in flow cytometry using fluorochrome-conjugated secondary antibodies for recombinant antibodies 14D4, 16I3, 16A22, 17D13, and 18J5 to the indicated antigens. These recombinant antibodies were evaluated for binding at a concentration of 1 μg / mL. [Figure 3C] Figure 1 shows the binding activity as measured by geometric mean in flow cytometry using fluorochrome-conjugated secondary antibodies for recombinant antibodies 14K23, 17K13, 17F22, 19F24, 20P19, and 20K24 to the indicated antigens. These recombinant antibodies were evaluated for binding at a concentration of 1 μg / mL. [Figure 4A] 1 is a bar graph showing the percent inhibition of binding of recombinant antibodies 5I17, 14F4, and 14K10 to beads coated with the 1851 RNAi construct by each of the indicated RNAi constructs, as determined by flow cytometry. Each of the indicated RNAi constructs was pre-incubated with the respective antibody at a 55:1 molar ratio (RNAi construct:antibody). A higher percent inhibition indicates that the antibody binds to a greater extent to the indicated RNAi construct. [Figure 4B] 1 is a bar graph showing the percent inhibition of binding of recombinant antibodies 17K13, 17F22, 19F24, 20P19, and 20K24 to beads coated with the 1851 RNAi construct by each of the indicated RNAi constructs, as determined by flow cytometry. Each of the indicated RNAi constructs was pre-incubated with the respective antibody at a 55:1 molar ratio (RNAi construct:antibody). A higher percent inhibition indicates that the antibody binds to a greater extent to the indicated RNAi construct. [Figure 5A] Schematic representation of one embodiment of the immunoassay method of the present invention. In this embodiment, a pan-siRNA antibody (Ab) of the present invention is immobilized on a solid surface (e.g., a microtiter plate) optionally via biotin-streptavidin interaction. A sample containing a GalNAc moiety (black triangle) conjugated to a chemically modified RNAi construct (GalNAc-siRNA conjugate) is contacted with the immobilized pan-siRNA antibody, which recognizes and binds to the double-stranded RNA component of the GalNAc-siRNA conjugate. Detection and quantification of the GalNAc-siRNA conjugate are then carried out using a labeled pan-siRNA antibody of the present invention, such as a ruthenium-labeled pan-siRNA antibody. The capture pan-siRNA antibody can be the same as the antibody used for the detection pan-siRNA antibody, or it can be a different pan-siRNA antibody. [Figure 5B]A second embodiment of the immunoassay method of the present invention is shown schematically. In this embodiment, a pan-siRNA antibody (Ab) of the present invention is immobilized on a solid surface (e.g., a microtiter plate) optionally via biotin-streptavidin interactions. A sample containing a GalNAc moiety (black triangle) conjugated to a chemically modified RNAi construct (GalNAc-siRNA conjugate) is contacted with the immobilized pan-siRNA antibody, which recognizes and binds to the double-stranded RNA component of the GalNAc-siRNA conjugate. Detection and quantification of intact GalNAc-siRNA conjugates is then carried out using a labeled GalNAc antibody of the present invention (e.g., a ruthenium-labeled GalNAc antibody), which binds to the GalNAc component of the GalNAc-siRNA conjugate. [Figure 5C] Schematic representation of a third embodiment of the immunoassay method of the present invention. In this embodiment, a pan-siRNA antibody (Ab) of the present invention is immobilized on a solid surface (e.g., a microtiter plate) optionally via biotin-streptavidin interactions. A sample containing an antibody-siRNA conjugate molecule is contacted with the immobilized pan-siRNA antibody, which recognizes and binds to the double-stranded RNA component of the antibody-siRNA conjugate molecule. Detection and quantification of intact antibody-siRNA conjugate molecules are then performed using a labeled binding partner (e.g., a ruthenium-labeled anti-Fc antibody) that specifically binds to the antibody component of the conjugate molecule. [Figure 6]Figure 5B is a line graph of various concentrations of GalNAc-conjugated RNAi constructs (construct nos. 16081, 16082, 16083, and 16084) in human serum plotted against the electrochemiluminescence signal in arbitrary units (MSD RFU) measured using either the whole drug assay format depicted in Figure 5A ("tot"; open symbols) or the intact drug assay format depicted in Figure 5B ("int"; filled symbols). The limit of detection (LOD) is shown for each assay format. [Figure 7A] A line graph of various concentrations of GalNAc-conjugated RNAi constructs (construct numbers 1907, 7213, 8172, and 10927) in human serum plotted against the electrochemiluminescence signal in arbitrary units (MSD RFU) measured using the intact drug assay format depicted in Figure 5B. [Figure 7B] A line graph of various concentrations of GalNAc-conjugated RNAi constructs (construct numbers 1907, 7213, 8172, and 10927) in cynomolgus monkey serum plotted against the electrochemiluminescence signal in arbitrary units (MSD RFU) measured using the intact drug assay format depicted in Figure 5B. [Figure 7C] A line graph of various concentrations of GalNAc-conjugated RNAi constructs (construct numbers 1907, 7213, 8172, and 10927) in rat serum plotted against the electrochemiluminescence signal in arbitrary units (MSD RFU) measured using the intact drug assay format depicted in Figure 5B. [Figure 7D] This is a superposition of the graphs depicted in Figures 7A-7C. [Figure 8]A line graph of various concentrations of the indicated GalNAc-conjugated RNAi constructs or their component sense and antisense strands in mouse serum plotted against the electrochemiluminescence signal in arbitrary units (MSD RFU) measured using the intact drug assay format depicted in Figure 5B. [Figure 9] Figure 5B shows the relationship between the concentration of monoclonal antibody-RNAi construct conjugate molecules in mouse serum and the electrochemiluminescence signal in arbitrary units (MSD RFU). The same RNAi construct was conjugated to a human monoclonal antibody that recognizes a cell surface receptor at various conjugation sites within the antibody, at an RNA-to-antibody ratio of 1 or 2. The conjugated molecules were evaluated in two different immunoassays. In assay 1, the 14K10 pan-specific RNAi construct antibody was used as the capture reagent, and a ruthenium-labeled anti-human Fc antibody was used as the detection reagent (see the format depicted in Figure 5C). Assay 2 had a similar format, except that the anti-GalNAc moiety antibody 14D4 was used as the capture reagent instead of the 14K10 antibody. A ruthenium-labeled anti-human Fc antibody was also used for detection in assay 2. [Figure 10A] 1 is a line graph of the relationship between serum concentrations of total drug ("total") or intact mAb-RNAi conjugate molecule ("intact") over time in mice administered intravenously with mAb-RNAi conjugate molecule 15722 or 15723. Mice were administered a single 12 mg / kg dose of either the 15722 or 15723 conjugate molecule at time 0, and serum samples were collected at various times after administration of these conjugate molecules. RAR = RNA-to-antibody ratio. [Figure 10B]1 is a line graph of the relationship between the concentration of total drug ("total") or intact mAb-RNAi conjugate molecule ("intact") in the pancreas over time in mice administered intravenously with mAb-RNAi conjugate molecule 15722 or 15723. Mice were administered a single 12 mg / kg dose of either the 15722 or 15723 conjugate molecule at time 0, and pancreatic samples were taken at various time points after administration of these conjugate molecules. RAR = RNA-to-antibody ratio. [Figure 10C] 1 is a line graph of the relationship between the concentration of total drug ("total") or intact mAb-RNAi conjugate molecule ("intact") in the liver over time in mice administered intravenously with mAb-RNAi conjugate molecule 15722 or 15723. Mice were administered a single 12 mg / kg dose of either the 15722 or 15723 conjugate molecule at time 0, and liver samples were taken at various time points after administration of these conjugate molecules. RAR = RNA-to-antibody ratio. [Figure 10D] 1 is a line graph of the relationship between kidney concentrations of total drug ("total") or intact mAb-RNAi conjugate molecule ("intact") over time in mice administered intravenously with mAb-RNAi conjugate molecule 15722 or 15723. Mice were administered a single 12 mg / kg dose of either the 15722 or 15723 conjugate molecule at time 0, and kidney samples were taken at various time points after administration of these conjugate molecules. RAR = RNA-to-antibody ratio. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention is based, in part, on the generation of monoclonal antibodies that specifically bind to chemically modified nucleic acids, and novel immunoassays incorporating such monoclonal antibodies. The monoclonal antibodies of the invention are useful for a variety of applications, including the detection of nucleic acid-based drugs, for example, to assess the pharmacokinetic properties, metabolism, and distribution of such nucleic acid-based drugs in body fluids and tissues.

[0023] Nucleic acids are notoriously poorly immunogenic, and it is difficult to generate antibodies against specific nucleic acids (see, e.g., Hu et al., Expert Rev. Mol. Diagn., Vol. 14:895-916, 2014, and Feederle and Schepers, RNA Biology, Vol. 14:1089-1098, 2017). Most antibodies capable of recognizing modified nucleotides target a single modified nucleotide and do not necessarily recognize the modified nucleotide when incorporated into a polynucleotide chain (Feederle and Schepers, 2017). The present invention provides a novel method for generating monoclonal antibodies that specifically bind to chemically modified nucleic acid molecules using multivalent nucleic acid-displaying nanobeads as immunogens. This method yields significantly higher antigen-specific antibody titers and antibodies with different binding specificities compared to methods using conventional immunogens composed of nucleic acids linked to carrier proteins (see Example 1). Furthermore, this method allows for the production of rabbit monoclonal antibodies, which are more difficult and less readily available (see Feederle and Schepers, 2017). In some embodiments, this method includes conjugating a plurality of nucleic acid molecules to beads to form an immunogen, each of which contains one or more modified nucleotides; administering the immunogen to an animal; obtaining splenocytes from the immunized animal; selecting splenocytes that are IgG-positive and bind to the chemically-modified nucleic acid molecules, thereby isolating cells that produce antigen-specific antibodies; plating the cells that produce the antigen-specific antibodies in single-cell culture; and isolating monoclonal antibodies from the single-cell culture.

[0024] The methods of the present invention can be used to generate monoclonal antibodies against any type of nucleic acid molecule. The term "nucleic acid molecule" refers to a molecule comprising a polymer of nucleotides, such as polynucleotides and oligonucleotides. A nucleic acid molecule can comprise ribonucleotides, deoxyribonucleotides, modified nucleotides, or a combination thereof. A nucleic acid molecule can be single-stranded, double-stranded, or can contain both single-stranded and double-stranded regions. In some embodiments, a nucleic acid molecule is an antisense oligonucleotide having a sequence complementary to a region of a target gene or mRNA sequence. In other embodiments, a nucleic acid molecule is an anti-miRNA oligonucleotide (e.g., an antagomir or anti-miR) having a sequence complementary to an miRNA. In still other embodiments, a nucleic acid molecule is messenger RNA (mRNA) or a fragment thereof. In certain embodiments, a nucleic acid molecule is an RNAi construct. As used herein, the term "RNAi construct" refers to an agent comprising a nucleic acid molecule that can downregulate the expression of a target gene via the RNA interference mechanism when introduced into a cell. RNA interference is a process in which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (e.g., an mRNA molecule) in a sequence-specific manner, for example, via the RNA-induced silencing complex (RISC) pathway. In some embodiments, an RNAi construct comprises a double-stranded nucleic acid molecule comprising two antiparallel strands of consecutive nucleotides that are sufficiently complementary to each other to hybridize and form a double-stranded region. "Hybridizing" or "hybridization" typically refers to the pairing of complementary polynucleotides via hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds) between complementary bases in two polynucleotides. A strand containing a region having a sequence substantially complementary to a target sequence (e.g., a target mRNA) is referred to as the "antisense strand." A "sense strand" refers to a strand containing a region substantially complementary to a region of the antisense strand. In some embodiments, the sense strand may contain a region having a sequence substantially identical to the target sequence.

[0025] A first sequence is "complementary" to a second sequence if, under certain conditions, such as physiological conditions, a polynucleotide comprising the first sequence can hybridize to a polynucleotide comprising the second sequence to form a double-stranded region. Other such conditions may include moderate or stringent hybridization conditions, which are known to those skilled in the art. A first sequence is considered to be fully complementary (100% complementary) to a second sequence if the polynucleotide comprising the first sequence base pairs with the polynucleotide comprising the second sequence without any mismatches along the entire length of one or both nucleotide sequences. A sequence is "substantially complementary" to a target sequence if the sequence is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the target sequence. The percent complementarity can be calculated by dividing the number of bases in a first sequence that are complementary to bases at corresponding positions in a second, or target, sequence by the total length of the first sequence. A sequence can also be said to be substantially complementary to another sequence if, when the two sequences hybridize, there are no more than 5, 4, 3, or 2 mismatches over a 30 base pair double-stranded region.

[0026] In some embodiments, the antisense strand region of the RNAi construct comprises a sequence that is completely complementary to a region of the target gene sequence (e.g., target mRNA). In such embodiments, the sense strand of the RNAi construct may comprise a sequence that is completely complementary to the antisense strand sequence. In other such embodiments, the sense strand may comprise a sequence that is substantially complementary to the antisense strand sequence, for example, may comprise a sequence with 1, 2, 3, 4, or 5 mismatches in the double-stranded region formed by the sense strand and the antisense strand. In certain embodiments, it is preferred that any mismatches occur within the terminal regions (e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5'-end and / or 3'-end of the strand). In one embodiment, any mismatches in the double-stranded region formed by the sense strand and the antisense strand occur within 6, 5, 4, 3, or 2 nucleotides of the 5'-end of the antisense strand.

[0027] In certain embodiments, an RNAi construct comprises a sense strand and an antisense strand, which are two separate molecules that hybridize to form a double-stranded region but are not otherwise connected. Such double-stranded nucleic acid molecules formed from two separate strands are referred to as "small interfering RNA" or "short interfering RNA" (siRNA). Thus, in some embodiments, the RNAi construct comprises an siRNA. In related embodiments, the RNAi construct comprises a microRNA (miRNA) or miRNA mimic. MiRNAs are endogenous double-stranded RNA molecules that regulate gene expression via the RNA interference pathway.

[0028] In other embodiments, an RNAi construct comprises a nucleic acid molecule having a partially self-complementary region that hybridizes to form a double-stranded region (i.e., the sense strand and antisense strand are part of the self-complementary region of a single nucleic acid molecule). Such nucleic acid molecules having at least a partially self-complementary region are referred to as "short hairpin RNAs" (shRNAs). shRNA molecules typically comprise a double-stranded region (also referred to as a stem region) and a loop region. The 3' end of the sense strand is connected to the 5' end of the antisense strand by a contiguous sequence of unpaired nucleotides, thereby forming the loop region. The loop region is typically sufficiently long to allow the RNA molecule to refold on itself so that the antisense strand can base pair with the sense strand to form the duplex or stem region. The loop region may comprise about 3 to about 25, about 5 to about 15, or about 8 to about 12 unpaired nucleotides. Thus, in certain embodiments, an RNAi construct comprises an shRNA. In a related embodiment, the RNAi construct comprises a precursor miRNA (pre-miRNA).

[0029] The length of the nucleic acid molecule will vary depending on the type of nucleic acid molecule. For the preparation of immunogens, as described in more detail below, the nucleic acid molecule will generally be about 15 to about 150 nucleotides in length. For example, each strand of a double-stranded siRNA molecule, miRNA molecule, or miRNA mimic molecule is typically about 15 to about 30 nucleotides in length, while single-stranded shRNA molecules and pre-miRNA molecules that fold back on themselves to form stem-loop or hairpin structures can be about 35 to about 120 nucleotides in length. Antisense oligonucleotides and anti-miRNA oligonucleotides are typically about 15 to about 25 nucleotides in length. In certain embodiments, the nucleic acid molecule is an RNAi construct comprising a sense strand and an antisense strand, each strand independently being about 19 to about 30 nucleotides in length, about 18 to about 28 nucleotides in length, about 19 to about 27 nucleotides in length, about 19 to about 25 nucleotides in length, about 19 to about 23 nucleotides in length, about 19 to about 21 nucleotides in length, about 21 to about 25 nucleotides in length, or about 21 to about 23 nucleotides in length.

[0030] In embodiments in which the RNAi construct comprises an siRNA, the sense strand and the antisense strand do not need to be the same length as the double-stranded region. A "double-stranded region" refers to two complementary or substantially complementary regions in a polynucleotide that base pair with each other through Watson-Crick base pairing or other hydrogen-bonding interactions to form a duplex between the two polynucleotides. For example, one or both strands may be longer than the double-stranded region and may have one or more unpaired nucleotides or mismatches adjacent to the double-stranded region. Thus, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, a "nucleotide overhang" refers to an unpaired nucleotide at the end of a strand or a nucleotide that extends beyond the double-stranded region. A nucleotide overhang is typically generated when the 3' end of one strand extends beyond the 5' end of the other strand, or when the 5' end of one strand extends beyond the 3' end of the other strand. The length of the nucleotide overhang is generally 1 to 6 nucleotides, 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, or 2 to 4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In a particular embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. In certain other embodiments, the nucleotide overhang comprises a single nucleotide.

[0031] The nucleotide overhangs may be present at the 5'-end or 3'-end of one or both strands. For example, in one embodiment, the RNAi construct comprises nucleotide overhangs at the 5'-end and 3'-end of the antisense strand. In another embodiment, the RNAi construct comprises nucleotide overhangs at the 5'-end and 3'-end of the sense strand. In some embodiments, the RNAi construct comprises nucleotide overhangs at the 5'-end of the sense strand and the 5'-end of the antisense strand. In other embodiments, the RNAi construct comprises nucleotide overhangs at the 3'-end of the sense strand and the 3'-end of the antisense strand.

[0032] An RNAi construct may comprise a nucleotide overhang at one end of the double-stranded RNA molecule and a blunt end at the other end. "Blunt end" means that the sense strand and the antisense strand are perfectly base-paired at the ends of the molecule, and there are no unpaired nucleotides extending beyond the double-stranded region. In some embodiments, an RNAi construct comprises a nucleotide overhang at the 3' end of the sense strand and a blunt end at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, an RNAi construct comprises a nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, an RNAi construct comprises blunt ends at both ends of the double-stranded RNA molecule. In such embodiments, the sense strand and the antisense strand have the same length, and the double-stranded region is the same length as the sense strand and the antisense strand (i.e., the molecule is double-stranded throughout its entire length).

[0033] The nucleic acid molecule is preferably a chemically modified nucleic acid molecule, and this chemically modified nucleic acid molecule refers to a nucleic acid molecule containing one or more modified nucleotides. "Modified nucleotide" refers to a nucleotide having one or more chemical modifications to the nucleoside, nucleobase, pentose ring, or phosphate group. As used herein, modified nucleotide does not include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate, or deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. However, the nucleic acid molecule used in the method of the present invention can contain a combination of modified nucleotides, deoxyribonucleotides, and ribonucleotides. In certain embodiments, all nucleotides in the nucleic acid molecule are modified nucleotides. In other embodiments, all nucleotides in the nucleic acid molecule are a combination of modified nucleotides and deoxynucleotides.

[0034] In certain embodiments, modified nucleotides have modifications of the ribose sugar. The sugar modifications can include modifications at the 2' and / or 5' positions of the pentose ring, as well as bicyclic sugar modifications. A 2'-modified nucleotide refers to a nucleotide having a pentose ring with a substituent other than H or OH at the 2' position. Such 2'-modifications include, but are not limited to, 2'-O-alkyl (e.g., O-C1-C2). 10 Or O-C1~C 10substituted alkyl), 2'-O-allyl (O-CHCH=CH), 2'-C-allyl, 2'-deoxy-2'-fluoro (also referred to as 2'-F or 2'-fluoro), 2'-O-methyl (OCH), 2'-O-methoxyethyl (O-(CH)OCH), 2'-OCF, 2'-O(CH)SCH, 2'-O-aminoalkyl, 2'-amino (e.g., NH), 2'-O-ethylamine, and 2'-azido. Modifications at the 5' position of the pentose ring include, but are not limited to, 5'-methyl (R or S); 5'-vinyl, and 5'-methoxy.

[0035] "Bicyclic sugar modification" refers to a modification of the pentose ring in which a bridge connects two atoms of the ring to form a second ring, resulting in a bicyclic sugar structure. In some embodiments, a bicyclic sugar modification comprises a bridge between the 4' and 2' carbons of the pentose ring. Nucleotides comprising a sugar moiety having a bicyclic sugar modification are referred to herein as bicyclic nucleic acids or BNAs. Exemplary bicyclic sugar modifications include, but are not limited to, α-L-methyleneoxy (4'-CH2-O-2') bicyclic nucleic acids (BNAs); β-D-methyleneoxy (4'-CH2-O-2') BNAs (also known as locked nucleic acids or LNAs); ethyleneoxy (4'-(CH2)2-O-2') BNAs; aminooxy (4'-CH2-ON(R)-2') BNAs; oxyamino (4'-CH2-N(R)-O-2') BNAs; methyl (methyleneoxy ) (4'-CH(CH3)-O-2') BNA (also known as constrained ethyl or cEt); methylene-thio (4'-CH2-S-2') BNA; methylene-amino (4'-CH2-N(R)-2') BNA; methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA; propylene carbocyclic (4'-(CH2)3-2') BNA; and methoxy(ethyleneoxy) (4'-CH(CHOMe)-O-2') BNA (also known as constrained MOE or cMOE). These and other sugar-modified nucleotides that can be incorporated into nucleic acid molecules for use in the methods of the present invention are described in U.S. Pat. No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated by reference in their entireties.

[0036] In some embodiments, a nucleic acid molecule comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), or combinations thereof. In certain embodiments, a nucleic acid molecule comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, or combinations thereof. In certain embodiments, a nucleic acid molecule comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or combinations thereof.

[0037] In certain embodiments, modified nucleotides incorporated into nucleic acid molecules for use in the methods of the invention have a nucleobase (also referred to herein as "base") modification. A "modified nucleobase" or "modified base" refers to a base other than the naturally occurring purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases may be synthetically or naturally occurring modifications, including, but not limited to, the universal bases 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine (X), hypoxanthine (I), 2-aminoadenine, 6-methyladenine, 6-methylguanine, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propyl ... ropynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0038] In some embodiments, the modified base is a universal base. "Universal base" refers to a base analog that indiscriminately base pairs with all natural bases in RNA and DNA without altering the double helix structure of the resulting double-stranded region. Universal bases are known to those skilled in the art and include, but are not limited to, inosine, C-phenyl, C-naphthyl and other aromatic derivatives, azole carboxamides, and nitroazole derivatives, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

[0039] Other suitable modified bases that can be incorporated into nucleic acid molecules for use in the methods of the present invention include those described in Herdewijn, Antisense Nucleic Acid Drug Dev., Vol.10:297-310,2000, and Peacock et al., J.Org.Chem., Vol.76:7295-7300,2011 (both of which are incorporated herein by reference in their entirety).Those skilled in the art will be well aware that guanine, cytosine, adenine, thymine, and uracil can be replaced with other nucleobases, such as the modified nucleobases described above, without substantially changing the base pairing properties of polynucleotides containing nucleotides with such substituted nucleobases.

[0040] In some embodiments, a nucleic acid molecule may contain one or more abasic nucleotides. An "abasic nucleotide" or "abasic nucleoside" is a nucleotide or nucleoside that lacks a nucleobase at the 1' position of the ribose sugar. In certain embodiments, an abasic nucleotide is incorporated at one or both ends of a nucleic acid molecule, for example, at the ends of the sense and / or antisense strands of an RNAi construct. In one embodiment, the sense strand contains an abasic nucleotide as the terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends. In another embodiment, the antisense strand contains an abasic nucleotide as the terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends. In such embodiments in which the abasic nucleotide is a terminal nucleotide, the terminal nucleotide may be an inverted nucleotide, i.e., linked to an adjacent nucleotide via a 3'-3' internucleotide linkage (if on the 3' end of the strand) or a 5'-5' internucleotide linkage (if on the 5' end of the strand) rather than the natural 3'-5' internucleotide linkage. The abasic nucleotide may also include a sugar modification, such as any of the sugar modifications described above. In certain embodiments, the abasic nucleotide includes a 2'-modification, such as a 2'-fluoro modification, a 2'-O-methyl modification, or a 2'-H (deoxy) modification. In one embodiment, the abasic nucleotide includes a 2'-O-methyl modification. In another embodiment, the abasic nucleotide includes a 2'-H modification (i.e., a deoxy abasic nucleotide).

[0041] Chemically modified nucleic acid molecules for use in or detected by the methods of the present invention may also contain one or more modified internucleotide linkages. As used herein, the term "modified internucleotide linkage" refers to an internucleotide linkage other than a natural 3'-5' phosphodiester linkage. In some embodiments, the modified internucleotide linkage is a phosphorus-containing internucleotide linkage, such as a phosphotriester, aminoalkylphosphotriester, alkylphosphonate (e.g., methylphosphonate, 3'-alkylenephosphonate), phosphinate, phosphoramidate (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), phosphorothioate (P=S), chiral phosphorothioate, phosphorodithioate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, and boranophosphate. In one embodiment, the modified internucleotide linkage is a 2'-5' phosphodiester linkage. In other embodiments, the modified internucleotide linkage is a non-phosphorus-containing internucleotide linkage and may therefore be referred to as a modified internucleoside linkage. Such non-phosphorus-containing linkages include, but are not limited to, morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane linkages (-O-Si(H)-O-); sulfide, sulfoxide, and sulfone linkages; formacetyl and thioformacetyl linkages; alkene-containing backbones; sulfamate backbones; methylenemethylimino (-CH-N(CH)-O-CH-) and methylenehydrazino linkages; sulfonate and sulfonamide linkages; amide linkages; and others having mixed N, O, S, and CH component moieties. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) to produce peptide nucleic acids (i.e., PNA), such as those described in U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262.Other suitable modified internucleotide and internucleoside linkages that can be used in chemically modified nucleic acid molecules for use in the methods of the present invention are described in U.S. Pat. Nos. 6,693,187, 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated by reference in their entireties.

[0042] In certain embodiments, chemically modified nucleic acid molecules contain one or more phosphorothioate internucleotide linkages. In embodiments in which the nucleic acid molecule is double-stranded (i.e., an RNAi construct comprising a sense strand and an antisense strand), the phosphorothioate internucleotide linkages may be present in the sense strand, the antisense strand, or both strands. For example, in some embodiments, the sense strand contains 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In other embodiments, the antisense strand contains 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In still other embodiments, both strands contain 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. A double-stranded nucleic acid molecule may contain one or more phosphorothioate internucleotide linkages at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand, the antisense strand, or both strands. For example, in certain embodiments where the nucleic acid molecule is an RNAi construct comprising a sense strand and an antisense strand, the RNAi construct comprises from about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6, or more) consecutive phosphorothioate internucleotide linkages at the 3'-end of the sense strand, the antisense strand, or both strands. In other embodiments, the RNAi construct comprises from about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6, or more) consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand, the antisense strand, or both strands. In any of the embodiments in which one or both strands comprise one or more phosphorothioate internucleotide linkages, the remaining internucleotide linkages within the strands can be natural 3'-5' phosphodiester linkages. For example, in some embodiments, each internucleotide linkage in the sense strand and the antisense strand is selected from phosphodiester and phosphorothioate, and at least one internucleotide linkage is phosphorothioate.

[0043] Chemically modified nucleic acid molecules, such as RNAi constructs, can be easily produced using techniques known in the art, for example, by conventional solid-phase nucleic acid synthesis.Polynucleotides can be constructed using a suitable nucleic acid synthesizer that uses standard nucleotide or nucleoside precursors (e.g., phosphoramidites).Automated nucleic acid synthesizers are commercially available from several vendors, including the DNA / RNA synthesizer from Applied Biosystems (Foster City, CA), the MerMade synthesizer from BioAutomation (Irving, TX), and the OligoPilot synthesizer from GE Healthcare Life Sciences (Pittsburgh, PA).

[0044] Oligonucleotides can be synthesized using phosphoramidite chemistry using a 2' silyl protecting group along with acid-labile dimethoxytrityl (DMT) at the 5' position of the ribonucleoside. Final deprotection conditions are known not to significantly degrade the RNA product. All syntheses can be carried out on large-, medium-, or small-scale in any automated or manual synthesizer. Synthesis can also be carried out in multiwell plates, columns, or glass slides.

[0045] The 2'-O-silyl group can be removed by exposure to fluoride ions, which can include any source of fluoride ions, such as salts containing fluoride ions paired with inorganic counterions (e.g., cesium fluoride and potassium fluoride), or salts containing fluoride ions paired with organic counterions (e.g., tetraalkylammonium fluoride). In the deprotection reaction, a crown ether catalyst can be used in combination with an inorganic fluoride. Preferred fluoride ion sources are tetrabutylammonium fluoride or aminohydrofluorides (e.g., aqueous HF combined with triethylamine in a dipolar aprotic solvent such as dimethylformamide).

[0046] The choice of protecting groups used for the phosphite triesters and phosphotriesters can alter the stability of the triesters to fluoride: methyl protection of the phosphotriester or phosphite triester can stabilize the bond to fluoride ions and improve process yields.

[0047] Because ribonucleosides have a reactive 2' hydroxyl substituent, it may be desirable to protect the reactive 2' position in RNA with a protecting group that is orthogonal to the 5'-O-dimethoxytrityl protecting group (e.g., a protecting group that is stable to acid treatment). Silyl protecting groups meet this requirement and can be easily removed in a final fluoride deprotection step, thereby minimizing RNA degradation.

[0048] In standard phosphoramidite coupling reactions, tetrazole catalysts can be used. Preferred catalysts include, for example, tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, and p-nitrophenyltetrazole.

[0049] As can be appreciated by those of skill in the art, additional methods for synthesizing the RNAi constructs described herein will be apparent to those of skill in the art. Additionally, the various synthetic steps may be performed in an alternate order or sequence to achieve the desired compound. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl, amino, etc. present in bases), and protecting group methodologies (protection and deprotection) useful in the synthesis of chemically modified nucleic acid molecules described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Buts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. Custom synthesis of chemically modified nucleic acid molecules is also available from several commercial vendors, such as Dharmacon, Inc. (Lafayette, CO), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, CA).

[0050] The nucleic acid molecule may be covalently linked to a ligand. As used herein, "ligand" refers to any compound or molecule that can directly or indirectly interact with another compound or molecule. The interaction of the ligand with another compound or molecule may elicit a biological response (e.g., triggering a signaling cascade, inducing receptor-mediated endocytosis) or may simply be a physical association. A ligand may alter one or more properties of the nucleic acid molecule to which it is attached (e.g., the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, endosomal release, charge, and / or clearance properties of the nucleic acid molecule).

[0051] Ligands include serum proteins (e.g., human serum albumin, low-density lipoproteins, globulins), cholesterol moieties, vitamins (biotin, vitamin E, vitamin B 12), folate moieties, steroids, bile acids (e.g., cholic acid), fatty acids (e.g., palmitic acid, myristic acid), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), glycosides, phospholipids, or antibodies or binding fragments thereof (e.g., antibodies or binding fragments that target nucleic acid molecules to specific cell types such as the liver). Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, and geranyloxyhexyl. groups, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl or phenoxazine, peptides (e.g., antennapedia peptide, Tat peptide, RGD peptide), alkylating agents, polymers such as polyethylene glycol (PEG) (e.g., PEG-40K), polyamino acids, and polyamines (e.g., spermine, spermidine).

[0052] In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or other steroid. Cholesterol-conjugated oligonucleotides have been reported to be more active than their unconjugated counterparts (Manoharan, Antisense Nucleic Acid Drug Development, Vol. 12:103-228, 2002). Ligands comprising cholesterol moieties and other lipids for conjugation to nucleic acid molecules are also described in U.S. Pat. Nos. 7,851,615; 7,745,608; and 7,833,992, all of which are incorporated herein by reference in their entireties. In another embodiment, the ligand comprises a folate moiety. Polynucleotides conjugated to a folate moiety can be taken up into cells via receptor-mediated endocytosis. Such folate-polynucleotide conjugates are described in U.S. Pat. No. 8,188,247, which is incorporated herein by reference in its entirety.

[0053] In certain embodiments, the ligand may target the nucleic acid molecule to a particular tissue or cell type, for example, if it is desired to restrict the activity of the nucleic acid molecule to that particular tissue or cell type. In one embodiment, the ligand may target specific delivery of the nucleic acid molecule to liver cells (e.g., hepatocytes) using various means, as described in more detail below. In certain embodiments, the nucleic acid molecule (e.g., an RNAi construct or an antisense oligonucleotide) is targeted to liver cells by a ligand that binds to the surface-expressed asialoglycoprotein receptor (ASGR) or its components (e.g., ASGR1, ASGR2).

[0054] In some embodiments, the ligand covalently linked to the nucleic acid molecule comprises a carbohydrate. "Carbohydrate" refers to a compound composed of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least six carbon atoms, with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. In some embodiments, the carbohydrate incorporated into the ligand is a monosaccharide selected from pentose, hexose, or heptose, as well as disaccharides and trisaccharides containing such monosaccharide units. In other embodiments, the carbohydrate incorporated into the ligand is an amino sugar, such as galactosamine, glucosamine, N-acetylgalactosamine, and N-acetylglucosamine.

[0055] In certain embodiments, the ligand comprises a hexose or hexosamine. The hexose may be selected from glucose, galactose, mannose, fucose, or fructose. The hexosamine may be selected from fructosamine, galactosamine, glucosamine, or mannosamine. In some embodiments, the ligand comprises glucose, galactose, galactosamine, or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine, or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine. In certain embodiments, the ligand comprises N-acetyl-galactosamine. Glucose-containing ligands, galactose-containing ligands, and N-acetyl-galactosamine (GalNAc)-containing ligands are particularly effective for targeting compounds to liver cells, as such ligands bind to ASGR, which is expressed on the surface of liver cells. See, e.g., D'Souza and Devarajan, J. Control Release, Vol. 203:126-139, 2015. Examples of GalNAc- or galactose-containing ligands that can be covalently linked to nucleic acid molecules used in the methods of the invention are described in U.S. Patent Nos. 7,491,805; 8,106,022; and 8,877,917; U.S. Patent Application Publication Nos. 20030130186 and 20170253875; and WO 2013166155, 2014179620, and 2018039647, all of which are incorporated by reference in their entireties.

[0056] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a "multivalent carbohydrate moiety" refers to a moiety containing two or more carbohydrate units that can independently bind or interact with other molecules. For example, a multivalent carbohydrate moiety contains two or more binding domains composed of carbohydrates that can bind to two or more different molecules or to two or more different sites on the same molecule. The valency of a carbohydrate moiety indicates the number of individual binding domains within the carbohydrate moiety. For example, the terms "monovalent," "divalent," "trivalent," and "tetravalent" with respect to a carbohydrate moiety refer to carbohydrate moieties having one, two, three, and four binding domains, respectively. A multivalent carbohydrate moiety can comprise a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetyl-galactosamine moiety, a multivalent N-acetyl-glucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the polyvalent carbohydrate moiety can be bivalent, trivalent, or tetravalent. In such embodiments, the polyvalent carbohydrate moiety can be biantennary or triantennary. In a particular embodiment, the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another particular embodiment, the polyvalent galactose moiety is trivalent or tetravalent. Exemplary trivalent GalNAc-containing ligands that can be linked to nucleic acid molecules used in the methods of the invention are described in Example 1 (TL01, TL02, and TL03 GalNAc moieties). Other examples of trivalent and tetravalent galactose- and GalNAc-containing ligands that can be linked to nucleic acid molecules used in the methods of the invention have been described previously. See, e.g., U.S. Pat. Nos. 7,491,805 and 8,106,022; U.S. Patent Application Publication No. 20170253875; and WO 2013166155, WO 2014179620, and WO 2018039647.

[0057] The ligand can be attached to or linked to the nucleic acid molecule directly or indirectly via a linker moiety. The ligand can be attached to the nucleobase, pentose sugar, or internucleotide linkage of the nucleic acid molecule. Conjugation or attachment to a purine nucleobase or its derivative can occur at any position, including endocyclic and exocyclic atoms. In certain embodiments, the 2-, 6-, 7-, or 8-position of a purine nucleobase is attached to the ligand. Conjugation or attachment to a pyrimidine nucleobase or its derivative can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be attached to the ligand. Conjugation or attachment to the pentose sugar of a nucleotide can occur at any carbon atom. Exemplary carbon atoms of the pentose sugar that can be attached to the ligand include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to the ligand, removing this nucleobase, such as in an abasic nucleotide. An internucleotide linkage can also facilitate ligand attachment. In the case of phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the ligand can be attached directly to the phosphorus atom or to an O atom, N atom, or S atom bonded to the phosphorus atom. In the case of amine- or amide-containing internucleoside linkages (e.g., PNA), the ligand can be attached to the nitrogen atom of the amine or amide, or to an adjacent carbon atom.

[0058] In certain embodiments, the ligand may be attached to the 3'-end or 5'-end of the nucleic acid molecule. For example, in embodiments in which the nucleic acid molecule is double-stranded, the ligand may be attached to the 3'-end or 5'-end of either strand (e.g., the sense or antisense strand of an RNAi construct). In some such embodiments, the ligand is covalently attached to the 5'-end of the sense strand. In such embodiments, the ligand is attached to the 5'-terminal nucleotide of the sense strand. In these and other embodiments, the ligand is attached at the 5'-position of the 5'-terminal nucleotide of the sense strand. In embodiments in which an inverted abasic nucleotide or inverted deoxyribonucleotide is the 5'-terminal nucleotide of the sense strand and is linked to the adjacent nucleotide via a 5'-5' internucleotide bond, the ligand may be attached at the 3'-position of the inverted abasic nucleotide or inverted deoxyribonucleotide. In other embodiments, the ligand is covalently attached to the 3'-end of the sense strand. For example, in some embodiments, the ligand is attached to the 3'-terminal nucleotide of the sense strand. In certain such embodiments, the ligand is attached at the 3' position of the 3'-terminal nucleotide of the sense strand. In embodiments in which an inverted abasic nucleotide or inverted deoxyribonucleotide is the 3'-terminal nucleotide of the sense strand and is linked to an adjacent nucleotide via a 3'-3' internucleotide linkage, the ligand can be attached at the 5' position of the inverted abasic nucleotide or inverted deoxyribonucleotide. In alternative embodiments, the ligand is attached near the 3' end of the sense strand but before one or more terminal nucleotides (i.e., before one, two, three, or four terminal nucleotides). In some embodiments, the ligand is attached at the 2' position of the sugar of the 3'-terminal nucleotide of the sense strand. In other embodiments, the ligand is attached at the 2' position of the sugar of the 5'-terminal nucleotide of the sense strand.

[0059] In certain embodiments, the ligand is attached to the nucleic acid molecule via a linker moiety. A "linker moiety" refers to an atom or group of atoms that covalently attaches the ligand to the nucleic acid molecule. The linker moiety can be about 1 to about 30 atoms in length, about 2 to about 28 atoms in length, about 3 to about 26 atoms in length, about 4 to about 24 atoms in length, about 6 to about 20 atoms in length, about 7 to about 20 atoms in length, about 8 to about 20 atoms in length, about 8 to about 18 atoms in length, about 10 to about 18 atoms in length, or about 12 to about 18 atoms in length. In some embodiments, the linker moiety can comprise a bifunctional linking moiety, which typically comprises an alkyl moiety having two functional groups. One of these functional groups is selected to bind to the nucleic acid molecule, and the other is selected to bind to essentially any selected group, such as the ligand described herein. In certain embodiments, the linker moiety comprises a chain structure or an oligomer of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups commonly used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups, and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturated (e.g., double or triple bond), and the like. Linker moieties that can be used to attach ligands to the nucleic acid molecules include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, substituted C1-C 10 Alkyl, substituted or unsubstituted C2-C 10 Alkenyl, or substituted or unsubstituted C2-C 10 Alkynyl. Preferred substituents for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0060] Other types of linker moieties suitable for attaching ligands to nucleic acid molecules are known in the art and may include those described in U.S. Pat. Nos. 7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551, all of which are incorporated herein by reference in their entireties.

[0061] In certain embodiments, the ligand covalently attached to the nucleic acid molecule comprises a GalNAc moiety, e.g., a multivalent GalNAc moiety. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 3' end of the nucleic acid molecule (e.g., at the 3' end of the sense strand of an RNAi construct). In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 5' end of the nucleic acid molecule (e.g., at the 5' end of the sense strand of an RNAi construct). In still other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3' end of the nucleic acid molecule (e.g., at the 3' end of the sense strand of an RNAi construct). In still other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5' end of the nucleic acid molecule (e.g., at the 5' end of the sense strand of an RNAi construct).

[0062] In certain embodiments, the nucleic acid molecule used in or detected according to the methods of the invention has the following structure: [ka] where "Ac" represents an acetyl group. In a preferred embodiment, a ligand comprising this structure is covalently attached to the 5' end of the nucleic acid molecule (e.g., at the 5' end of the sense strand of an RNAi construct) via a linker moiety, such as those described herein.

[0063] In one embodiment, a nucleic acid molecule used in or detected according to the methods of the invention is covalently attached to a TL01 GalNAc moiety. In another embodiment, a nucleic acid molecule used in or detected according to the methods of the invention is covalently attached to a TL02 GalNAc moiety. In yet another embodiment, a nucleic acid molecule used in or detected according to the methods of the invention is covalently attached to a TL03 GalNAc moiety. The structures of the TL01 GalNAc moiety, TL02 GalNAc moiety, and TL03 GalNAc moiety are shown in Example 1.

[0064] In certain embodiments, methods of the present invention for generating monoclonal antibodies against chemically modified nucleic acid molecules use multivalent nucleic acid-presenting nanobeads as immunogens. In some such embodiments, the methods include conjugating a plurality of chemically modified nucleic acid molecules to beads to form an immunogen. A plurality of chemically modified nucleic acid molecules refers to more than two molecules, typically 10 or more, 50 or more, 100 or more, 500 or more, or 1000 or more molecules. The beads can be made from any number of materials, including, but not limited to, latex, polystyrene, polypropylene, polycarbonate, polyvinylidene fluoride, silica, or other polymers with properties similar to any of the aforementioned polymers. In certain embodiments, the beads are polystyrene beads. In other embodiments, the beads are silica beads. The average diameter of the beads can be about 20 nm to about 5 μm, or about 50 nm to 2 μm. In preferred embodiments, the beads have an average diameter of at least 70 nm. Without being bound by theory, it is believed that beads at least this size or larger may have a reduced in vivo clearance rate, thereby increasing exposure of the immunogen to the immune system. In some embodiments, the beads have an average diameter of about 0.1 μm to about 5 μm. In other embodiments, the beads have an average diameter of about 0.1 μm to about 1 μm. In one embodiment, the beads have an average diameter of about 0.1 μm.

[0065] The chemically modified nucleic acid molecule can be conjugated to beads by various methods known to those skilled in the art (e.g., covalent bonding, adsorption, and affinity binding). The chemically modified nucleic acid molecule can be directly bound to the beads via functional groups present on the surface of the beads. Suitable functional groups can include carboxyl, amino, hydroxyl, hydrazide, and chloromethyl groups in the case of polymer beads, and silanol and carboxyl groups in the case of silica beads. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC)-mediated binding is often used to covalently immobilize nucleic acid molecules on carboxyl-functionalized beads. A linker can be used to function as a spacer between the nucleic acid molecule and the surface of the beads. Suitable linkers are described in the art (see, for example, Hermanson, Bioconjugate techniques, San Diego: Academic Press (1996); Jones, IVD Technology, Vol. Nov / Dec. 39, 2001; and Carmon et al., BioTechniques, Vol. 32: 410-420, 2002). Nucleic acid molecules can also be passively adsorbed to the surface of silica beads bearing hydroxyl or silanol groups. Methods for covalently attaching nucleic acid molecules to solid supports are known to those skilled in the art and include those described in Andreadis and Chrisey, Nucleic Acids Res., Vol. 28:e5, 2000; Armstrong et al., Cytometry, Vol 20:102-108, 2000; Spiro et al., Appl. Environ. Microbiol., Vol. 66:4258-4265, 2000; Beaucage, Curr. Med. Chem., Vol. 8:1213-1244, 2001; Taylor et al., BioTechniques, Vol. 30:661-666, 668-669, 2001; and Walsh et al., J. Biochem. Biophys. Methods, Vol. 47:221-231, 2001.

[0066] Alternatively, the chemically modified nucleic acid molecule can be attached to beads via affinity binding interactions. For example, the beads can be coated with an affinity binding protein (e.g., streptavidin) that interacts with a binding partner (e.g., biotin) bound to the nucleic acid molecule. In certain embodiments, the beads are coated with streptavidin, and the chemically modified nucleic acid molecule is biotinylated (e.g., biotinylated at the 5' and / or 3' ends of one or both strands). Streptavidin-coated beads, as well as beads containing various functional groups described herein that are suitable for use in the methods of the invention, are commercially available from several vendors, including polystyrene microspheres and silica microspheres from Bangs Laboratories, Inc.

[0067] The chemically modified nucleic acid molecule can be conjugated to beads in various orientations. In embodiments in which the chemically modified nucleic acid molecule is covalently linked to a ligand, the nucleic acid molecule is preferably conjugated to the bead at a position within the molecule remote from the ligand, such that the ligand is located away from the surface of the bead. For example, a nucleic acid molecule covalently linked to a ligand at its 5' end will preferably be conjugated to a bead at its 3' end. In embodiments in which the nucleic acid molecule is double-stranded (e.g., an RNAi construct comprising a sense strand and an antisense strand), either strand can be conjugated to a bead at its 5' end or 3' end. In one embodiment, the sense strand is conjugated to a bead at its 5' end. In another embodiment, the antisense strand is conjugated to a bead at its 5' end. In yet another embodiment, the sense strand is conjugated to a bead at its 3' end. In yet another embodiment, the antisense strand is conjugated to a bead at its 3' end. In certain embodiments, the nucleic acid molecule is an RNAi construct comprising a sense strand and an antisense strand, wherein the sense strand is covalently bound to a ligand (e.g., a GAlANc-containing ligand) at its 5' end, and the antisense strand is conjugated to a bead (e.g., via biotin) at its 5' end. In certain other embodiments, the nucleic acid molecule is an RNAi construct comprising a sense strand and an antisense strand, wherein the sense strand is covalently bound to a ligand (e.g., a GAlANc-containing ligand) at its 3' end, and the antisense strand is conjugated to a bead (e.g., via biotin) at its 3' end. An exemplary orientation of a GalNAc-conjugated RNAi construct on a bead is shown in Figure 1.

[0068] The density of the chemically modified nucleic acid molecules on the beads can vary depending on the type of nucleic acid molecule used, for example, the density will be higher for nucleic acid molecules with lower molecular weights and lower for nucleic acid molecules with higher molecular weights. A suitable density of the chemically modified nucleic acid molecules on the beads is about 10 2 molecules / μm 2 ~about 10 6molecules / μm 2 , about 10 4 molecules / μm 2 ~about 10 6 molecules / μm 2 , about 10 3 molecules / μm 2 ~about 10 5 molecules / μm 2 , or approximately 5 × 10 4 molecules / μm 2 ~Approx. 8×10 5 molecules / μm 2 In embodiments in which the chemically modified nucleic acid molecule is a double-stranded RNAi construct (e.g., an siRNA molecule), a bead having a diameter of about 0.1 μm can have about 2,000 to 6,400 RNAi constructs conjugated to the bead.

[0069] After preparation of the immunogen, according to some embodiments of the method of the present invention, the immunogen is administered to an animal to generate an immune response to the immunogen. Any immunocompetent animal may be used, such as a mouse, rabbit, rat, goat, non-human primate (e.g., cynomolgus monkey or rhesus monkey), or other mammal. In certain embodiments of the method of the present invention, the animal to which the immunogen is administered is a rabbit. In certain other embodiments of the method of the present invention, the animal to which the immunogen is administered is a mouse. Splenocytes are then obtained from the immunized animal and selected for IgG surface expression (i.e., IgG positive) and the ability to bind to the chemically modified nucleic acid molecule used in the immunogen to identify cells that produce antigen-specific antibodies. IgG-positive and antigen-specific splenocytes may be identified using a labeled version of the antibody (e.g., a fluorescently labeled chemically modified nucleic acid molecule) and a labeled (e.g., fluorescently labeled) anti-IgG antibody (e.g., an anti-rabbit IgG antibody) specific for IgG molecules from the immunized animal. Cells positive for both markers (i.e., cells that produce antigen-specific antibodies) are then plated in single-cell culture using methods known in the art (e.g., limiting dilution, fluorescence-activated cell sorting, and microfluidics). In certain embodiments of the methods of the present invention, cells that produce antigen-specific antibodies are selected and plated in single culture using fluorescence-activated cell sorting techniques, such as those described in Example 1 herein.

[0070] From the single cell culture, the monoclonal antibody can be isolated and, optionally, purified using any technique known in the art (e.g., protein A chromatography). The monoclonal antibody can also optionally be screened to confirm or further characterize the binding properties of the antibody, for example, using an ELISA or competitive binding assay as described in Example 1 herein. In some embodiments, the method further comprises lysing B cells obtained from the single cell culture and sequencing the antibody genes from the clonal B cells. The sequences can then be used to recombinantly produce the monoclonal antibody in cell culture, as further described herein.

[0071] In an alternative embodiment of the method of the present invention, hybridomas can be generated from splenocytes obtained from an animal immunized with the bead-based immunogen described herein. For example, an animal (e.g., a rabbit, rat, mouse, or other mammal) is immunized with a bead-based immunogen containing multiple chemically modified nucleic acid molecules of interest described herein; splenocytes are collected from the immunized animal; the collected splenocytes are fused with a myeloma cell line, thereby producing hybridoma cells; hybridoma cell lines are established from the hybridoma cells; and hybridoma cell lines that produce antibodies that bind to the chemically modified nucleic acid molecules of interest are identified to generate hybridoma cell lines. Methods for generating hybridoma cell lines by fusing splenocytes obtained from immunized animals with myeloma cells are known in the art. See, for example, Antibodies; Harlow and Lane, Cold Spring Harbor Laboratory Press, 1st Edition (e.g., from 1988) or 2nd Edition (e.g., from 2014). Myeloma cells for use in hybridoma-producing fusion procedures are preferably non-antibody-producing, have high fusion efficiency, and possess enzyme deficiencies that prevent the cells from growing in certain selective media that support the growth of only the desired fused cells (hybridomas). Examples of cell lines suitable for use in fusion with mouse cells include, but are not limited to, Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XXO B1. Examples of cell lines suitable for use in fusion with rat cells include, but are not limited to, R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusion are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[0072] The present invention includes monoclonal antibodies produced by any of the methods described herein. These monoclonal antibodies find use in a variety of applications, including the detection and isolation of chemically modified nucleic acid molecules in biological fluids and tissues using, for example, immunoassay, immunoprecipitation, and immunohistochemistry techniques. Detection methods using the monoclonal antibodies of the invention can be used, for example, to evaluate the pharmacokinetic properties (e.g., bioavailability), metabolism, and distribution of therapeutic molecules containing chemically modified nucleic acids. Labeled forms of the monoclonal antibodies of the invention can be used in competitive assay formats to detect antibodies against nucleic acid-based drugs in samples from subjects administered the drugs. The monoclonal antibodies of the invention can also be used as positive antibodies in such anti-drug antibody assays.

[0073] In some embodiments, the present invention provides antibodies that specifically bind to chemically modified nucleic acid molecules (e.g., panspecific antibodies) independent of nucleotide sequence. Such antibodies have binding specificity for chemically modified double-stranded nucleic acid molecules and do not significantly bind to or cross-react with endogenous nucleic acid molecules. In some embodiments, the antibodies have higher binding affinity for double-stranded nucleic acid molecules compared to single-stranded nucleic acid molecules.

[0074] In certain embodiments, the present invention provides antibodies that sequence-specifically bind to an RNAi construct comprising the nucleotide sequence of SEQ ID NO: 192. Such antibodies specifically bind to the 1851 RNAi construct molecules described herein and do not significantly bind to or cross-react with other chemically modified nucleic acid molecules that differ in nucleotide sequence. In other embodiments, the present invention provides antibodies that specifically bind to N-acetyl-galactosamine (GalNAc) moieties, such as those described herein. In some such embodiments, the GalNAc moiety is a multivalent GalNAc moiety. In one embodiment, the GalNAc moiety is a trivalent GalNAc moiety. In another embodiment, the GalNAc moiety has the structure of Structure 1.

[0075] An antibody is a protein that contains an antigen-binding fragment that specifically binds to an antigen and a scaffold or framework portion that enables the antigen-binding fragment to assume a conformation that promotes binding of the antibody to the antigen. As used herein, the term "antibody" generally refers to a tetrameric immunoglobulin protein containing two light chain polypeptides (each approximately 25 kDa) and two heavy chain polypeptides (each approximately 50-70 kDa). The term "light chain" or "immunoglobulin light chain" refers to a polypeptide that contains, from the amino terminus to the carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). The immunoglobulin light chain constant domain (CL) can be a human kappa (κ) constant domain or a human lambda (λ) constant domain. The term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising, from amino to carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. In some species (e.g., humans), IgG and IgA class antibodies are each further divided into subclasses, i.e., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three constant domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four constant domains (CH1, CH2, CH3, and CH4). Immunoglobulin heavy chain constant domains can be derived from any immunoglobulin isotype, including subtypes. Antibody chains are linked to each other via interpolypeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of the two antibody heavy chains.

[0076] The term "monoclonal antibody" (or "mAb"), as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring variants that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a distinct antigenic site or epitope, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different epitopes.

[0077] The present invention also includes antigen-binding fragments of the monoclonal antibodies described herein. An "antigen-binding fragment," used interchangeably herein with "binding fragment" or "fragment," is a portion of an antibody that lacks at least some of the amino acids present in the full-length heavy and / or light chains, yet is still capable of specifically binding to an antigen. Antigen-binding fragments include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., the VH domain of camelid heavy chain antibodies; VHH fragments; see Cortez-Retamozo et al., Cancer Research, Vol. 64:2853-57, 2004), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity-determining region (CDR) fragments, and may be derived from any mammalian source, such as human, mouse, rat, rabbit, or camel. An antigen-binding fragment can compete with an intact antibody for binding to a target antigen, and can be produced by modification of an intact antibody (e.g., enzymatic or chemical cleavage) or can be synthesized de novo using recombinant DNA technology or peptide synthesis. In some embodiments, an antigen-binding fragment comprises at least one CDR from an antibody that binds to the antigen, for example, the heavy chain CDR3 from an antibody that binds to the antigen. In other embodiments, an antigen-binding fragment comprises all three CDRs from the heavy chain of an antibody that binds the antigen, or all three CDRs from the light chain of an antibody that binds the antigen. In yet other embodiments, an antigen-binding fragment comprises all six CDRs (three from the heavy chain and three from the light chain) from an antibody that binds the antigen.

[0078] An "isolated molecule" (where the molecule is, for example, a polypeptide, polynucleotide, nucleic acid molecule, antibody, or antigen-binding fragment) is a molecule that, by virtue of its origin or source, (1) is not associated with naturally associated components that accompany it in its natural state; (2) is substantially free from other molecules from the same species; (3) is expressed by cells from a different species; or (4) is not naturally occurring. Thus, a molecule that is chemically synthesized or expressed in a cellular system other than the cell in which it naturally occurs will be "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by isolation using purification techniques known in the art. The purity or homogeneity of a molecule can be assessed by many means known in the art. For example, the purity of a polypeptide sample can be assessed using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide using techniques known in the art. For certain purposes, higher resolution can be provided by using HPLC or other purification means known in the art.

[0079] An antibody or antigen-binding fragment "specifically binds" to a target antigen if, under similar binding assay conditions, it has a significantly higher binding affinity compared to its affinity for other, unrelated molecules, such that it can distinguish between the antigens. An antibody or antigen-binding fragment that specifically binds to an antigen has an equilibrium dissociation constant (K D ) is 1×10 -6 The antibody or antigen-binding fragment may be K D is 1×10 -8 M or less, it specifically binds to the antigen with "high affinity."

[0080] Affinity is determined using a variety of techniques, one example of which is an affinity ELISA assay. In various embodiments, affinity is determined by a surface plasmon resonance assay (e.g., a BIAcore®-based assay). Using this methodology, the association rate constant (k a Unit: M -1 s -1 ) and dissociation rate constant (kd Unit: s -1 The equilibrium dissociation constant (K D The ratio of the kinetic rate constants (k d / k a In some embodiments, affinity is determined by a kinetic method, such as the equilibrium exclusion binding assay (KExA) as described in Rathanaswami et al., Analytical Biochemistry, Vol. 373:52-60, 2008. The KinExA assay can be used to determine the equilibrium dissociation constant (K D Unit: M) and association rate constant (k a Unit: M -1 s -1 The dissociation rate constant (k d Unit: s -1 ) to these values ​​(K D ×k a In other embodiments, affinity is determined by biolayer interferometry, such as that described in Kumaraswamy et al., Methods Mol. Biol., Vol. 1278:165-82, 2015, and used in the Octet® system (Pall ForteBio). The rate constant (k a and k d ) and affinity constant (K D ) can be calculated in real time using biolayer interferometry. In some embodiments, the antibodies or antigen-binding fragments described herein exhibit desirable properties, e.g., at about 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 s -1 k for each of the following target antigens d (dissociation rate constant) (lower values ​​indicate higher binding affinity), and / or -7 , 10 -8 , 10 -9 , 10 -10, 10 -11 , 10 -12 K for each target antigen below M D The binding affinity as measured by the equilibrium dissociation constant (equilibrium dissociation constant) is shown (lower values ​​indicate higher binding affinity).

[0081] The antibody or antigen-binding fragment of the present invention may comprise one or more complementarity-determining regions (CDRs) from the light and heavy chain variable regions of the monoclonal antibodies described herein. The term "CDR" refers to a complementarity-determining region (also called a "minimal recognition unit" or "hypervariable region") within an antibody variable sequence. There are three heavy chain variable region CDRs (CDRH1, CDRH2, and CDRH3) and three light chain variable region CDRs (CDRL1, CDRL2, and CDRL3). The term "CDR region" as used herein refers to a group of three CDRs (i.e., three light chain CDRs or three heavy chain CDRs) present within a single variable region. The CDRs in each of the two chains are typically aligned by framework regions (FRs) to form a structure that specifically binds to a specific epitope or domain of a target antigen. From N-terminus to C-terminus, naturally occurring light and heavy chain variable regions typically correspond to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised for assigning numbers to the amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The complementarity-determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using this system. Other numbering systems for amino acids in immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

[0082] In certain embodiments, an antibody or antigen-binding fragment of the invention comprises at least one light chain variable region comprising CDRL1, CDRL2, and CDRL3, and at least one heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, derived from any of the monoclonal antibodies described herein. The monoclonal antibodies of the invention can be classified into three categories based on their binding specificity: (i) monoclonal antibodies that specifically bind to chemically modified nucleic acid molecules independent of nucleotide sequence ("pan-specific mAbs"), (ii) monoclonal antibodies that sequence-specifically bind to an RNAi construct comprising the nucleotide sequence of SEQ ID NO: 192 ("1851 RNAi construct-specific mAbs"), and (iii) monoclonal antibodies that specifically bind to GalNAc moieties ("GalNAc moiety-specific mAbs"). The light and heavy chain variable regions and associated CDRs of exemplary monoclonal antibodies in each of these three categories are shown in Tables 1A and 1B, respectively, below.

[0083] [Table 1]

[0084] [Table 2]

[0085] [Table 3]

[0086] [Table 4]

[0087] Antibodies or antigen-binding fragments of the invention may comprise one or more of the light chain CDRs (i.e., CDRLs) and / or heavy chain CDRs (i.e., CDRHs) set forth in Tables 1A and 1B. For example, in some embodiments, antibodies or antigen-binding fragments of the invention (e.g., panspecific antibodies or antigen-binding fragments thereof) that specifically bind to chemically modified nucleic acid molecules independent of nucleotide sequence include CDRL1 comprising a sequence selected from SEQ ID NOs: 1-3; CDRL2 comprising a sequence selected from SEQ ID NOs: 14-16; CDRL3 comprising a sequence selected from SEQ ID NOs: 25-27; CDRH1 comprising a sequence selected from SEQ ID NOs: 51-53; CDRH2 comprising a sequence selected from SEQ ID NOs: 64-66; and CDRH3 comprising a sequence selected from SEQ ID NOs: 77-79. In one embodiment, a pan-specific antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 1, 14, and 25, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 51, 64, and 77, respectively. In another embodiment, a pan-specific antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 2, 15, and 26, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 52, 65, and 78, respectively. In yet another embodiment, the pan-specific antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 3, 16, and 27, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 53, 66, and 79, respectively.

[0088] In certain embodiments, antibodies or antigen-binding fragments of the present invention that sequence-specifically bind to an RNAi construct comprising the nucleotide sequence of SEQ ID NO: 192 (e.g., 1851 RNAi construct-specific antibodies or antigen-binding fragments thereof) include CDRL1 comprising a sequence selected from SEQ ID NOs: 4 to 8; CDRL2 comprising a sequence selected from SEQ ID NOs: 17 to 20; CDRL3 comprising a sequence selected from SEQ ID NOs: 28 to 32; CDRH1 comprising a sequence selected from SEQ ID NOs: 54 to 58; CDRH2 comprising a sequence selected from SEQ ID NOs: 67 to 71; and CDRH3 comprising a sequence selected from SEQ ID NOs: 80 to 84. In one embodiment, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 4, 17, and 28, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 54, 67, and 80, respectively. In another embodiment, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 18, and 29, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 55, 68, and 81, respectively. In another embodiment, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the present invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 6, 19, and 30, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 56, 69, and 82, respectively.In yet another embodiment, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 7, 20, and 31, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 57, 70, and 83, respectively. In yet another embodiment, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 8, 17, and 32, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 58, 71, and 84, respectively.

[0089] In some embodiments, antibodies or antigen-binding fragments of the invention that specifically bind to a GalNAc moiety (e.g., GalNAc moiety-specific antibodies or antigen-binding fragments thereof) comprise a CDRL1 comprising a sequence selected from SEQ ID NOs: 9-13; a CDRL2 comprising a sequence selected from SEQ ID NOs: 19 and 21-24; a CDRL3 comprising a sequence selected from SEQ ID NOs: 33-37; a CDRH1 comprising a sequence selected from SEQ ID NOs: 59-63; a CDRH2 comprising a sequence selected from SEQ ID NOs: 72-76; and a CDRH3 comprising a sequence selected from SEQ ID NOs: 85-89. In one embodiment, a GalNAc moiety-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 9, 21, and 33, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 59, 72, and 85, respectively. In another embodiment, a GalNAc moiety-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 10, 22, and 34, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 60, 73, and 86, respectively. In another embodiment, a GalNAc moiety-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 11, 19, and 35, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 61, 74, and 87, respectively.In yet another embodiment, a GalNAc moiety-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 12, 23, and 36, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 62, 75, and 88, respectively. In yet another embodiment, a GalNAc moiety-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 13, 24, and 37, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 63, 76, and 89, respectively.

[0090] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL) from one of the monoclonal antibodies described herein. The term "variable region," as used interchangeably herein with "variable domain" (light chain variable region (VL) and heavy chain variable region (VH)), refers to the region in each of the light and heavy immunoglobulin chains that is directly involved in binding the antibody to the antigen. As discussed above, the variable light and heavy chain regions have the same general structure, and each region contains four framework (FR) regions, the sequences of which are widely conserved and connected by three CDRs. The framework regions adopt a β-sheet structure, and the CDRs may form loops connecting this β-sheet structure. The CDRs within each chain are held in a three-dimensional structure by the framework regions and, together with the CDRs from the other chain, form the antigen-binding site. Thus, in some embodiments, the antibodies and antigen-binding fragments of the invention may comprise a light chain variable region shown in Table 1A and / or a heavy chain variable region shown in Table 1, or variants of these light and heavy chain variable regions.

[0091] In certain embodiments, the pan-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising a sequence selected from SEQ ID NOs: 38-40, or a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 38-40. In these and other embodiments, the pan-specific antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 90-92, or a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 90-92. In one embodiment, the pan-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 38 and a heavy chain variable region comprising the sequence of SEQ ID NO: 90. In another embodiment, the pan-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 39 and a heavy chain variable region comprising the sequence of SEQ ID NO: 91. In another embodiment, the pan-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 40 and a heavy chain variable region comprising the sequence of SEQ ID NO: 92.

[0092] In other embodiments, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising a sequence selected from SEQ ID NOs: 41-45, or a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 41-45. In these and other embodiments, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 93-97, or a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 93-97. In one embodiment, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 41 and a heavy chain variable region comprising the sequence of SEQ ID NO: 93. In another embodiment, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 42 and a heavy chain variable region comprising the sequence of SEQ ID NO: 94. In another embodiment, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 43 and a heavy chain variable region comprising the sequence of SEQ ID NO: 95. In yet another embodiment, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 44 and a heavy chain variable region comprising the sequence of SEQ ID NO: 96. In yet another embodiment, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 45 and a heavy chain variable region comprising the sequence of SEQ ID NO: 97.

[0093] In some embodiments, GalNAc moiety-specific antibodies or antigen-binding fragments of the invention comprise a light chain variable region comprising a sequence selected from SEQ ID NOs: 46-50, or a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 46-50. In these and other embodiments, GalNAc moiety-specific antibodies or antigen-binding fragments of the invention comprise a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 98-102, or a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 98-102. In one embodiment, GalNAc moiety-specific antibodies or antigen-binding fragments of the invention comprise a light chain variable region comprising the sequence of SEQ ID NO: 46 and a heavy chain variable region comprising the sequence of SEQ ID NO: 98. In another embodiment, GalNAc moiety-specific antibodies or antigen-binding fragments of the invention comprise a light chain variable region comprising the sequence of SEQ ID NO: 47 and a heavy chain variable region comprising the sequence of SEQ ID NO: 99. In another embodiment, a GalNAc moiety-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 48 and a heavy chain variable region comprising the sequence of SEQ ID NO: 100. In yet another embodiment, a GalNAc moiety-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 49 and a heavy chain variable region comprising the sequence of SEQ ID NO: 101. In yet another embodiment, a GalNAc moiety-specific antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 50 and a heavy chain variable region comprising the sequence of SEQ ID NO: 102.

[0094] The term "identity," as used herein, refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity," as used herein, refers to the percent of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule being compared. For this calculation, gaps in the alignment, if any, must be addressed by a specific mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. For example, sequence identity can be determined by standard methods commonly used to compare the similarity at amino acid positions of two polypeptides.Using a computer program such as BLAST or FASTA, two polypeptide sequences or two polynucleotide sequences are aligned so that their respective residues are optimally matched (along the entire length of one or both sequences, or along a predetermined portion of one or both sequences). This program provides a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 (Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3, 1978) or BLOSUM62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) can be used with this computer program. Then, for example, the percent identity can be calculated as follows: the total number of perfect matches is multiplied by 100, and then divided by the sum of the length of the longer sequence in the matched span and the number of gaps introduced into the longer sequence to align the two sequences. In calculating percent identity, the sequences being compared are aligned to maximize the match between the sequences.

[0095] The GCG program package is a computer program that can be used to determine percent identity, and this package includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or two polynucleotides for which the percent sequence identity is to be determined. These sequences are aligned so that their respective amino acids or nucleotides are optimally matched (the "match span" determined by the algorithm). This algorithm is used with a gap opening penalty (calculated as 3 x average diagonal, where "average diagonal" refers to the average of the diagonals of the comparison matrix used; "diagonal" refers to the score or number assigned to each perfect amino acid match by a particular comparison matrix), a gap extension penalty (usually 1 / 10 of the gap opening penalty), and a comparison matrix such as PAM 250 or BLOSUM 62. In certain embodiments, the algorithm also uses standard comparison matrices (for the PAM 250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM 62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0096] Suggested parameters for determining percent identity of polypeptide or nucleotide sequences using the GAP program include the following: Algorithm: Needleman et al. 1970, J. Mol. Biol. 48:443-453; Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, referenced above; Gap penalty: 12 (but no penalty for end gaps) Gap length penalty: 4 Similarity threshold: 0.

[0097] Certain alignment schemes for aligning two amino acid sequences may result in matching only short regions of the two sequences, and this small aligned region may have very high sequence identity despite the lack of significant relationship between the two full-length sequences. Thus, the selected alignment method (GAP program) may be adjusted as necessary to result in alignment over at least 50 consecutive amino acids of the target polypeptide.

[0098] The antibodies of the present invention may comprise any immunoglobulin constant region. The term "constant region," used interchangeably with "constant domain" herein, refers to all domains of an antibody other than the variable region. The constant region is not directly involved in antigen binding but exerts various effector functions. As explained above, antibodies are divided into specific isotypes (IgA, IgD, IgE, IgG, and IgM) and subtypes (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) depending on the amino acid sequence of the constant region of their heavy chain. Antibodies provided herein that contain a constant region from one subclass or a species (e.g., rabbit) can be modified using recombinant DNA technology to contain a constant region from another subclass or another species (e.g., mouse or human). Therefore, an antibody containing a rabbit IgG constant region can be converted to an antibody containing an IgG constant region from another species, such as mouse or human, depending on the desired use of the antibody. Immunoglobulin constant regions from various subclasses of antibodies from several species are known in the art and can be combined with the variable region sequences shown in Tables 1A and 1B to form complete antibody light and heavy chains with a desired isotype. Furthermore, the heavy and light chain sequences so generated can be combined to form a complete antibody structure (e.g., an antibody structure comprising two light chains and two heavy chains).

[0099] The amino acid sequences of the full-length light chains and full-length heavy chains of exemplary monoclonal antibodies of the invention (eg, pan-specific antibodies, 1851 RNAi construct-specific antibodies, and GalNAc moiety-specific antibodies) are shown in Table 2 below.

[0100] [Table 5]

[0101] [Table 6]

[0102] [Table 7]

[0103] [Table 8]

[0104] [Table 9]

[0105] In certain embodiments, antibodies or antigen-binding fragments of the invention may comprise a light chain selected from the light chains shown in Table 2 and / or a heavy chain selected from the heavy chains shown in Table 2, or variants of these light and heavy chains. In some embodiments, pan-specific antibodies or antigen-binding fragments of the invention comprise: (a) a light chain comprising the sequence of SEQ ID NO: 103 and a heavy chain comprising the sequence of SEQ ID NO: 116; (b) a light chain comprising the sequence of SEQ ID NO: 104 and a heavy chain comprising the sequence of SEQ ID NO: 117; or (c) a light chain comprising the sequence of SEQ ID NO: 105 and a heavy chain comprising the sequence of SEQ ID NO: 118. In certain embodiments, an 1851 RNAi construct-specific antibody or antigen-binding fragment of the present invention comprises: (a) a light chain comprising the sequence of SEQ ID NO: 106 and a heavy chain comprising the sequence of SEQ ID NO: 119; (b) a light chain comprising the sequence of SEQ ID NO: 107 and a heavy chain comprising the sequence of SEQ ID NO: 120; (c) a light chain comprising the sequence of SEQ ID NO: 108 and a heavy chain comprising the sequence of SEQ ID NO: 121; (d) a light chain comprising the sequence of SEQ ID NO: 109 and a heavy chain comprising the sequence of SEQ ID NO: 122; or (e) a light chain comprising the sequence of SEQ ID NO: 110 and a heavy chain comprising the sequence of SEQ ID NO: 123. In certain other embodiments, a GalNAc moiety-specific antibody or antigen-binding fragment of the invention comprises: (a) a light chain comprising the sequence of SEQ ID NO: 111 and a heavy chain comprising the sequence of SEQ ID NO: 124; (b) a light chain comprising the sequence of SEQ ID NO: 112 and a heavy chain comprising the sequence of SEQ ID NO: 125; (c) a light chain comprising the sequence of SEQ ID NO: 113 and a heavy chain comprising the sequence of SEQ ID NO: 126; (d) a light chain comprising the sequence of SEQ ID NO: 114 and a heavy chain comprising the sequence of SEQ ID NO: 127; or (e) a light chain comprising the sequence of SEQ ID NO: 115 and a heavy chain comprising the sequence of SEQ ID NO: 128. In any of the embodiments described above, the antibody may be a rabbit monoclonal antibody, and in particular, a rabbit IgG antibody.

[0106] Variants of the antibodies disclosed herein are also contemplated. For example, a variant of the present pan-specific antibody may comprise a light chain comprising a sequence at least 80%, 85%, 90%, or 95% identical to a sequence selected from SEQ ID NOs: 103-105, and a heavy chain comprising a sequence at least 80%, 85%, 90%, or 95% identical to a sequence selected from SEQ ID NOs: 116-118. In some embodiments, the present 1851 RNAi construct-specific antibody may comprise a light chain comprising a sequence at least 80%, 85%, 90%, or 95% identical to a sequence selected from SEQ ID NOs: 106-110, and a heavy chain comprising a sequence at least 80%, 85%, 90%, or 95% identical to a sequence selected from SEQ ID NOs: 119-123. In certain embodiments, the GalNAc moiety-specific antibody may comprise a light chain comprising a sequence at least 80%, 85%, 90%, or 95% identical to a sequence selected from SEQ ID NOs: 111-115, and a heavy chain comprising a sequence at least 80%, 85%, 90%, or 95% identical to a sequence selected from SEQ ID NOs: 124-128.

[0107] The antibodies or antigen-binding fragments of the present invention can be produced using the methods for producing the monoclonal antibodies of the present invention described above. The antibodies or antigen-binding fragments of the present invention can also be produced using recombinant expression methods known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988). The relevant amino acid sequence from an immunoglobulin or a region thereof (e.g., variable region, constant region, etc.) can be determined by direct protein sequencing, and a suitable encoding nucleotide sequence can be designed according to a universal codon table. Alternatively, genomic or cDNA encoding a monoclonal antibody of the invention or a binding fragment thereof can be isolated and sequenced from a cell (e.g., a clonal B cell or a hybridoma) that produces such an antibody using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody).

[0108] Table 3 provides exemplary nucleic acid sequences encoding the variable regions of the light and heavy chains of pan-specific, 1851 RNAi construct-specific, and GalNAc moiety-specific antibodies of the invention, and Table 4 lists exemplary nucleic acid sequences encoding the full-length light and heavy chains of antibodies of the invention. Polynucleotides encoding the variable regions and full chains can be used to recombinantly express the antibodies or variants thereof described herein.

[0109] [Table 10]

[0110] Table 11

[0111] Table 12

[0112] Table 13

[0113] Table 14

[0114] Table 15

[0115] Table 16

[0116] Table 17

[0117] Table 18

[0118] Table 19

[0119] Table 20

[0120] Table 21

[0121] [Table 22]

[0122] [Table 23]

[0123] [Table 24]

[0124] [Table 25]

[0125] [Table 26]

[0126] [Table 27]

[0127] The nucleic acid sequences shown in Tables 3 and 4 are exemplary only. As will be appreciated by those of skill in the art, due to the degeneracy of the genetic code, a large number of nucleic acids can be generated, all of which encode the CDRs, variable regions, and heavy and light chains of the antibodies described herein. Thus, having identified a particular amino acid sequence, one of skill in the art could generate any number of different nucleic acids by altering the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. Thus, isolated polynucleotides encoding antibodies and antigen-binding fragments of the invention can comprise a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to any of the nucleotide sequences listed in Tables 3 and 4.

[0128] In some embodiments, the isolated polynucleotide encoding the light chain variable region of the pan-specific antibody comprises a sequence at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 129-131. In other embodiments, the isolated polynucleotide encoding the light chain variable region of the pan-specific antibody comprises a sequence selected from SEQ ID NOs: 129-131. In certain embodiments, the isolated polynucleotide encoding the light chain of the pan-specific antibody comprises a sequence at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 155-157. In certain other embodiments, the isolated polynucleotide encoding the light chain of the pan-specific antibody comprises a sequence selected from SEQ ID NOs: 155-157. In these and other embodiments, the isolated polynucleotide encoding the heavy chain variable region of the pan-specific antibody comprises a sequence at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 142-144. In some embodiments, the isolated polynucleotide encoding the heavy chain variable region of the pan-specific antibody comprises a sequence selected from SEQ ID NOs: 142-144. In related embodiments, the isolated polynucleotide encoding the heavy chain of the pan-specific antibody comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 168-170. In some embodiments, the isolated polynucleotide encoding the heavy chain of the pan-specific antibody comprises a sequence selected from SEQ ID NOs: 168-170.

[0129] In certain embodiments, the isolated polynucleotide encoding the light chain variable region of the 1851 RNAi construct-specific antibody comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 132-136. In certain other embodiments, the isolated polynucleotide encoding the light chain variable region of the 1851 RNAi construct-specific antibody comprises a sequence selected from SEQ ID NOs: 132-136. In some embodiments, the isolated polynucleotide encoding the light chain of the 1851 RNAi construct-specific antibody comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 158-162. In other embodiments, the isolated polynucleotide encoding the light chain of the 1851 RNAi construct-specific antibody comprises a sequence selected from SEQ ID NOs: 158-162. In these and other embodiments, the isolated polynucleotide encoding the heavy chain variable region of the 1851 RNAi construct-specific antibody comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 145-149. In some embodiments, the isolated polynucleotide encoding the heavy chain variable region of the 1851 RNAi construct-specific antibody comprises a sequence selected from SEQ ID NOs: 145-149. In related embodiments, the isolated polynucleotide encoding the heavy chain of the 1851 RNAi construct-specific antibody comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 171-175. In some embodiments, the isolated polynucleotide encoding the heavy chain of the 1851 RNAi construct-specific antibody comprises a sequence selected from SEQ ID NOs: 171-175.

[0130] In certain other embodiments, the isolated polynucleotide encoding the light chain variable region of the GalNAc moiety-specific antibody comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 137-141. In some embodiments, the isolated polynucleotide encoding the light chain variable region of the GalNAc moiety-specific antibody comprises a sequence selected from SEQ ID NOs: 137-141. In other embodiments, the isolated polynucleotide encoding the light chain of the GalNAc moiety-specific antibody comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 163-167. In still other embodiments, the isolated polynucleotide encoding the light chain of the GalNAc moiety-specific antibody comprises a sequence selected from SEQ ID NOs: 163-167. In these and other embodiments, the isolated polynucleotide encoding the heavy chain variable region of the GalNAc moiety-specific antibody comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 150-154. In some embodiments, the isolated polynucleotide encoding the heavy chain variable region of the GalNAc moiety-specific antibody comprises a sequence selected from SEQ ID NOs: 150-154. In related embodiments, the isolated polynucleotide encoding the heavy chain of the GalNAc moiety-specific antibody comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 176-180. In other related embodiments, the isolated polynucleotide encoding the heavy chain of the GalNAc moiety-specific antibody comprises a sequence selected from SEQ ID NOs: 176-180.

[0131] Expression vectors containing one or more polynucleotides encoding the antibodies of the present invention or portions thereof can be constructed from the polynucleotide sequences described above and used to transform host cells to produce the antibodies or antigen-binding fragments of the present invention. The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information into a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. The term "expression vector" or "expression construct," as used herein, refers to a recombinant nucleic acid molecule containing a desired coding sequence and appropriate nucleic acid control sequences required for the expression of an operably linked coding sequence in a particular host cell. Expression vectors may include, but are not limited to, sequences that affect or control transcription, translation, and, if present, introns, sequences that affect RNA splicing of the operably linked coding region. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, an optional operator sequence, a ribosome binding site, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. Similarly, a secretory signal peptide sequence may optionally be encoded by an expression vector operably linked to a coding sequence of interest so that the expressed polypeptide may be secreted by the recombinant host cell, if desired, so that the polypeptide of interest can be more easily isolated from the cell.

[0132] Typically, expression vectors used in host cells to produce the antibodies and antigen-binding fragments of the invention contain sequences for cloning and expression of exogenous nucleotide sequences encoding components of the antibody or antigen-binding fragment. In certain embodiments, such sequences, collectively referred to as "flanking sequences," typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element.

[0133] Expression and cloning vectors typically contain a promoter that is recognized by a host cell and is operably linked to a nucleic acid molecule encoding a polypeptide. The term "operably linked," as used herein, refers to the joining of two or more nucleic acid sequences to produce a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule. For example, a control sequence in a vector "operably linked" to a protein-coding sequence is ligated to the protein-coding sequence such that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence. More specifically, a promoter and / or enhancer sequence (including any combination of cis-acting transcriptional control elements) is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Numerous promoters recognized by a variety of potential host cells are known. A suitable promoter is operably linked to a polynucleotide encoding, for example, the heavy chain, light chain, or other component of an antibody or antigen-binding fragment of the invention by removing the promoter from the source nucleic acid by restriction enzyme digestion and inserting the desired promoter sequence into the vector.

[0134] Expression vectors can be constructed from a starting vector, such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. If one or more of the desired flanking sequences are not initially present in the vector, they can be obtained individually and ligated into the vector. Methods used to obtain each of the flanking sequences are known to those skilled in the art. The expression vector can be introduced into a host cell, thereby producing antibodies and antigen-binding fragments encoded by the nucleic acid present in the vector.

[0135] After constructing a vector and inserting one or more nucleic acid molecules encoding components of the antibodies and antigen-binding fragments described herein into the appropriate sites in the vector, the completed vector can be inserted into a suitable host cell for amplification and / or polypeptide expression. The term "host cell," as used herein, refers to a cell that has been transformed with a nucleic acid or is capable of being transformed with a nucleic acid and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not identical in morphology or genetic make-up to the original parent cell, so long as the gene of interest is present. A host cell containing an isolated polynucleotide of the invention, preferably operably linked to at least one expression control sequence (e.g., promoter or enhancer), is a "recombinant host cell."

[0136] Transformation of a selected host cell with an expression vector for an antibody or antigen-binding fragment of the invention can be accomplished by known methods (e.g., transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques). The method selected will depend, in part, on the type of host cell used.

[0137] When cultured under appropriate conditions, host cells synthesize antibodies, antigen-binding fragments, or antigen-binding proteins, which can then be recovered from the culture medium (if the host cells secrete them into the medium) or directly from the producing host cells (if they are not secreted). The selection of an appropriate host cell will depend on various factors, such as the desired expression level, polypeptide modifications (e.g., glycosylation or phosphorylation) that are desirable or necessary for activity, and the ease of folding into a biologically active molecule. Suitable host cells include, but are not limited to, prokaryotic cells (e.g., E. coli, B. subtilis), yeast cells (Saccharomyces cerevisiae, Pichia pastoris), and mammalian cells (e.g., Chinese hamster ovary (CHO), human embryonic kidney (HEK)).

[0138] The host cells used to produce the antibodies and antigen-binding fragments of the invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) are suitable for culturing the host cells. Any of these media may be supplemented, as needed, with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., Gentamicin™ drugs), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary nutritional supplements may also be included at appropriate concentrations that would be known to one of skill in the art. Culture conditions, such as temperature, pH, and the like, will be those previously used with the host cell chosen for expression and will be apparent to one of skill in the art.

[0139] When host cells are cultured, antibodies or antigen-binding fragments can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody or antigen-binding fragment is produced intracellularly, as a first step, the particulate debris (i.e., either host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. The antibody or antigen-binding fragment can be purified from the culture medium, culture supernatant, or other liquid after the recovery step using, for example, hydroxyapatite chromatography, cation or anion exchange chromatography, preferably affinity chromatography using the antigen of interest or Protein A or Protein G as the affinity ligand.

[0140] In certain embodiments, the antibodies or antigen-binding fragments of the invention are used in diagnostic or analyte detection methods, such as those described herein. Accordingly, in some embodiments, the antibodies or antigen-binding fragments of the invention are conjugated to a detectable label. The detectable label can be any molecular entity capable of producing a detectable signal under a specific set of conditions. Any conventional label capable of providing a detectable signal, alone or in combination with other compositions or compounds, can be used. The detectable label can be a radiolabel, an enzyme, a fluorophore, a chromophore, a chemiluminescent label, an electrochemiluminescent (ECL) luminophore, a metal nanoparticle, or a metal nanoshell.

[0141] In one embodiment, the detectable label conjugated to the binding partner is a metal nanoparticle or metal nanoshell. Metal nanoparticles or metal nanoshells suitable for use as detectable labels include, but are not limited to, gold nanoparticles, silver nanoparticles, copper nanoparticles, platinum nanoparticles, cadmium nanoparticles, composite nanoparticles (e.g., silver and gold, or copper and silver), gold hollow spheres, gold-coated silica nanoshells, and silica-coated gold shells. In another embodiment, the detectable label conjugated to the binding partner is an enzyme capable of converting a substrate into a detectable signal (e.g., a colored, fluorescent, or chemiluminescent product). Non-limiting examples of enzymes suitable for conjugation to the antibodies or antigen-binding fragments of the invention include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, beta-lactamase, galactose oxidase, lactoperoxidase, luciferase, myeloperoxidase, and amylase. In another embodiment, the detectable label conjugated to the antibodies or antigen-binding fragments of the invention is a fluorophore. Exemplary fluorescent molecules suitable for use as detectable labels include fluorescein, Texas Red, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, Alexa dye molecules, rhodamine dye molecules, and the like. In yet another embodiment, the detectable label conjugated to the antibody or antigen-binding fragment of the invention is a radiolabel. Suitable radiolabels include, but are not limited to: 125 I, 131 I, 3 H, 14 C. 13 N, 18 F, and 35 S.

[0142] In certain embodiments, the detectable label conjugated to the antibody or antigen-binding fragment of the invention is an ECL luminophore. ECL luminophores that can be conjugated to the antibody or antigen-binding fragment of the invention include, but are not limited to, ruthenium complexes (e.g., tri-2,2'-bipyridyl ruthenium(II) [Ru(bpy)3], 2+]), iridium complexes, aluminum complexes, chromium complexes, copper complexes, europium complexes, osmium complexes, platinum complexes, and rhenium complexes, such as those described in Richter, Chem. Rev., Vol. 104:3003-3036, 2004; Liu et al., Chem. Soc. Rev., Vol. 44, 3117-3142, 2015; and Zhou et al., Dalton Trans., Vol. 46, 355-363, 2017. In certain embodiments, the ECL luminophore conjugated to the antibody or antigen-binding fragment of the invention is a ruthenium complex.

[0143] Methods for conjugating a detectable label to a protein, such as an antibody or antigen-binding fragment of the invention, are known in the art and may include passive adsorption (e.g., when a metal nanoparticle or nanoshell is the detectable label) and conjugation chemistries such as succinimide ester conjugation to primary amines and maleimide conjugation to sulfhydryl groups. Other methods for conjugating a macromolecule to a detectable label are known to those skilled in the art, and an appropriate method may be selected based on the type of desired detectable label used.

[0144] Any of the antibodies or antigen-binding fragments described herein or produced by the methods of the invention can be incorporated into immunoassays to detect chemically modified nucleic acid molecules in various samples, such as biological samples. Accordingly, the present invention provides a method for detecting chemically modified nucleic acid molecules in a sample. In one embodiment, the method includes providing a surface comprising a capture antibody or antigen-binding fragment thereof that specifically binds to the chemically modified nucleic acid molecule; contacting the surface with the sample under conditions that allow the chemically modified nucleic acid molecule, if present in the sample, to bind to the capture antibody or antigen-binding fragment thereof on the surface; contacting the surface with a detection reagent, where the detection reagent comprises a detectable label conjugated to a binding partner that specifically binds to the chemically modified nucleic acid molecule; and detecting a signal from the detectable label.

[0145] In certain embodiments of the detection methods of the present invention, the capture antibody that specifically binds to the chemically modified nucleic acid molecule is one of the pan-specific antibodies described herein, such as the 14K10 antibody, the 14F4 antibody, or the 5I17 antibody. In one embodiment, the capture antibody comprises a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 14, a CDRL3 of SEQ ID NO: 25, a CDRH1 of SEQ ID NO: 51, a CDRH2 of SEQ ID NO: 64, and a CDRH3 of SEQ ID NO: 77. In another embodiment, the capture antibody comprises a CDRL1 of SEQ ID NO: 2, a CDRL2 of SEQ ID NO: 15, a CDRL3 of SEQ ID NO: 26, a CDRH1 of SEQ ID NO: 52, a CDRH2 of SEQ ID NO: 65, and a CDRH3 of SEQ ID NO: 78. In another embodiment, the capture antibody comprises a CDRL1 of SEQ ID NO: 3, a CDRL2 of SEQ ID NO: 16, a CDRL3 of SEQ ID NO: 27, a CDRH1 of SEQ ID NO: 53, a CDRH2 of SEQ ID NO: 66, and a CDRH3 of SEQ ID NO: 79. In some embodiments, the capture antibody comprises (a) a light chain variable region comprising the sequence of SEQ ID NO: 38 and a heavy chain variable region comprising the sequence of SEQ ID NO: 90; (b) a light chain variable region comprising the sequence of SEQ ID NO: 39 and a heavy chain variable region comprising the sequence of SEQ ID NO: 91; or (c) a light chain variable region comprising the sequence of SEQ ID NO: 40 and a heavy chain variable region comprising the sequence of SEQ ID NO: 92. In other embodiments, the capture antibody comprises (a) a light chain comprising the sequence of SEQ ID NO: 103 and a heavy chain comprising the sequence of SEQ ID NO: 116; (b) a light chain comprising the sequence of SEQ ID NO: 104 and a heavy chain comprising the sequence of SEQ ID NO: 117; or (c) a light chain comprising the sequence of SEQ ID NO: 105 and a heavy chain comprising the sequence of SEQ ID NO: 118.

[0146] In some embodiments of the detection methods of the present invention, in which the chemically modified nucleic acid molecule to be detected is an 1851 RNAi construct, the capture antibody can be any of the 1851 RNAi construct-specific antibodies described herein, such as the 17K13 antibody, the 17F22 antibody, the 20K24 antibody, the 20P19 antibody, or the 19F24 antibody. In one such embodiment, the capture antibody comprises a CDRL1 of SEQ ID NO:4, a CDRL2 of SEQ ID NO:17, a CDRL3 of SEQ ID NO:28, a CDRH1 of SEQ ID NO:54, a CDRH2 of SEQ ID NO:67, and a CDRH3 of SEQ ID NO:80. In another embodiment, the capture antibody comprises a CDRL1 of SEQ ID NO:5, a CDRL2 of SEQ ID NO:18, a CDRL3 of SEQ ID NO:29, a CDRH1 of SEQ ID NO:55, a CDRH2 of SEQ ID NO:68, and a CDRH3 of SEQ ID NO:81. In another embodiment, the capture antibody comprises a CDRL1 of SEQ ID NO: 6, a CDRL2 of SEQ ID NO: 19, a CDRL3 of SEQ ID NO: 30, a CDRH1 of SEQ ID NO: 56, a CDRH2 of SEQ ID NO: 69, and a CDRH3 of SEQ ID NO: 82. In yet another embodiment, the capture antibody comprises a CDRL1 of SEQ ID NO: 7, a CDRL2 of SEQ ID NO: 20, a CDRL3 of SEQ ID NO: 31, a CDRH1 of SEQ ID NO: 57, a CDRH2 of SEQ ID NO: 70, and a CDRH3 of SEQ ID NO: 83. In yet another embodiment, the capture antibody comprises a CDRL1 of SEQ ID NO: 8, a CDRL2 of SEQ ID NO: 17, a CDRL3 of SEQ ID NO: 32, a CDRH1 of SEQ ID NO: 58, a CDRH2 of SEQ ID NO: 71, and a CDRH3 of SEQ ID NO: 84.

[0147] In some embodiments of the detection method, the capture antibody comprises (a) a light chain variable region comprising the sequence of SEQ ID NO: 41 and a heavy chain variable region comprising the sequence of SEQ ID NO: 93; (b) a light chain variable region comprising the sequence of SEQ ID NO: 42 and a heavy chain variable region comprising the sequence of SEQ ID NO: 94; (c) a light chain variable region comprising the sequence of SEQ ID NO: 43 and a heavy chain variable region comprising the sequence of SEQ ID NO: 95; (d) a light chain variable region comprising the sequence of SEQ ID NO: 44 and a heavy chain variable region comprising the sequence of SEQ ID NO: 96; or (e) a light chain variable region comprising the sequence of SEQ ID NO: 45 and a heavy chain variable region comprising the sequence of SEQ ID NO: 97. In other embodiments of the detection method of the present invention, the capture antibody comprises (a) a light chain comprising the sequence of SEQ ID NO: 106 and a heavy chain comprising the sequence of SEQ ID NO: 119; (b) a light chain comprising the sequence of SEQ ID NO: 107 and a heavy chain comprising the sequence of SEQ ID NO: 120; (c) a light chain comprising the sequence of SEQ ID NO: 108 and a heavy chain comprising the sequence of SEQ ID NO: 121; (d) a light chain comprising the sequence of SEQ ID NO: 109 and a heavy chain comprising the sequence of SEQ ID NO: 122; or (e) a light chain comprising the sequence of SEQ ID NO: 110 and a heavy chain comprising the sequence of SEQ ID NO: 123.

[0148] The capture antibody used in the methods of the invention is preferably attached to or immobilized on a surface. This surface can be a bead or particle (e.g., magnetic beads or particles including silica, latex, polystyrene, polycarbonate, polyacrylate, or polyvinylidene fluoride (PVDF)), a membrane (e.g., PVDF, nitrocellulose, polyethylene, or nylon membrane), a tube, a resin, a column, an electrode, or a well in an assay plate (e.g., a well in a microtiter plate). Such surfaces can include glass, cellulose-based materials, thermoplastic polymers such as polyethylene, polypropylene, or polyester, sintered structures made of particulate materials (e.g., glass or various thermoplastic polymers), or cast membrane films made of nitrocellulose, nylon, polysulfone, or the like. All of these surface materials can be used in any suitable form, such as a film, sheet, or plate, or they can be coated on, bound to, or laminated to a suitable inert support such as paper, glass, plastic film, or fabric.

[0149] The capture antibody can be immobilized on or attached to a surface by a variety of procedures known to those skilled in the art. The capture antibody can be striped, deposited, or printed onto a surface, followed by drying the surface to promote immobilization. Immobilization of the capture antibody can occur via adsorption or covalent binding. Depending on the nature of the surface, derivatization methods can be used to promote covalent bond formation between the surface and the capture antibody. Derivatization methods can include treating the surface with compounds such as glutaraldehyde or carbodiimide and applying the capture antibody. The capture reagent can also be indirectly attached to a surface via a moiety conjugated to the capture antibody (e.g., a moiety with high affinity for the surface-attached component) that allows for covalent or non-covalent binding. For example, the capture reagent can be conjugated to biotin, and the surface-attached component can be avidin, streptavidin, or neutravidin (see, e.g., Figures 5-5C). Other physical, chemical, or biological methods of directly or indirectly immobilizing antibodies to a surface are known in the art and may be used to immobilize or attach the capture antibody to a surface.

[0150] After contacting the sample with the surface containing the capture antibody and any optional washing steps to remove unbound molecules, the detection methods of the present invention include contacting the surface with a detection reagent and detecting a signal from a detectable label in the detection reagent. The detection reagent comprises a detectable label conjugated to a binding partner that specifically binds to the chemically modified nucleic acid molecule. The binding partner in the detection reagent can be an antibody or antigen-binding fragment thereof, an aptamer, a polynucleotide that hybridizes to the chemically modified nucleic acid molecule, or another molecule that can specifically bind to the chemically modified nucleic acid molecule.

[0151] In certain embodiments, the binding partner in the detection reagent is an antibody or antigen-binding fragment that specifically binds to the chemically modified nucleic acid molecule. In some such embodiments, the antibody in the detection reagent is a polyclonal antibody. Polyclonal antibodies that bind to the chemically modified nucleic acid molecule of interest can be generated by immunizing an immunocompetent animal (e.g., a mouse, rabbit, rat, goat, or other mammal) with a chemically modified nucleic acid molecule conjugated to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin, in the absence or presence of an adjuvant. In other embodiments, the antibody in the detection reagent is one of the pan-specific antibodies described herein, such as the 14K10 antibody, the 14F4 antibody, or the 5I17 antibody. In some such embodiments, both the capture antibody immobilized on the surface and the antibody in the detection reagent are pan-specific antibodies described herein. See, e.g., Figure 5A. In such embodiments, the capture antibody and the antibody in the detection reagent can be the same pan-specific antibody (e.g., the 14K10 antibody) or can be different antibodies. In one particular embodiment, the antibody in the detection reagent comprises a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 14, a CDRL3 of SEQ ID NO: 25, a CDRH1 of SEQ ID NO: 51, a CDRH2 of SEQ ID NO: 64, and a CDRH3 of SEQ ID NO: 77. In another particular embodiment, the antibody in the detection reagent comprises a light chain variable region comprising the sequence of SEQ ID NO: 38 and a heavy chain variant region comprising the sequence of SEQ ID NO: 90. In yet another particular embodiment, the antibody in the detection reagent comprises a light chain comprising the sequence of SEQ ID NO: 103 and a heavy chain comprising the sequence of SEQ ID NO: 116.

[0152] In embodiments of the methods of the invention in which the chemically modified nucleic acid molecule to be detected is an 1851 RNAi construct, the antibody in the detection reagent can be any of the 1851 RNAi construct-specific antibodies described herein, such as the 17K13 antibody, the 17F22 antibody, the 20K24 antibody, the 20P19 antibody, or the 19F24 antibody. In one such embodiment, the antibody in the detection reagent comprises a CDRL1 of SEQ ID NO:4, a CDRL2 of SEQ ID NO:17, a CDRL3 of SEQ ID NO:28, a CDRH1 of SEQ ID NO:54, a CDRH2 of SEQ ID NO:67, and a CDRH3 of SEQ ID NO:80. In another embodiment, the antibody in the detection reagent comprises a CDRL1 of SEQ ID NO:5, a CDRL2 of SEQ ID NO:18, a CDRL3 of SEQ ID NO:29, a CDRH1 of SEQ ID NO:55, a CDRH2 of SEQ ID NO:68, and a CDRH3 of SEQ ID NO:81. In another embodiment, the antibody in the detection reagent comprises a CDRL1 of SEQ ID NO: 6, a CDRL2 of SEQ ID NO: 19, a CDRL3 of SEQ ID NO: 30, a CDRH1 of SEQ ID NO: 56, a CDRH2 of SEQ ID NO: 69, and a CDRH3 of SEQ ID NO: 82. In some embodiments of the detection method, the antibody in the detection reagent comprises (a) a light chain variable region comprising the sequence of SEQ ID NO: 41 and a heavy chain variable region comprising the sequence of SEQ ID NO: 93; (b) a light chain variable region comprising the sequence of SEQ ID NO: 42 and a heavy chain variable region comprising the sequence of SEQ ID NO: 94; or (c) a light chain variable region comprising the sequence of SEQ ID NO: 43 and a heavy chain variable region comprising the sequence of SEQ ID NO: 95. In other embodiments of the detection method of the invention, the antibody in the detection reagent comprises (a) a light chain comprising the sequence of SEQ ID NO: 106 and a heavy chain comprising the sequence of SEQ ID NO: 119; (b) a light chain comprising the sequence of SEQ ID NO: 107 and a heavy chain comprising the sequence of SEQ ID NO: 120; or (c) a light chain comprising the sequence of SEQ ID NO: 108 and a heavy chain comprising the sequence of SEQ ID NO: 121.

[0153] For detection of 1851 RNAi constructs in biological samples, any of the present 1851 RNAi construct-specific antibodies may be used as a capture antibody, an antibody in a detection reagent, or both. For example, in some embodiments of the detection methods of the present invention, a pan-specific antibody described herein (e.g., 14K10 antibody) is used as a capture antibody, and an 1851 RNAi construct-specific antibody described herein (e.g., 17K13 antibody, 17F22 antibody, or 20K24 antibody) is used as an antibody in a detection reagent. In other embodiments of the detection methods of the present invention, an 1851 RNAi construct-specific antibody described herein (e.g., 17K13 antibody, 17F22 antibody, or 20K24 antibody) is used as a capture antibody, and a pan-specific antibody described herein (e.g., 14K10 antibody) is used as an antibody in a detection reagent. In certain embodiments of the detection methods of the present invention, an 1851 RNAi construct-specific antibody described herein is used as both a capture antibody and an antibody in a detection reagent. In such embodiments, the same 1851 RNAi construct-specific antibody may be used as both the capture antibody and the antibody in the detection reagent, or different antibodies may be used as the capture antibody and the antibody in the detection reagent, for example, the 17K13 antibody may be used as the capture antibody and the 20K24 antibody may be used as the antibody in the detection reagent.

[0154] In certain embodiments, the chemically modified nucleic acid molecule to be detected is covalently linked to a ligand containing a GalNAc moiety, such as any of the GalNAc moieties described herein. In such embodiments, the binding partner in the detection reagent can be a molecule that specifically binds to a GalNAc residue (e.g., a lectin, a ligand-binding domain of the ASGR receptor, or an antibody or antigen-binding fragment thereof). In certain embodiments, the binding partner in the detection reagent is one of the GalNAc moiety-specific antibodies described herein, such as the 14D4, 16I3, 16A22, 17D13, or 18J5 antibodies. In some such embodiments, the capture antibody immobilized on the surface is one of the pan-specific antibodies described herein (e.g., the 14K10 antibody), and the antibody in the detection reagent is one of the GalNAc moiety-specific antibodies described herein (e.g., the 14D4 antibody). See, e.g., Figure 5B. In one such embodiment, the antibodies in the detection reagent comprise a CDRL1 of SEQ ID NO:9, a CDRL2 of SEQ ID NO:21, a CDRL3 of SEQ ID NO:33, a CDRH1 of SEQ ID NO:59, a CDRH2 of SEQ ID NO:72, and a CDRH3 of SEQ ID NO:85. In another embodiment, the antibodies in the detection reagent comprise a CDRL1 of SEQ ID NO:10, a CDRL2 of SEQ ID NO:22, a CDRL3 of SEQ ID NO:34, a CDRH1 of SEQ ID NO:60, a CDRH2 of SEQ ID NO:73, and a CDRH3 of SEQ ID NO:86. In another embodiment, the antibodies in the detection reagent comprise a CDRL1 of SEQ ID NO:11, a CDRL2 of SEQ ID NO:19, a CDRL3 of SEQ ID NO:35, a CDRH1 of SEQ ID NO:61, a CDRH2 of SEQ ID NO:74, and a CDRH3 of SEQ ID NO:87. In yet another embodiment, the antibodies in the detection reagent comprise a CDRL1 of SEQ ID NO:12, a CDRL2 of SEQ ID NO:23, a CDRL3 of SEQ ID NO:36, a CDRH1 of SEQ ID NO:62, a CDRH2 of SEQ ID NO:75, and a CDRH3 of SEQ ID NO:88. In yet another embodiment, the antibodies in the detection reagent comprise CDRL1 of SEQ ID NO: 13, CDRL2 of SEQ ID NO: 24, CDRL3 of SEQ ID NO: 37, CDRH1 of SEQ ID NO: 63, CDRH2 of SEQ ID NO: 76, and CDRH3 of SEQ ID NO: 89.

[0155] In some embodiments of the detection method, the antibody in the detection reagent comprises (a) a light chain variable region comprising the sequence of SEQ ID NO: 46 and a heavy chain variable region comprising the sequence of SEQ ID NO: 98; (b) a light chain variable region comprising the sequence of SEQ ID NO: 47 and a heavy chain variable region comprising the sequence of SEQ ID NO: 99; (c) a light chain variable region comprising the sequence of SEQ ID NO: 48 and a heavy chain variable region comprising the sequence of SEQ ID NO: 100; (d) a light chain variable region comprising the sequence of SEQ ID NO: 49 and a heavy chain variable region comprising the sequence of SEQ ID NO: 101; or (e) a light chain variable region comprising the sequence of SEQ ID NO: 50 and a heavy chain variable region comprising the sequence of SEQ ID NO: 102. In other embodiments of the detection method of the present invention, the antibody in the detection reagent comprises (a) a light chain comprising the sequence of SEQ ID NO: 111 and a heavy chain comprising the sequence of SEQ ID NO: 124; (b) a light chain comprising the sequence of SEQ ID NO: 112 and a heavy chain comprising the sequence of SEQ ID NO: 125; (c) a light chain comprising the sequence of SEQ ID NO: 113 and a heavy chain comprising the sequence of SEQ ID NO: 126; (d) a light chain comprising the sequence of SEQ ID NO: 114 and a heavy chain comprising the sequence of SEQ ID NO: 127; or (e) a light chain comprising the sequence of SEQ ID NO: 115 and a heavy chain comprising the sequence of SEQ ID NO: 128.

[0156] In certain embodiments, the chemically modified nucleic acid molecule to be detected is conjugated to an antibody or antigen-binding fragment thereof. In such embodiments, the binding partner in the detection reagent can be the target antigen (or a fragment of the antigen containing the epitope) of the antibody or antigen-binding fragment. In other such embodiments, the binding partner in the detection reagent is an anti-idiotypic antibody. An anti-idiotypic antibody is an antibody that binds to the idiotype of another antibody. The idiotype of an antibody is the specific combination of idiotopes present in the variable region of the antibody. In still other such embodiments, the binding partner in the detection reagent is a protein that specifically binds to the Fc region of an antibody, such as an anti-Fc region antibody, protein A, or protein G. In some such embodiments, the capture antibody immobilized on the surface is one of the pan-specific antibodies described herein (e.g., the 14K10 antibody), and the antibody in the detection reagent is an anti-Fc region antibody. See, e.g., Figure 5C. In certain embodiments of the detection methods of the present invention, the chemically modified nucleic acid molecule to be detected is conjugated to a protein, and the binding partner in the detection reagent is an antibody or antigen-binding fragment thereof that specifically binds to this protein.

[0157] The detectable label in the detection reagent can be any of the detectable labels described above. In some embodiments, the detectable label in the detection reagent is a radiolabel, an enzyme, a fluorophore, a chromophore, a chemiluminescent label, an electrochemiluminescent (ECL) luminophore, a metal nanoparticle, or a metal nanoshell. In certain embodiments, the detectable label in the detection reagent is a fluorophore (e.g., fluorescein, rhodamine, Alexa dye molecules, etc.), a metal nanoparticle (e.g., gold nanoparticles, silver nanoparticles, composite nanoparticles, etc.), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, etc.), a radiolabel ( 125 I, 131 I, 3 H, 35S, etc.), or an ECL luminophore (e.g., a ruthenium complex, an iridium complex, etc.). In one particular embodiment, the detectable label in the detection reagent is an ECL luminophore, such as a ruthenium complex.

[0158] In some embodiments, the detection reagent may include a second molecule that links a detectable label to a binding partner that specifically binds to the chemically modified nucleic acid molecule. For example, in embodiments in which the binding partner that specifically binds to the chemically modified nucleic acid molecule is an antibody, the antibody does not need to be directly covalently linked to a detectable label. Rather, a second antibody specific to the antibody of the species from which the first antibody is derived may be covalently linked to a detectable label. For example, if the binding partner that specifically binds to the chemically modified nucleic acid molecule is one of the rabbit monoclonal antibodies described herein, a second antibody specific to the rabbit antibody may be covalently linked to a detectable label.

[0159] After contacting the surface containing the captured chemically modified nucleic acid molecule with a detection reagent, the method of the present invention includes detecting or measuring a signal from a detectable label in the detection reagent. The signal from the detectable label indicates the presence of the target chemically modified nucleic acid molecule in the sample. In some embodiments in which the chemically modified nucleic acid molecule is conjugated to another molecule (e.g., a GalNAc moiety or a protein), the signal from the detectable label can also indicate that the target chemically modified nucleic acid molecule conjugate is intact, i.e., the nucleic acid molecule remains covalently linked to the conjugate partner (i.e., a GalNAc moiety or a protein). The detected signal depends on the type of detection label used. For example, signals from metal nanoparticle or nanoshell labels can be detected by measuring the amount of light scattering or light absorption. Signals from fluorophores or ECL luminophores can be detected or measured as light intensity at a specific emission wavelength. When the detectable label is an enzyme, a signal is generated by adding a substrate for the enzyme (e.g., a chromogenic, fluorogenic, or chemiluminescent substrate) that produces a detectable signal. Instruments such as spectrophotometers, fluorescence / luminescence plate readers, and other instruments capable of detecting spectral and electrochemical changes are commercially available and known to those of skill in the art. In certain embodiments, detecting the signal from the detectable label provides a qualitative assessment (i.e., that the chemically modified nucleic acid molecule is present in the sample). In other embodiments, detecting the signal from the detectable label provides a quantitative measurement of the amount of the chemically modified nucleic acid molecule in the sample. For example, in certain embodiments, the amount of the chemically modified nucleic acid molecule in a sample can be quantitatively determined, e.g., by measuring light scattering, light absorption, or fluorescence / luminescence emission. Such quantification can be achieved by measuring the signal from the detectable label in samples containing known amounts of the chemically modified nucleic acid molecule, constructing a calibration curve from this data, and determining the amount of the chemically modified nucleic acid molecule in a test sample from this calibration curve.

[0160] The detection methods of the invention can be used to assess the pharmacokinetic properties (e.g., bioavailability), metabolism, and distribution of chemically modified nucleic acid molecules. For example, samples taken at various time points from a subject administered a chemically labeled nucleic acid molecule can be assessed with the detection methods of the invention to determine the half-life of the molecule in a particular fluid or tissue, or the period during which the molecule is distributed to a particular tissue compartment. Thus, in certain embodiments, the invention provides a method for assessing the pharmacokinetic profile of a chemically modified nucleic acid molecule in a subject. In one embodiment, the method includes administering a chemically modified nucleic acid molecule to a subject; obtaining at least one sample from the subject after administration of the chemically modified nucleic acid molecule; contacting the sample with at least one monoclonal antibody or antigen-binding fragment thereof (e.g., a panspecific antibody) of the invention under conditions that allow the chemically modified nucleic acid molecule, if present in the sample, to bind to the monoclonal antibody or antigen-binding fragment thereof, thereby forming a complex; and detecting the complex. Any of the assay formats described herein can be used to detect complexes between a monoclonal antibody or antigen-binding fragment thereof (e.g., a panspecific antibody) of the present invention and a chemically modified nucleic acid molecule. Similarly, immunohistochemistry methods using labeled forms of the monoclonal antibody or antigen-binding fragment thereof described herein can be used to detect complexes between the monoclonal antibody or antigen-binding fragment thereof and a chemically modified nucleic acid molecule. In some embodiments, multiple samples are obtained from the subject at various time points after administration of the chemically modified nucleic acid molecule. In such embodiments, the level or concentration of the chemically modified nucleic acid molecule in these samples can be measured to determine changes in the concentration or level of the chemically modified nucleic acid molecule over time. The sample can be a bodily fluid, such as serum, plasma, urine, or blood. In other embodiments, the sample is a tissue sample or tissue homogenate, such as a tissue sample or tissue homogenate from the liver, kidney, pancreas, or other organ. The subject can be a mammal, such as a mouse, rat, dog, pig, or non-human primate (e.g., a cynomolgus monkey). In some embodiments, the subject is a human.

[0161] The monoclonal antibodies or antigen-binding fragments of the present invention can also be used in methods for detecting anti-drug antibodies in subjects administered chemically-modified nucleic acid-based drugs. For example, labeled forms of the monoclonal antibodies or antigen-binding fragments thereof (e.g., panspecific antibodies) of the present invention can be used in competitive immunoassays to detect the presence of anti-drug antibodies in samples from subjects administered chemically-labeled nucleic acid molecules. In such competitive assays, chemically-modified nucleic acid drug molecules are immobilized on a surface (e.g., the wells of a microtiter plate) and contacted with a sample obtained from a subject administered the chemically-modified nucleic acid molecules. A labeled form of the monoclonal antibody of the present invention (e.g., a panspecific monoclonal antibody conjugated to a detectable label) is then added to the surface. If few or no anti-drug antibodies are present in the sample from the subject, the labeled monoclonal antibody can bind to the available chemically-modified nucleic acid molecules immobilized on the surface. If anti-drug antibodies are present in the sample, the anti-drug antibodies will bind to the immobilized chemically-modified nucleic acid molecules, and there will be few or no immobilized chemically-modified nucleic acid molecules available to bind to the labeled monoclonal antibody. Therefore, any anti-drug antibodies present in the sample will compete with the labeled monoclonal antibody for binding to the immobilized chemically modified nucleic acid molecule. The signal from the detectable label on the monoclonal antibody of the present invention is inversely correlated with the amount of anti-drug antibodies in the sample of interest (i.e., a decrease in the signal from the detectable label compared to a control without sample indicates that the test sample contains anti-drug antibodies). Methods for immobilizing nucleic acid molecules on a surface are known to those skilled in the art and may include any of the methods described above for attaching nucleic acid molecules to nanobeads.

[0162] Thus, the present invention includes a method for detecting anti-drug antibodies against a chemically modified nucleic acid molecule in a subject. In some embodiments, the method includes providing a surface comprising the chemically modified nucleic acid molecule; contacting the surface with a sample obtained from a subject administered the chemically modified nucleic acid molecule; contacting the surface with a detection reagent, the detection reagent comprising a monoclonal antibody of the present invention conjugated to a detectable label; and detecting a signal from the detectable label, the signal from the detectable label indicating the absence of anti-drug antibodies in the sample. In certain embodiments, the detection reagent comprises any one of the pan-specific antibodies of the present invention conjugated to a detectable label. In one particular embodiment, the detection reagent comprises a 14K10 antibody conjugated to a detectable label. In other embodiments, the chemically modified nucleic acid molecule is an 1851 RNAi construct, and the detection reagent comprises any one of the 1851 RNAi construct-specific antibodies of the present invention conjugated to a detectable label. In some embodiments of the anti-drug antibody detection method of the present invention, the sample is a serum sample or a plasma sample.

[0163] The methods of the present invention can be used to detect or measure chemically modified nucleic acid molecules in various types of samples. In some embodiments, the sample is a bodily fluid, such as blood, serum, plasma, cerebrospinal fluid, saliva, or urine. In other embodiments, the sample is a tissue (e.g., a tissue homogenate), a cell lysate, or a subcellular fraction. The tissue can be from any organ, including, but not limited to, the liver, kidney, spleen, lung, or skin. In certain embodiments, the sample (a bodily fluid sample, a tissue sample, or a cell sample) is obtained from an animal or human subject to which the chemically modified nucleic acid molecule has been administered. The sample can be obtained from the subject before, during, or after treatment with the chemically modified nucleic acid molecule. In some embodiments, the sample is obtained from a cell culture that has been exposed to the chemically modified nucleic acid molecule. In such embodiments, the sample can be the supernatant of the cell culture, a lysate of cells in the culture, or a subcellular fraction of cells in the culture.

[0164] According to the methods of the present invention, any type of chemically modified nucleic acid molecule, such as those described herein, can be detected or measured in a sample. In some embodiments, the chemically modified nucleic acid molecule comprises one or more modified nucleotides selected from 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, BNAs, or combinations thereof. The chemically modified nucleic acid molecule can also comprise one or more phosphorothioate internucleotide linkages.

[0165] In certain embodiments, the chemically modified nucleic acid molecule detected or measured according to the methods of the present invention is double-stranded. In some such embodiments, the chemically modified nucleic acid molecule is an RNAi construct comprising a sense strand and an antisense strand. The sense strand and antisense strand of the RNAi construct can each independently be about 19 to about 30 nucleotides in length, about 18 to about 28 nucleotides in length, about 19 to about 27 nucleotides in length, about 19 to about 25 nucleotides in length, about 19 to about 23 nucleotides in length, about 19 to about 21 nucleotides in length, about 21 to about 25 nucleotides in length, or about 21 to about 23 nucleotides in length. In some embodiments, the RNAi construct is therapeutic (i.e., targets a gene or RNA molecule associated with a disease or disorder). The RNAi construct can be any of the RNAi constructs listed in Table 6. In one embodiment, the RNAi construct is the 1851 RNAi construct (sense strand comprising the sequence of SEQ ID NO: 181, and antisense strand comprising the sequence of SEQ ID NO: 192).

[0166] In some embodiments, a chemically modified nucleic acid molecule detected or measured according to the methods of the invention is covalently linked to a ligand, such as any of the ligands described above. In some such embodiments, the ligand comprises a GalNAc moiety. The GalNAc moiety can be a multivalent GalNAc moiety, such as a trivalent or tetravalent GalNAc moiety. In one embodiment, the GalNAc moiety has the structure of Structure 1. In another embodiment, the GalNAc moiety is a TL01 GalNAc moiety. In yet another embodiment, the GalNAc moiety is a TL02 GalNAc moiety. In yet another embodiment, the GalNAc moiety is a TL03 GalNAc moiety.

[0167] The present invention includes a kit for detecting chemically modified nucleic acid molecules in a sample. In one embodiment, the kit includes a capture antibody immobilized on a surface that specifically binds to the chemically modified nucleic acid molecule; a detection reagent comprising a detectable label conjugated to a binding partner that specifically binds to the chemically modified nucleic acid molecule; and instructions for contacting the sample with the immobilized capture antibody and detection reagent and for detecting a signal from the detectable label. Any of the capture antibodies and binding partners described above for use in the methods of the present invention can be the capture antibodies and binding partners included in the kit.

[0168] In certain embodiments of the kits of the invention, the capture antibody is one of the pan-specific antibodies described herein, such as the 14K10 antibody, the 14F4 antibody, or the 5I17 antibody. In one embodiment, the capture antibody comprises a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 14, a CDRL3 of SEQ ID NO: 25, a CDRH1 of SEQ ID NO: 51, a CDRH2 of SEQ ID NO: 64, and a CDRH3 of SEQ ID NO: 77. In a related embodiment, the capture antibody comprises a light chain variable region comprising the sequence of SEQ ID NO: 38 and a heavy chain variable region comprising the sequence of SEQ ID NO: 90. In another related embodiment, the capture antibody comprises a light chain comprising the sequence of SEQ ID NO: 103 and a heavy chain comprising the sequence of SEQ ID NO: 116.

[0169] In some embodiments, the detection reagent of the kit comprises one of the pan-specific antibodies described herein (e.g., the 14K10 antibody, the 14F4 antibody, or the 5I17 antibody) conjugated to a detectable label. In one such embodiment, the antibody conjugated to a detectable label comprises a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 14, a CDRL3 of SEQ ID NO: 25, a CDRH1 of SEQ ID NO: 51, a CDRH2 of SEQ ID NO: 64, and a CDRH3 of SEQ ID NO: 77. In another such embodiment, the antibody conjugated to a detectable label comprises a light chain variable region comprising the sequence of SEQ ID NO: 38 and a heavy chain variable region comprising the sequence of SEQ ID NO: 90. In yet another such embodiment, the antibody conjugated to a detectable label comprises a light chain comprising the sequence of SEQ ID NO: 103 and a heavy chain comprising the sequence of SEQ ID NO: 116.

[0170] In other embodiments, the detection reagent of the kit comprises one of the GalNAc moiety-specific antibodies described herein (e.g., the 14D4, 16I3, 16A22, 17D13, or 18J5 antibodies) conjugated to a detectable label. In one such embodiment, the antibody conjugated to a detectable label comprises a CDRL1 of SEQ ID NO:9, a CDRL2 of SEQ ID NO:21, a CDRL3 of SEQ ID NO:33, a CDRH1 of SEQ ID NO:59, a CDRH2 of SEQ ID NO:72, and a CDRH3 of SEQ ID NO:85. In another further embodiment, the antibody conjugated to a detectable label comprises a light chain variable region comprising the sequence of SEQ ID NO:46 and a heavy chain variable region comprising the sequence of SEQ ID NO:98. In yet another such embodiment, the antibody conjugated to a detectable label comprises a light chain comprising the sequence of SEQ ID NO:111 and a heavy chain comprising the sequence of SEQ ID NO:124.

[0171] The detectable label conjugated to the antibody can be any of the detectable labels described herein. In some embodiments, the detectable label is a fluorophore, a metal nanoparticle, an enzyme, a radiolabel, or an ECL luminophore. In one particular embodiment, the detectable label in the detection reagent is an ECL luminophore, such as a ruthenium complex.

[0172] In certain embodiments of the kits of the invention, the surface comprising the capture antibody can be a bead or particle, a membrane, a tube, a resin, a column, an electrode, or a well in an assay plate (e.g., a well in a microtiter plate). In one particular embodiment, the surface is a well in a microtiter plate.

[0173] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims. [Example]

[0174] Example 1. Generation of monoclonal antibodies against chemically modified RNAi constructs To generate monoclonal antibodies against trivalent N-acetyl-galactosamine (GalNAc)-conjugated RNAi constructs, two different immunogens were designed and synthesized. The first immunogen contained GalNAc-conjugated RNAi construct 1851 conjugated to keyhole limpet hemocyanin (KLH) protein (KLH-1851 immunogen). The second immunogen contained streptavidin nanobeads coated with biotinylated RNAi construct 1851 (beads-1851 immunogen). Specifically, the 1851 RNAi construct was biotinylated at the 5' end of the antisense strand via a PEG-4-carbon-6 linker and mixed with streptavidin nanobeads (Bangs Laboratories, Inc.; catalog number CP01000) with an average diameter of 0.1 μm at a 1:4 (w / w) ratio (RNAi construct:nanobeads) (Figure 1). The second immunogen was designed to present the oligonucleotide antigen in a multivalent form with a specific orientation and spacing. The nanobeads used to generate the second immunogen contained approximately 1600-2000 streptavidin groups per bead, which resulted in a similar density and rigidity of antigen presentation that could not be achieved using conjugation to carrier proteins such as KLH. The size and shape of the nanobeads were also thought to reduce the in vivo clearance rate of the immunogen and increase its exposure to the immune system.

[0175] The nucleotide sequences of the sense and antisense strands of RNAi construct 1851, as well as the structure of the GalNAc moiety, are shown in Table 6 below. The RNAi construct was synthesized using solid-phase phosphoramidite chemistry. Synthesis was performed on a MerMade12 (Bioautomation) instrument. After automated synthesis, a trivalent GalNAc moiety was conjugated to the 5' end of the sense strand. After purification by cation exchange chromatography and desalting by size exclusion chromatography, the sense and antisense strands were mixed in an equimolar ratio, heated to 90°C for 5 minutes, and then allowed to cool to room temperature, allowing the strands to anneal to form a duplex.

[0176] Rabbits were immunized subcutaneously with either of these two immunogens on days 0, 7, 21, 42, 63, 91, 119, and 147. For the first injection, animals received 100–200 μg of immunogen in combination with complete Freund's adjuvant. For all subsequent injections, animals received 25–100 μg of immunogen in combination with incomplete Freund's adjuvant. Starting on day 28, animals were bled 7 and 14 days after each injection to determine antibody titers and antigen specificity by ELISA. At the end of the immunization protocol, spleens were harvested, dissociated, and frozen. Thawed rabbit cells were single-cell sorted into 384-well plates using a FACS Aria III instrument with biotinylated RNAi construct 1851 (detected with streptavidin conjugated to Alexa Fluor 647) and anti-rabbit IgG antibody conjugated to Alexa Fluor 488 to select for IgG-positive antigen-specific cells. Each well of the microtiter plate into which cells were sorted contained RPMI medium supplemented with fetal bovine serum (FBS), 10% activated rabbit splenocyte supernatant, and feeder cell culture. After 7 days of monoclonal culture and B cell expansion, culture supernatants were harvested and then subjected to the assays described in more detail below.

[0177] Culture supernatants from rabbit B cell proliferation were screened for specificity using a standard colorimetric ELISA format. Four biotin-conjugated RNAi constructs were used in this screening assay: (i) the 1851 RNAi construct (with a GalNAc moiety), (ii) the 1851 RNAi construct without a GalNAc moiety, (iii) the 6189 RNAi construct (with a GalNAc moiety), and (iv) the 6189 RNAi construct without a GalNAc moiety. The 6189 RNAi construct has the same GalNAc moiety, chemical modification pattern, and format as the 1851 RNAi construct, but differs in the nucleotide sequences of the sense and antisense strands. The structure of the 6189 RNAi construct is shown in Table 6. For the RNAi construct containing a trivalent GalNAc moiety at the 5' end of the sense strand, biotin was covalently attached to the 5' end of the antisense strand via a PEG 4-carbon 6 linker. For RNAi constructs lacking the GalNAc moiety, biotin was covalently attached to the 5' end of the sense strand via a PEG4-carbon-6 linker. The biotinylated molecules were captured on neutravidin-coated microtiter plates and blocked with 1% nonfat dry milk in phosphate-buffered saline. Culture supernatants were diluted 1:5 and added to each well. Antibody binding was determined using a secondary anti-rabbit IgG Fc antibody conjugated to horseradish peroxidase and TMB substrate (3,3',5,5'-tetramethylbenzidine), followed by quenching with hydrochloric acid. Supernatants were first screened with the 1851 RNAi construct (containing the GalNAc moiety) to confirm antigen-specific binding. Following this primary screening, supernatants were screened with all four antigens described above in secondary screening assays to more specifically confirm the antibody binding properties.

[0178] The results of the ELISA secondary screening assay revealed that these monoclonal antibodies fell into three distinct categories in terms of binding specificity. The first category of antibodies appeared to specifically recognize the GalNAc moiety (Figure 2A). The second category of antibodies appeared to specifically recognize the 1851 RNAi construct, and the third category of antibodies appeared to recognize the double-stranded RNA portion of the construct regardless of sequence (Figure 2B). A summary of the results of this ELISA screening assay, showing the binding specificity for rabbit monoclonal antibodies obtained from three different animals, is shown in Table 5 below.

[0179] [Table 28]

[0180] As shown in Table 5, the bead-based format of the RNAi constructs was much more efficient at generating antigen-specific antibodies than the RNAi constructs conjugated to the KLH carrier protein, because the bead-based immunogen produced approximately five times more antigen-specific antibodies. Furthermore, although the antibodies generated from the bead-1851 immunogen had a variety of binding specificities, the antibodies generated from the KLH-1851 immunogen primarily recognized the GalNAc moiety of the RNAi construct. All but one of the 1851 RNAi construct-specific antibodies was generated using the bead-based format of the antigen. Additionally, robust antibody titers were observed from rabbits immunized with the bead-1851 immunogen as early as 42 days after the first immunization. These antibody titers were 70- to 250-fold higher than the titers observed for rabbits immunized with the KLH-1851 immunogen at the same time point (day 42) (data not shown). Therefore, this immunization method using multivalent nucleic acid-presenting nanobeads as immunogens has proven to be a particularly effective method for producing monoclonal antibodies that bind to various aspects of nucleic acid molecules, which are known to have low immunogenicity.

[0181] Rabbit B cells producing the top antibody binders in each of the three binding specificity categories (e.g., GalNAc moiety-specific, 1851 RNAi construct-specific, and pan-RNAi construct-specific) were lysed, and the antibodies were sequenced and cloned. Monoclonal antibodies were recombinantly expressed in HEK 293-6E cells and purified. The amino acid sequences of selected antibodies in each binding specificity category are shown in Tables 1A-1B (CDRs and variable regions) and Table 2 (full-length heavy and light chains). The nucleotide sequences encoding the antibody variable regions and full chains are shown in Tables 3 and 4, respectively. The purified antibodies were evaluated in a further validation screen to confirm binding specificity. In this validation screen, each antibody was tested at three different concentrations (5, 1, and 0.2 μg / mL) for binding to each of the following antigens attached by biotinylation to streptavidin-coated beads: · (i) 1851 RNAi construct (with GalNAc moiety); · (ii) 1851 RNAi construct without GalNAc moiety; · (iii) 6189 RNAi construct (with GalNAc moiety); (iv) a 6189 RNAi construct that does not have a GalNAc moiety; and (v) β-GalNAc3 dendrimer.

[0182] The β-GalNAc3 dendrimer has the following structure: [ka] It was composed of:

[0183] Antibody binding was detected with an anti-rabbit IgG antibody conjugated to Alexa Fluor 488 and analyzed using an Intellicyt iQue Screener flow cytometer. Regarding pan-RNAi construct-specific antibodies, which are expected to recognize the double-stranded RNA structure of molecules regardless of nucleotide sequence, at a concentration of 1 μg / mL, one of the four recombinant monoclonal antibodies tested (14K10 antibody) bound approximately equally to the four antigens containing double-stranded RNA structures and did not bind to the β-GalNAc3 dendrimer antigen, which lacks an RNA component (Figure 3A). All five GalNAc moiety-specific recombinant monoclonal antibodies specifically recognized all antigens containing a GalNAc moiety and did not bind to the two antigens lacking a GalNAc moiety (Figure 3B). Note that the lack of binding of the 18J5 antibody to the 1851 GalNAc antigen in Figure 3B was the result of a technical error, as binding of this antibody was observed at the other two antibody concentrations (data not shown). Regarding antibodies that appeared specific to the 1851 RNAi construct in the initial screening, five of the six recombinant monoclonal antibodies evaluated were confirmed to specifically bind to the 1851 RNAi construct and not to the 6189 RNAi construct, which differs in the nucleotide sequence of each strand (Figure 3C). The 19F24 and 20P19 antibodies recognized the 1851 RNAi construct only when a GalNAc moiety was present, suggesting that the epitopes of these two antibodies may contain some aspect of the GalNAc moiety in combination with part of the nucleobase structure of the RNAi construct. Interestingly, neither 19F24 nor 20P19 bound to the 6189 RNAi construct or the β-GalNAc3 dendrimer, which contain a GalNAc moiety, arguing that these two antibodies bind exclusively to the GalNAc moiety.

[0184] To further evaluate the binding specificity of the pan-specific and 1851 RNAi construct-specific antibodies, each of these recombinant antibodies was evaluated in a competitive binding assay. Three different RNAi constructs (1851, 1907, and 1418; their sequences are shown in Table 6) were preincubated with each antibody at a 55:1 molar ratio (RNAi construct:antibody) and then exposed to streptavidin beads coated with biotinylated 1851 RNAi construct. The 1851 and 1907 RNAi constructs had identical core nucleotide sequences but different terminal structures: the 1851 construct had two blunt ends, while the 1907 construct had two nucleotide overhangs at the 3' end of each strand. The 1418 construct differed in nucleotide sequence from the 1851 and 1907 constructs, as well as in the GalNAc moiety. Antibody binding to beads coated with the 1851 RNAi construct was detected with an anti-rabbit IgG antibody conjugated to Alexa Fluor 488 and analyzed on an Intellicyt iQue Screener flow cytometer. When the antibody bound to the RNAi construct during preincubation and thus competed with the 1851 RNAi construct-coated beads for antibody binding, a decrease in this binding signal was observed compared to the binding signal to beads in the absence of the RNAi construct during preincubation, and this is represented in the figures as percent inhibition. A higher percent inhibition reflects antibody binding to the competing RNAi construct.

[0185] The results of the competition assays for the pan-specific and 1851 RNAi construct-specific antibodies are shown in Figures 4A and 4B, respectively. Of the three recombinant pan-specific antibodies tested, all three RNAi constructs effectively inhibited the binding of the 14K10 antibody to beads coated with the 1851 RNAi construct, confirming that this antibody recognizes some features of double-stranded RNA structure independent of the nucleotide sequence (Figure 4A). The 5I17 antibody was also inhibited by all three RNAi constructs, but to a lesser extent than the 14K10 antibody. The 14F4 antibody showed significant binding to the 1851 and 1418 RNAi constructs, which differ in nucleotide sequence and GalNAc moiety. However, 14F4 showed weak binding to the 1907 RNAi construct, which has a core nucleotide sequence similar to that of the 1851 RNAi construct.

[0186] As shown in Figure 4B, the binding of all five 1851 RNAi construct-specific antibodies to antigen-coated beads was inhibited by the 1851 RNAi construct but not by the 1418 RNAi construct, which has a different nucleotide sequence. The binding of four of these five antibodies to antigen-coated beads was also not inhibited by the 1907 RNAi construct, suggesting that these four antibodies are specific for the 1851 RNAi construct. The binding of the 20K24 antibody to antigen-coated beads was inhibited not only by the 1907 RNAi construct but also by the 1851 RNAi construct. Because these two constructs share a common core sequence, this result suggests that the 20K24 antibody recognizes a specific contiguous nucleotide sequence common to both constructs.

[0187] The results of the experiments described in this example demonstrate that the described immunization method, which uses multivalent nucleic acid-presenting nanobeads as immunogens, can effectively generate monoclonal antibodies that bind to unique structural features of chemically modified RNAi constructs. Antibodies with three different binding specificities were produced: (i) antibodies that bind to the double-stranded RNA structure of the RNAi construct regardless of nucleotide sequence (i.e., "pan-specific antibodies"), (ii) antibodies that specifically recognize the 1851 RNAi construct, and (iii) antibodies that specifically bind to the GalNAc moiety of the RNAi construct. These antibodies can be used in various assays to detect chemically modified RNAi constructs or their metabolites in biological samples, as described in more detail in Example 2.

[0188] RNAi constructs Table 6 below lists the modifications on the sense and antisense strands, as well as the structure and conjugation site of the GalNAc moiety, for each of the GalNAc-conjugated RNAi constructs used in the experiments described in the Examples. The nucleotide sequences in Table 6 are listed according to the following notation: A, U, G, and C = corresponding ribonucleotides; dT, dA, dG, and dC = corresponding deoxyribonucleotides; a, u, g, and c = corresponding 2'-O-methylribonucleotides; Af, Uf, Gf, and Cf = corresponding 2'-deoxy-2'-fluoro ("2'-fluoro") ribonucleotides; Phos = terminal nucleotide with a monophosphate group at its 5' end; invAb = inverted abasic nucleotide (i.e., if on the 3' end of the strand, adjacent to the 3'-position via a substituent). (3'-3' linkage) or, if on the 5'-end of the strand, linked to an adjacent nucleotide via a substituent at its 5'-position (5'-5' internucleotide linkage); and invdX = inverted deoxyribonucleotide (i.e., a deoxyribonucleotide that is linked to an adjacent nucleotide via a substituent at its 3'-position if on the 3'-end of the strand (3'-3' linkage) or, if on the 5'-end of the strand, linked to an adjacent nucleotide via a substituent at its 5'-position (5'-5' internucleotide linkage)). The insertion of an "s" in a sequence indicates that two adjacent nucleotides are connected by a phosphorothiodiester group (e.g., a phosphorothioate internucleotide linkage). Unless otherwise indicated, all other nucleotides are connected by a 3'-5' phosphodiester group. GalNAc structures are shown in Table 6 below. The TL01 and TL03 GalNAc moieties were conjugated to the sense strand of the RNAi construct by a phosphorothioate linkage, whereas the TL02 GalNAc moiety was conjugated to the sense strand by a phosphodiester linkage.

[0189] [Table 29]

[0190] The structure of TL01 GalNAc moiety is [ka] where the arrow indicates the site of conjugation to the nucleic acid molecule by a phosphorothiodiester bond.

[0191] The structure of the TL02 GalNAc moiety is [ka] where the arrow indicates the site of conjugation to the nucleic acid molecule via a phosphodiester bond. "Ac" represents an acetyl group.

[0192] TL03 The structure of the GalNAc moiety is [ka] where the arrow indicates the site of conjugation to the nucleic acid molecule via a phosphorothiodiester bond. "Ac" represents an acetyl group.

[0193] Example 2. Immunoassay for detection of chemically modified RNAi constructs Three different immunoassays using one or more of the antibodies described in Example 1 were developed to detect chemically modified RNAi constructs in biological samples such as serum or tissue homogenates.

[0194] All drug assays The first such assay (referred to as the whole-drug assay) uses the pan-RNAi construct-specific antibody (e.g., 14K10 antibody) described in Example 1 as both the capture and detection reagent to detect and quantitate chemically modified RNAi constructs in various samples, regardless of nucleotide sequence. One embodiment of this whole-drug assay is shown schematically in Figure 5A. In this embodiment, the 14K10 pan-RNAi construct-specific antibody was biotinylated and placed into the wells of a streptavidin-coated gold microtiter plate (Meso Scale Diagnostics (MSD) GOLD™ Streptavidin Plate) at a concentration of 1 μg / mL in blocking buffer (5% nonfat dry milk in Tris-buffered saline (Blocker™ BLOTTO, ThermoFisher Scientific)), and the plate was shaken for 30 minutes at room temperature. The plate was then washed with wash buffer (imidazole-buffered saline and Tween 20; supplied by KPL Inc. as a 20X wash solution concentrate). Biological samples (serum, plasma, or tissue homogenate) were diluted 1:20 in blocking buffer, added to the wells of the microtiter plate, and incubated at room temperature for 1 hour. The plate was washed again with wash buffer. Ruthenium-labeled 14K10 pan-RNAi construct-specific antibody was then added to each sample in blocking buffer at a concentration of 0.5 μg / mL and incubated at room temperature for 1 hour. After washing the plate with wash buffer, the signal from the ruthenium label was read using an MSD electrochemiluminescence reader (MSD Sector 5 600) and MSD Read Buffer T containing detergent.

[0195] Intact drug assay for GalNAc-conjugated RNAi constructs Another immunoassay incorporating the antibodies of the present invention can be used to detect and quantitate intact GalNAc-conjugated RNAi constructs (i.e., with the GalNAc moiety still conjugated to the RNA strand) in various biological samples. In this assay format, a pan-RNAi construct-specific antibody, such as the 14K10 antibody, is used as the capture reagent, and a GalNAc moiety-specific antibody, such as the 14D4 antibody, is used as the detection reagent. This assay format is particularly useful for pharmacokinetic and metabolic studies to track when and where the GalNAc moiety is removed from chemically modified RNAi constructs. One embodiment of this intact GalNAc-RNAi construct assay is shown schematically in Figure 5B.

[0196] The assay procedure is similar to that described above for the whole drug assay, except that the GalNAc moiety-specific antibody of the present invention is used as the detection antibody. Specifically, biotinylated 14K10 pan-specific antibody was added to the wells of a streptavidin-coated gold microtiter plate (MSD GOLD™ Streptavidin Plate) at a concentration of 1 μg / mL in blocking buffer, and the plate was shaken at room temperature for 30 minutes. The plate was then washed with washing buffer. Biological samples (serum, plasma, or tissue homogenate) were diluted 1:20 in blocking buffer, added to the wells of the microtiter plate, and incubated at room temperature for 1 hour. The plate was washed again with washing buffer. 14D4 antibody (which specifically binds to the GalNAc moiety of the chemically modified RNAi construct) was labeled with ruthenium and added to each sample at a concentration of 0.5 μg / mL in blocking buffer, and incubated at room temperature for 1 hour. After washing the plate with wash buffer, the signal from the ruthenium label was read using an MSD electrochemiluminescence reader (MSD Sector 5 600) and MSD Read Buffer T containing detergent.

[0197] Both assay formats described above were evaluated with four different GalNAc-conjugated RNAi constructs (construct numbers 16081, 16082, 16083, and 16084) to determine the assay's ability to detect both total and GalNAc-intact drugs in human serum. The sequences of the GalNAc-conjugated RNAi constructs are listed in Table 6 above. Each of the GalNAc-conjugated RNAi constructs was diluted in human serum to generate an 11-point standard curve (100 nM to 1.7 pM). The results of these two assays are shown in Figure 6. The limit of detection (LOD) of the intact drug assay for all four constructs was less than 1.7 pM, while the LOD of the whole drug assay ranged from approximately 15 to 45 pM for the various constructs. These results demonstrate that both assay formats are capable of detecting picomolar concentrations of RNAi constructs in biological matrices, regardless of the nucleotide sequence of the RNAi construct.

[0198] The GalNAc intact assay was further evaluated for its ability to detect additional GalNAc-conjugated RNAi constructs with different nucleotide sequences in matrices from three different species. Specifically, GalNAc-conjugated RNAi constructs 1907, 7213, 8172, and 10927 (these sequences are shown in Table 6 above) were diluted in serum from human, cynomolgus monkey, or rat to generate an 11-point standard curve (5000 ng / mL to 84 pg / mL). The assay was performed as described above. The results of this assay are shown in Figures 7A-7D and demonstrate that the assay can detect various GalNAc-conjugated RNAi constructs in serum samples from all three different species, even though these RNAi constructs have different nucleotide sequences and chemical modification patterns. Furthermore, construct 1907 gave similar results to the other three RNAi constructs in this assay, even though the 1907 construct had a different GalNAc moiety.

[0199] To explore the strand specificity of the GalNAc intact assay, two different GalNAc-conjugated RNAi constructs and their separate sense and antisense strands were tested in mouse serum at different concentrations. The sequences and structures of the GalNAc moieties are shown in Table 6 above. The structure of the GalNAc moiety in the 1851 RNAi construct was different from that in the 8172 RNAi construct. The sequences of the sense and antisense strands of these RNAi constructs were also different. The sense strand used in the experiment was conjugated to a GalNAc moiety, while the antisense strand was not. As shown in Figure 8, the results of this experiment demonstrate that the GalNAc intact assay is specific for double-stranded constructs at concentrations below 1 nM, since single-stranded GalNAc-conjugated sense strands were only detectable at concentrations above this threshold. As expected, no signal was observed in this assay for the single-stranded antisense strand lacking the GalNAc moiety. These results further demonstrate the broad applicability of this assay in detecting intact GalNAc-conjugated double-stranded RNA molecules, regardless of the nucleotide sequence, chemical modification pattern, and structure of the GalNAc moiety.

[0200] Any of the 1851 RNAi construct-specific antibodies described in Example 1 (e.g., antibodies 17K13, 17F22, 19F24, 20P19, and 20K24) can be substituted for a pan-RNAi construct-specific antibody in the whole drug or intact GalNAc assays described above and shown in Figures 5A and 5B to create an assay specific for the 1851 RNAi construct. Such an assay is particularly useful for assessing the pharmacokinetic profile, distribution, and / or metabolism of the 1851 RNAi construct in subjects receiving the 1851 RNAi construct.

[0201] Intact drug assay for antibody-RNAi construct conjugate molecules The antibodies of the present invention were incorporated into a third immunoassay to detect and quantify intact antibody-RNAi construct conjugate molecules in biological samples. In this assay format, a pan-RNAi construct-specific antibody of the present invention (e.g., the 14K10 antibody) is used to capture the antibody-RNAi construct conjugate molecule in the sample solution by binding to the RNA component of the conjugate molecule, and the conjugate is immobilized on a solid surface. The conjugate is then detected and quantified using a labeled binding partner that specifically recognizes the antibody component of the conjugate molecule (e.g., an antibody that specifically binds to the human Fc region). This assay format is shown schematically in Figure 5C.

[0202] Biotinylated 14K10 pan-specific RNAi construct antibody was added to the wells of a streptavidin-coated gold microtiter plate (MSD GOLD™ Streptavidin Plate) at a concentration of 1 μg / mL in blocking buffer (5% nonfat dry milk in Tris-buffered saline), and the plate was shaken for 30 minutes at room temperature. The plate was then washed with wash buffer (imidazole-buffered saline and Tween 20). Biological samples (serum, plasma, or tissue homogenate) were diluted 1:20 in blocking buffer, added to the wells of the microtiter plate, and incubated for 1 hour at room temperature. The plate was washed again with wash buffer. A ruthenium-labeled mouse monoclonal antibody directed against the Fc region of human immunoglobulin (anti-human Fc antibody; 0.5 μg / mL) was then added to each sample in blocking buffer and incubated for 1 hour at room temperature. After washing the plate with wash buffer, the signal from the ruthenium label was read using an MSD Sector 5 600 electrochemiluminescence reader and MSD Read Buffer T containing surfactant.

[0203] Antibody-RNAi construct conjugate molecules were prepared by covalently attaching an RNAi construct containing a sense strand with the sequence of SEQ ID NO:190 and an antisense strand with the sequence of SEQ ID NO:201 to a human monoclonal antibody (mAb) directed against a cell surface receptor. A cysteine ​​residue was substituted at position D70 in the light chain or position E272 or T359 in the heavy chain of the mAb (amino acid position numbering according to the EU numbering scheme) to create specific conjugation sites for the RNAi construct. The mAb containing the cysteine ​​substitution (cys mAb) was incubated with a solution of 2.5 mM cystamine and 2.5 mM cysteamine in 40 mM HEPES buffer, pH 7.5-8.5, for 15-20 hours at room temperature, followed by purification to obtain a bis-cysteamine-capped cys mAb. The sense strand of this RNAi construct contained a homoserine-aminohexanoic acid modification at its 5' end, which was further functionalized with a bromoacetyl group using succinimidyl bromoacetate. The bis-cysteamine-capped cys mAb intermediate was partially reduced using tris(2-carboxyethyl)phosphine (TCEP) or triphenylphosphine-3,3',3''-trisulfonic acid trisodium salt (TPPTS). The partially reduced cys mAb was then oxidized with dehydroascorbic acid (DHAA) at room temperature until only traces of reduced mAb species were observed. The bromoacetyl-RNAi construct was then added to the reaction mixture, and alkylation was carried out at room temperature for 15–48 h. Prior to purification, 10 equivalents of N-ethylmaleimide was added to the reaction mixture to cap any unreacted cysteines. Anion exchange chromatography was used to separate antibody-RNAi construct conjugate molecules with RNA-to-antibody ratios (RAR) of 1 and 2. A description of each conjugate molecule is provided in Table 7 below.

[0204] [Table 30]

[0205] Each of the antibody-RNAi construct conjugate molecules in Table 7 was diluted in mouse serum to generate an 11-point standard curve (500 nM to 8.5 pM) and measured using the intact drug assay for antibody-RNAi construct conjugate molecules described above and shown in Figure 5C (designated assay 1 in Figure 9). As a negative control, the conjugate molecules were also evaluated in an immunoassay in which the 14D10 pan-specific RNAi construct antibody was replaced with the 14D4 anti-GalNAc partial antibody as the capture reagent in the assay shown in Figure 5C (i.e., capture by 14D4 Ab and detection by anti-human Fc antibody; designated assay 2 in Figure 9). The results of these two assays are shown in Figure 9. All five conjugates were detectable in mouse serum by the intact antibody-construct conjugate assay (capture by 14K10 Ab and detection by anti-human Fc Ab), and the assay was linear over most of the concentration range tested. Neither the conjugation site of the RNAi construct to the antibody nor the number of RNAi constructs conjugated to the antibody (i.e., RAR1 vs. RAR2) appeared to affect the performance of this assay. As expected, the antibody-RNAi construct conjugate molecules did not contain a GalNAc moiety and therefore did not produce a signal in an immunoassay using an anti-GANAc antibody as the capture reagent (captured by 14D4 Ab and detected by an anti-human Fc antibody).

[0206] To demonstrate one use of the immunoassay of the present invention, serum and tissue samples from mice treated with mAb-RNAi construct conjugate molecules were analyzed using the intact antibody-RNAi construct assay described above and shown in Figure 5C. C57B1 / 6 wild-type mice were intravenously injected with either the 15722 or 15723 mAb-RNAi conjugate molecules described in Table 7 at a dose of 12 mg / kg. The only difference between the 15722 and 15723 conjugates was the number of RNAi constructs conjugated to the antibody: the 15722 conjugate contained one RNAi construct (i.e., RAR1), while the 15723 conjugate contained two RNAi construct molecules (i.e., RAR2). Serum was collected from the animals at 5, 15, and 30 minutes, and 1, 2, 4, 8, 24, 96, 360, and 528 hours after administration of the conjugated molecules. Tissues, including liver, spleen, and kidney, were collected from the animals at 5, 15 minutes, and 4, 8, 24, 96, 360, and 528 hours after administration of the conjugated molecules.

[0207] The amount of intact mAb-RNAi construct conjugate molecules in serum and tissue samples collected from animals treated with the conjugate molecules was measured using the intact assay method for antibody-RNAi construct conjugate molecules described above. Samples were diluted 1:20 in blocking buffer and placed in wells of a streptavidin-coated gold microtiter plate containing biotinylated 14K10 pan-specific RNAi construct antibody and incubated at room temperature for 1 hour. After washing the plate with the wash buffer described above, captured conjugate molecules were detected using a ruthenium-labeled anti-human Fc mAb, and the electrochemiluminescence signal was read using an MSD Sector 5 600 electrochemiluminescence reader. An anti-Fc / anti-Fc sandwich ELISA assay was used to measure total conjugate molecules in serum and liver samples. In the whole conjugate molecule ELISA assay, a first biotinylated anti-human Fc antibody was used to capture any human mAb in the sample, and a second ruthenium-labeled anti-human Fc antibody that binds to a different epitope than the first anti-human Fc antibody was used to detect the captured human mAb. The whole conjugate molecule assay detects naked human mAb (i.e., mAb without an RNAi construct) and mAbs to which one or two RNAi constructs are linked.

[0208] The results of the analysis of serum samples are shown in Figure 10A, and the results of the analysis of spleen, liver, and kidney samples are shown in Figures 10B-10D, respectively. Both conjugate molecules remained intact in the blood compartment for extended periods of time (Figure 10A). These conjugate molecules also remained intact in the spleen for extended periods of time, with the intact form of the RAR1 conjugate persisting longer than the intact form of the RAR2 conjugate (Figure 10B). These conjugate molecules rapidly lost their RNAi construct components in the liver and kidney (Figures 10C and 10D). These experimental results demonstrate that the assay method and antibodies of the present invention can be used in pharmacokinetic and drug metabolism studies to evaluate the clearance profile and metabolic degradation of antibody-RNAi construct conjugate molecules in vivo.

[0209] All publications, patents, and patent applications discussed and cited herein are incorporated herein by reference in their entirety. It is understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the appended claims.

[0210] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A method for producing a monoclonal antibody that specifically binds to a chemically modified nucleic acid molecule, comprising: (a) conjugating a plurality of nucleic acid molecules to beads to form an immunogen, each of the nucleic acid molecules comprising one or more modified nucleotides; (b) administering the immunogen to an animal; (c) obtaining splenocytes from the immunized animal; (d) selecting splenocytes that are IgG positive and bind to the chemically modified nucleic acid molecule, thereby isolating cells that produce antigen-specific antibodies; (e) plating the antigen-specific antibody-producing cells in single cell culture; and (f) isolating the monoclonal antibody from the single cell culture. A method comprising:

2. The method of claim 1 , wherein the beads have an average diameter of at least 70 nm.

3. The method of claim 1, wherein the beads have an average diameter of about 50 nm to about 2 μm.

4. The method of any one of claims 1 to 3, wherein the nucleic acid molecule is double-stranded.

5. The method of claim 4 , wherein the nucleic acid molecule is an RNAi construct comprising a sense strand and an antisense strand.

6. 6. The method of claim 5, wherein the sense strand and the antisense strand are each independently about 19 to about 30 nucleotides in length.

7. The method of any one of claims 1 to 3, wherein the nucleic acid molecule is single-stranded.

8. 8. The method of any one of claims 1 to 7, wherein the nucleic acid molecules each comprise one or more modified nucleotides selected from 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), or combinations thereof.

9. The method of any one of claims 1 to 8, wherein each of the nucleic acid molecules is covalently linked to a carbohydrate-containing ligand.

10. 10. The method of claim 9, wherein the carbohydrate is galactose, galactosamine, or N-acetyl-galactosamine.

11. The method of claim 10, wherein the ligand comprises a multivalent galactose moiety or a multivalent N-acetyl-galactosamine moiety.

12. The method of claim 11, wherein the multivalent galactose or N-acetyl-galactosamine moiety is trivalent or tetravalent.

13. The method of any one of claims 1 to 12, wherein the animal to which the immunogen is administered is a rabbit.

14. A monoclonal antibody produced by the method according to any one of claims 1 to 13.

15. An isolated monoclonal antibody that specifically binds to a chemically modified nucleic acid molecule independent of nucleotide sequence, said monoclonal antibody comprising: (i) a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3; and (ii) a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3; (a) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 1, 14, and 25, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 51, 64, and 77, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 2, 15, and 26, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 52, 65, and 78, respectively; or (c) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 3, 16, and 27, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 53, 66, and 79, respectively; Isolated monoclonal antibodies.

16. The monoclonal antibody is (a) a light chain variable region comprising the sequence of SEQ ID NO: 38 and a heavy chain variable region comprising the sequence of SEQ ID NO: 90; (b) a light chain variable region comprising the sequence of SEQ ID NO: 39 and a heavy chain variable region comprising the sequence of SEQ ID NO: 91; or (c) a light chain variable region comprising the sequence of SEQ ID NO: 40 and a heavy chain variable region comprising the sequence of SEQ ID NO: 92; 16. The isolated monoclonal antibody of claim 15.

17. 1. An isolated monoclonal antibody that sequence-specifically binds to an RNAi construct comprising the nucleotide sequence of SEQ ID NO: 192, said monoclonal antibody comprising: (i) a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3; and (ii) a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3; (a) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 4, 17, and 28, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 54, 67, and 80, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 18, and 29, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 55, 68, and 81, respectively; (c) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 6, 19, and 30, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 56, 69, and 82, respectively; (d) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 7, 20, and 31, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 57, 70, and 83, respectively; or (e) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 8, 17, and 32, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 58, 71, and 84, respectively; Isolated monoclonal antibodies.

18. The monoclonal antibody is (a) a light chain variable region comprising the sequence of SEQ ID NO: 41 and a heavy chain variable region comprising the sequence of SEQ ID NO: 93; (b) a light chain variable region comprising the sequence of SEQ ID NO: 42 and a heavy chain variable region comprising the sequence of SEQ ID NO: 94; (c) a light chain variable region comprising the sequence of SEQ ID NO: 43 and a heavy chain variable region comprising the sequence of SEQ ID NO: 95; (d) a light chain variable region comprising the sequence of SEQ ID NO: 44 and a heavy chain variable region comprising the sequence of SEQ ID NO: 96; or (e) a light chain variable region comprising the sequence of SEQ ID NO: 45 and a heavy chain variable region comprising the sequence of SEQ ID NO: 97; 18. The isolated monoclonal antibody of claim 17.

19. 1. An isolated monoclonal antibody that specifically binds to an N-acetyl-galactosamine (GalNAc) moiety, said monoclonal antibody comprising: (i) a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3; and (ii) a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3; (a) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 9, 21, and 33, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 59, 72, and 85, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 10, 22, and 34, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 60, 73, and 86, respectively; (c) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 11, 19, and 35, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 61, 74, and 87, respectively; (d) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 12, 23, and 36, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 62, 75, and 88, respectively; or (e) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 13, 24, and 37, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 63, 76, and 89, respectively; Isolated monoclonal antibodies.

20. The monoclonal antibody is (a) a light chain variable region comprising the sequence of SEQ ID NO: 46 and a heavy chain variable region comprising the sequence of SEQ ID NO: 98; (b) a light chain variable region comprising the sequence of SEQ ID NO: 47 and a heavy chain variable region comprising the sequence of SEQ ID NO: 99; (c) a light chain variable region comprising the sequence of SEQ ID NO: 48 and a heavy chain variable region comprising the sequence of SEQ ID NO: 100; (d) a light chain variable region comprising the sequence of SEQ ID NO: 49 and a heavy chain variable region comprising the sequence of SEQ ID NO: 101; or (e) a light chain variable region comprising the sequence of SEQ ID NO: 50 and a heavy chain variable region comprising the sequence of SEQ ID NO: 102; 20. The isolated monoclonal antibody of claim 19.

21. The monoclonal antibody of any one of claims 14 to 20, wherein the antibody is conjugated to a detectable label.

22. 22. The monoclonal antibody of claim 21, wherein the detectable label is a fluorophore, a metal nanoparticle, an enzyme, a radiolabel, or an electrochemiluminescence (ECL) luminophore.

23. The monoclonal antibody according to any one of claims 15 to 22, wherein the monoclonal antibody is a rabbit IgG antibody.

24. 1. A method for detecting chemically modified nucleic acid molecules in a sample, comprising: (a) providing a surface comprising a capture antibody that specifically binds to the chemically modified nucleic acid molecule, wherein the capture antibody is any one of the monoclonal antibodies of claims 15 or 16; (b) contacting the surface with the sample under conditions that allow the chemically modified nucleic acid molecule, if present in the sample, to bind to the capture antibody on the surface; (c) contacting the surface with a detection reagent, wherein the detection reagent comprises a detectable label conjugated to a binding partner that specifically binds to the chemically modified nucleic acid molecule; and (d) detecting a signal from the detectable label. A method comprising:

25. 25. The method of claim 24, wherein the binding partner is a second antibody that specifically binds to the chemically modified nucleic acid molecule.

26. 26. The method of claim 25, wherein the second antibody is any one of the monoclonal antibodies of claim 15 or 16.

27. 25. The method of claim 24, wherein the chemically modified nucleic acid molecule is an 1851 RNAi construct and the binding partner is any one of the monoclonal antibodies of claim 17 or 18.

28. The method of claim 24, wherein the chemically modified nucleic acid molecule is covalently linked to a ligand containing a GalNAc moiety, and the binding partner is any one of the monoclonal antibodies described in claim 19 or 20.

29. 29. The method of claim 28, wherein the chemically modified nucleic acid molecule is covalently linked to a ligand comprising a multivalent GalNAc moiety.

30. 30. The method of claim 29, wherein the multivalent GalNAc moiety is trivalent or tetravalent.

31. 25. The method of claim 24, wherein the chemically modified nucleic acid molecule is conjugated to an antibody and the binding partner is a target antigen of the antibody, an anti-Fc region antibody, or an anti-idiotype antibody.

32. 25. The method of claim 24, wherein the chemically modified nucleic acid molecule is conjugated to a protein and the binding partner is an antibody that specifically binds to the protein.

33. 33. The method of any one of claims 24 to 32, wherein the detectable label is a fluorophore, a metal nanoparticle, an enzyme, a radiolabel, or an ECL luminophore.

34. 34. The method of claim 33, wherein the ECL luminophore is a ruthenium complex.

35. 35. The method of any one of claims 24 to 34, wherein the chemically modified nucleic acid molecule comprises one or more modified nucleotides selected from 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNAs, or a combination thereof.

36. The method according to any one of claims 24 to 35, wherein the chemically modified nucleic acid molecule is an RNAi construct comprising a sense strand and an antisense strand.

37. 37. The method of claim 36, wherein the sense strand and the antisense strand are each independently about 19 to about 30 nucleotides in length.

38. The method of any one of claims 24 to 37, wherein the sample is serum, plasma, or tissue homogenate.

39. 39. The method of any one of claims 24 to 38, wherein the sample is obtained from the subject before, during, or after treatment with the chemically modified nucleic acid molecule.

40. 1. A kit for detecting chemically modified nucleic acid molecules in a sample, comprising: (a) a capture antibody immobilized on a surface, the capture antibody specifically binding to the chemically modified nucleic acid molecule, and being any one of the monoclonal antibodies of claim 15 or 16; (b) a detection reagent comprising a detectable label conjugated to a binding partner that specifically binds to the chemically modified nucleic acid molecule; and (c) instructions for contacting the sample with the immobilized capture antibody and detection reagent, and instructions for detecting a signal from the detectable label. Kit including:

41. 41. The kit of claim 40, wherein the capture antibody comprises (i) a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, and (ii) a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 1, 14, and 25, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 51, 64, and 77, respectively.

42. 42. The kit of claim 40 or 41, wherein the detection reagent comprises a detectable label conjugated to a monoclonal antibody, the monoclonal antibody comprising (i) a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, and (ii) a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 1, 14, and 25, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 51, 64, and 77, respectively.

43. 42. The kit of claim 40 or 41, wherein the detection reagent comprises a detectable label conjugated to a monoclonal antibody, the monoclonal antibody comprising (i) a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, and (ii) a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 9, 21, and 33, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 59, 72, and 85, respectively.

44. 44. The kit of any one of claims 40 to 43, wherein the detectable label is a fluorophore, a metal nanoparticle, an enzyme, a radiolabel, or an ECL luminophore.

45. 45. The kit of claim 44, wherein the ECL luminophore is a ruthenium complex.

46. 46. ​​The kit of any one of claims 40 to 45, wherein the surface is a well of a microtiter plate.

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