Monoclonal antibody panels for detection of chemically modified oligonucleotides

Abstract

The present disclosure relates to monoclonal antibodies specifically recognizing a nucleic acid chemical modification and use of the monoclonal antibodies to detect, quantify, enrich or purify oligonucleotides comprising the chemical modification.

Description

[0001] MONOCLONAL ANTIBODY PANELS FOR DETECTION OF CHEMICALLY MODIFIED OLIGONUCLEOTIDES

[0002] PRIORITY

[0003] This Application claims the benefit of, and priority to, US Application No. 63 / 547,067, filed November 2, 2023, which is hereby incorporated by reference in its entirety.

[0004] BACKGROUND

[0005] The development of oligonucleotide therapeutics as drugs to affect various diseases is hindered by the lack of available analytical tools. Analytical assays are essential to satisfy the requirements of regulatory agencies to ensure the safety and performance of these drugs, for example by providing information on the adsorption, distribution, metabolism, excretion, and toxicology (ADMET) of the drug in pre-clinical and non-clinical studies, patient-based clinical trials, ongoing quality assurance of manufactured drug, and patient stratification and response studies. While custom-made antibodies have been developed for specific oligonucleotide therapeutics, the resultant antibody likely recognizes a plethora of epitopes such as the nucleic acid sequence itself, conformation, modification, other possible epitopes and their combinations. Indeed, since the size of a typical antibody epitope is generally equivalent to 5-15 amino acids or 3-4 sugar residues, a typical epitope is expected to encompass more than a single chemical modification. Kaiser, G. (2008), 12,2: Antigens and Epitopes, in Microbiology, LIBRETEXTS Biology, Community College of Baltimore County (CCBC). Therefore, the custom-made antibodies do not have utility across oligonucleotide drugs.

[0006] Therefore, reagents of general utility that recognize specific chemical modifications in a largely sequence-independent manner, and use of such antibodies to assay oligonucleotide drugs, are desired.

[0007] Further, chemical modifications introduced while preparing oligonucleotide drugs create chiral centers. For example, phosphorothioate (PS), which protects against exonuclease degradation in vivo, is a chiral entity that has right-handed (R) and left-handed (S) enantiomers. The chiral make-up of oligonucleotide drugs are difficult to determine with available analytical assays. Further, the presence and location of specific enantiomers may affect the biological activity of the drug. See, e.g., Jahns et al.. Chirality matters: stereo- defined phosphorothioate linkages at the termini of small interfering RNAs improve pharmacology in vivo. Nucleic Acids Research 50(3): 1221-1240 (2022). However, the introduction and retention of the chiral information during synthesis remains a very challenging task. See, e.g., Lemouzy et al., Chiral phosphorus compounds by catalytic asymmetric C-P coupling. Nat Catal 5, 8-9 (2022).

[0008] Therefore, methods of characterizing stereochemistry of oligonucleotide therapeutics, and methods for stereospecific synthesis of oligonucleotide therapeutics, are needed.

[0009] SUMMARY OF THE DISCLOSURE

[0010] The present disclosure provides monoclonal antibodies that specifically recognize chemical modifications of oligonucleotide therapeutics (ONTs). In embodiments, the antibodies recognize chemically-modified oligonucleotides largely independent of the nucleobase sequence or strandedness. Aspects of the present disclosure relate to monoclonal antibodies specifically recognizing a nucleic acid chemical modification, methods for producing the monoclonal antibodies, and use of the monoclonal antibodies to detect, quantify, enrich or purify oligonucleotides comprising the chemical modification.

[0011] In an aspect, the disclosure provides a method for making a monoclonal antibody specifically recognizing a nucleic acid chemical modification. The method comprises providing an immunogen comprising an oligonucleotide or nucleotide containing the chemical modification, where the oligonucleotide or nucleotide is conjugated to a carrier protein to illicit an immune response in a mouse. The method further comprises immunizing a mouse with the immunogen, and screening antibody-producing B lymphocytes isolated from the mouse and / or hybridomas generated therefrom against one or more positive and negative control oligonucleotides. In embodiments, the method further comprises selecting one or more monoclonal antibodies that display binding specificity for the positive control oligonucleotide(s) over the negative control oligonucleotides.

[0012] In embodiments, the chemical modification comprises a nucleotide backbone modification. In embodiments, the backbone modification can include one or a mix of enantiomers of the backbone modification. In embodiments, the backbone modification is phosphorothioate intemucleotide linkages. Other backbone modification include phosphoacetate linkage (PACE), methylphosphonate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, phosphoramidite, phosphorodiamidate, phosphorodithioate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, thiomorpholino, peptide nucleic acid, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, or sulfone intemucleoside linkages.

[0013] In embodiments, the chemical modification comprises a 2' sugar modification of DNA or RNA nucleotides. In embodiments, the 2' sugar modification is selected from 2'-0 methoxyethyl (MOE), 2'-0 methyl (2'-methoxy), 2'-0 ethyl, 2'-Fluoro, 2'-Methyl, and 2'- Ethyl. In embodiments, the 2' sugar modification is a bridged nucleotide, such as a 2' to 4' bridge. In embodiments, the bridged nucleotide has a methylene bridge (LNA), ethylene bridge (ENA), or a constrained ethyl bridge (cEt). In embodiments, the immunogen comprises a nucleotide or oligonucleotide with the 2' sugar modification conjugated to the carrier protein.

[0014] In embodiments, the chemical modification comprises a nucleobase modification. In embodiments, the chemical modification is 5-methylcytosine, pseudouridine, or Nl- methylpseudouridine. In embodiments, the immunogen comprises a nucleotide or oligonucleotide with the nucleobase modification conjugated to the carrier protein.

[0015] In embodiments, the oligonucleotide conjugated to the carrier protein is an antisense oligonucleotide, and is optionally a gapmer, or is a PNA, morpholino, or thiomorpholino oligonucleotide. In still other embodiments, the oligonucleotide conjugated to the immunogen is an siRNA, miRNA, or aptamer. In still other embodiments, the oligonucleotide is an mRNA. In various embodiments, the oligonucleotide is single stranded or double stranded, and optionally comprises one or more of RNA:RNA, RNA-DNA, and DNA:DNA hydrogen-bonded nucleotides, which are optionally Watson-Crick base-pairing.

[0016] In embodiments, the carrier protein is keyhole limpet hemocyanin (KLH). For example, the chemically-modified nucleotide or oligonucleotide is conjugated to the carrier protein using a linking reagent. An exemplary bivalent linking reagent is m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). In embodiments, the linking reagent conjugates to free primary amines of the carrier protein. In embodiments, the linking reagent conjugates to a reduced sulfhydryl group on the oligonucleotide or nucleotide, which is optionally on a terminus of a chemically-modified oligonucleotide. In embodiments, the oli onucleotide is conjugated to the carrier protein at a ratio of from about 20: 1 to about 1 : 1, such as from about 12: 1 to about 2: 1, or from about 10: 1 to about 4: 1.

[0017] In embodiments, the positive control oligonucleotides comprise a panel with varying amount of the chemical modification, and the negative control(s) lack the chemical modification. In embodiments, the positive control oligonucleotides comprise different nucleotide sequences, and / or contain the chemical modification at different positions. In embodiments, the negative control oligonucleotide(s) comprise other chemical modifications not included in the immunogen or no chemical modification, and wherein at least one negative control has the same nucleobase sequence as the oligonucleotide used for the immunogen. Positive and negative control oligonucleotides can be screened using any process, such as but not limited to ELISA and dot blot assays. In embodiments, antibodies are selected with the desired specificity for positive controls over negative controls. Antibodies can be further characterized by one or more of: antibody isotype, amino acid sequence analysis, epitope characterization (including characterization of stereochemistry), binding affinity for positive controls, and binding affinity for negative controls.

[0018] In various embodiments, the candidate monoclonal antibodies (e.g., selected monoclonal antibodies) recognize a single chemical modification in a substantially sequence-non-specific manner.

[0019] In various embodiments, the candidate monoclonal antibodies (e.g., selected monoclonal antibodies) recognize a particular enantiomer of the chemical modification, such as a phosphorothioate linkage. In embodiments, the candidate monoclonal antibody specifically or preferentially recognizes a right-handed (R) enantiomer of phosphorothioate linkage. In embodiments, the candidate monoclonal antibody specifically or preferentially recognizes a left-handed (S) enantiomer of phosphorothioate linkage.

[0020] In embodiments, antibodies are cloned, and produced in any desired format, including but not limited to full monoclonal antibody, single chain variable fragment (ScFv), heavy chain only antibody, as well as antigen binding fragments (e.g., Fab or F(ab')2).

[0021] In an aspect, the present disclosure provides monoclonal antibodies prepared by the methods described herein.

[0022] In other aspects, the disclosure provides a method for detecting a chemically- modified oligonucleotide. The method comprises providing a sample suspected of comprising the chemically-modified oligonucleotide, and detecting the presence or absence or amount of the chemically modified oligonucleotide in the sample by contacting the sample with an antibody of this disclosure (e.g., recognizing the chemical modification). The presence, absence, or amount of the chemically-modified oligonucleotide may be detected using an assay selected from immunohistochemistry, immunofluorescence microscopy, ELISA, Western blot, dot blot, flow cytometry / FACS analysis, and immunoprecipitation. In embodiments, the sample is a test sample from a subject that was administered the chemically-modified oligonucleotide, such as a biological fluid or organ or tissue samples. In such embodiments, the method is useful for characterizing the pharmacokinetics or biodistribution of the chemically-modified oligonucleotide. In other embodiments, the sample is a manufactured batch of the chemically-modified oligonucleotide. In such embodiments, the method is useful for quality control.

[0023] In aspects, the present disclosure provides a method of quantifying or assaying phosphorothioate (PS) stereochemistry in a PS-modified oligonucleotide. In these aspects, the method comprises contacting the oligonucleotide with a monoclonal antibody against a PS modification of this disclosure, and quantifying the binding of the oligonucleotide to the candidate monoclonal antibody. In embodiments, binding to the chemically-modified oligonucleotide is quantified using an assay selected from immunohistochemistry, immunofluorescence microscopy, ELISA, southern blot, northern blot, flow cytometry / FACS analysis, and immunoprecipitation.

[0024] In aspects, the present disclosure provides a method of enriching or purifying an oligonucleotide comprising an enantiomer of phosphorothioate (PS) linkage. In such embodiments, the method comprises contacting a sample comprising the oligonucleotide with a monoclonal antibody against a PS modification of this disclosure, to produce a complex of the monoclonal antibody and the oligonucleotide. Bound complexes can be sequestered or removed, or bound oligonucleotide may be eluting or recovered from the complex. In embodiments, the monoclonal antibody is bound directly or indirectly to a solid support such as but not limited to a bead (e.g., a magnetic bead), a chromatography column, or any other support.

[0025] In an aspect, the present disclosure provides a method of stereospecific oligonucleotide synthesis with respect to phosphorothioate (PS) linkage. In this aspect, the method comprises contacting a synthetic intermediate of the oligonucleotide with a monoclonal antibody recognizing an enantiomer of a PS linkage (e.g., according to this disclosure) to produce a complex of the monoclonal antibody and the intermediate comprising a specific enantiomer of PS linkage. The intermediate bound to the monoclonal antibody can be eluted (recovered) or otherwise sequestered (e.g., removed). In embodiments, the monoclonal antibody is bound directly or indirectly to a solid support, such as a bead (e.g., a magnetic bead), a chromatography column, or any other support. In embodiments, the method further comprises subjecting the eluted intermediate to further synthesis, or subjecting the intermediate after sequestration (e.g., removal) of a specific enantiomer of PS linkage to further synthesis. In embodiments, the oligonucleotide synthesis is chemical synthesis, biochemical synthesis, or a combination thereof.

[0026] Further aspects and embodiments of the present disclosure will be apparent from the following detailed description.

[0027] BRIEF DESCRIPTION OF THE FIGURES

[0028] FIG. 1 shows illustrative, non-limiting chemical modifications used to enhance oligonucleotide therapeutics (ONTs), and for which antibodies may be generated according to the present disclosure.

[0029] FIG. 2 shows illustrative, non-limiting oligonucleotide therapeutics that can be detected and / or assayed using antibodies of the present disclosure.

[0030] FIG. 3 is a radar chart showing the aggregate nucleotide base specificity of anti-2'- MOE clones by relative ELISA titer.

[0031] FIG. 4 are immunofluorescence images showing intracellular localization of an antisense oligonucleotide (ASO), detected using anti-phosphorothioate (PS) monoclonal antibody clone PS05.

[0032] FIG. 5 shows co-localization of ONT with endosomal marker using clone PS03.

[0033] FIG. 6 is a line graph showing the quantification of fully PS modified ASO by ELISA using anti-PS monoclonal antibody (clone PS04). Non-modified PO oligonucleotide of the same sequence was used as a negative control (PO Oligo).

[0034] FIG. 7A and FIG. 7B show results of ELISA of selected anti-PS monoclonal antibodies against candidate ONT drugs. FIG. 7A shows sequence / chemistry reactivity of anti-PS antibody clone PS04, clone PS05, and clone PS03 against a panel of six (6) candidate ONT drugs. FIG. 7B shows the reactivity of each of these antibodies against PS linkages in a PS modified ONT drug when the degree of PS modification is lowered from 100% to 95%, 75%, 50%, 25% and 5%.

[0035] FIG. 8 shows the biodistribution of an ONT drug having different levels of PS modifications, and detected using selected anti-PS monoclonal antibodies (PS03, PS04, PS05). Human hepatocytes were treated with an ASO by free uptake and then stained by immunofluorescence microscopy with DAP I, phalloidin, and anti-PS antibody.

[0036] FIG. 9 shows immunofluorescence microscopy using anti-PS monoclonal antibody clone PS03.

[0037] FIG. 10 shows the quantification of fully PS modified ASO by ELISA using anti-PS monoclonal antibody (clone PS04).

[0038] FIG. 11 shows the results of an assay to characterize selectivity for the stereochemistry of the PS bond recognized by the panel of anti-PS antibodies.

[0039] FIG. 12 shows a generalized (not limiting) workflow for the development of monoclonal antibodies.

[0040] DETAILED DESCRIPTION

[0041] The present disclosure provides monoclonal antibodies that specifically recognize chemical modifications of oligonucleotide therapeutics (ONTs), such as but not limited to PS, 2'-M0E or 2'-OMe modifications. In embodiments, the antibodies recognize chemically- modified oligonucleotides largely independent of the nucleobase sequence or strandedness. The antibodies of this disclosure have the ability to localize and quantify oligonucleotide therapeutics, among others, in biological specimens. The performance of these antibodies is demonstrated by various in vitro assays that show the utility of these reagents to facilitate the collection of analytical data to support ADMET studies useful to ensure ONT approval by regulatory agencies.

[0042] In aspects and embodiments, the present disclosure provides antibodies that recognize phosphorothioate (PS) modifications of oligonucleotides, and which are specific to right-handed (R) enantiomers or are specific to left-handed (S) enantiomers. Accordingly, in various aspects, provided herein are methods of detecting, quantifying, enriching or purifying oligonucleotides having R or S enantiomers of PS internucleotide linkages.

[0043] Drug properties of oligonucleotide therapeutics differ significantly from those of traditional small -molecule therapeutics. Therefore, several challenges must be addressed to further advance the use of oligonucleotide therapeutics for a variety of diseases. These challenges include better understandings of toxicity, targeting of extrahepatic tissues for therapy, and the efficacy of drug adsorbed into the patient’s system relative to the dose administered. These challenges require collection of analytical data to answer questions regarding tissue localization, uptake of drug, clearance of drug by the liver and kidney, sequestration and release, secretion rates, off-target binding, assessing the biological effect of the drug treatment, and many other parameters. In general terms, analytical data is needed to assess the pharmacodynamic, pharmacokinetic, and toxicological properties of these drugs.

[0044] To date, analytical assays deployed to collect analytical data relating to ONTs have been primarily based on physiochemical assays. Liquid chromatography (LC), mass spectroscopy (MS) and / or hybridization assays are the most commonly used analytical assays to determine the localization and quantification of oligonucleotide drugs, likely due to the lack of alternative and / or potentially superior assays. While the currently available analytical assays have high specificity and sensitivity in highly exposed tissues like liver, sensitivity is limiting in other tissues such as plasma and urine. These methods also are expensive to perform, require specialized equipment, and a high degree of technical expertise to execute the assay and interpret the resultant data.

[0045] Hybridization assays, sometimes referred to as “ISH” assays, are based on heterogeneous noncompetitive binding of the oligonucleotide drug to a chemically synthesized template probe, followed by addition of signal probe via ligation and detection using a fluorescence microtiter plate reader. Probes used in hybridization assays must be designed for each specific oligonucleotide drug sequence as Watson-Crick base pairing is the mechanism by which the probe binds to the oligonucleotide drug. As a consequence, this method is costly and time consuming as it does not lend itself to the use of a universal detection reagent, since unique probes must be designed and synthesized for each oligonucleotide drug candidate. This is a considerable challenge as often large oligonucleotide libraries are screened in early upstream drug development. Furthermore, certain short oligonucleotide sequences, iterative nucleic acid sequences, and base pair combinations do not perform well in this assay.

[0046] Fluorescent tag labeling and radioactive isotope labeling are other methods of analysis for oligonucleotide drugs, especially useful to assess early-stage drug candidates, tissue distribution, metabolite scouting, and quantification in vivo in animal studies. However, the use of radioactivity to assess drug efficacy is limited due to the inherent difficulties and safety of working with radioactivity. Fluorescent labeling also has limitations in that introduction of a fluorescent tag introduces a new chemistry that may change the chemical, physical, or binding properties of the oligonucleotide drug. Other assays such as affinity capillary electrophoresis, flow cytometry, and others have been reported to be used to collect analytical data, but all of these methods have limitations that offset the value of the data collected and limit their widespread use, or limit the likelihood that these assays would be recognized as an accepted methodology by regulatory agencies.

[0047] According to this disclosure, antibodies are generated to recognize chemical modifications of oligonucleotide drugs. The antibodies according to this disclosure can be used for the essentially universal detection of oligonucleotide drugs possessing the chemical modification, that is, largely independent of nucleic acid sequence, drug platform, or structure. This disclosure demonstrates that a sensitive and specific immune response can be generated against chemical moieties commonly employed in ONTs. This disclosure demonstrates generating such an immune response to these moieties, and screening of a large pool of potential monoclonal antibodies to select a clone or clones demonstrating the desired properties needed for immunoassays.

[0048] The monoclonal antibodies produced according to this disclosure are useful in numerous types of immunoassays that can provide data pertaining to the tissue localization of drug, the precise intracellular location of drug taken up by cells, the ranking of drug candidates, as well as determination of drug potency and immunogenicity, the development of anti -drug-antibody assays, assays to assess drug binding to tissue and serum proteins, and other analytical assays. Immunoassays such as immunohistochemistry, immunofluorescence microscopy, ELISA, Western blot, dot blot, flow cytometry / FACS analysis and immunoprecipitation are all enabled by this disclosure. Oligonucleotide drugs, such as antisense oligonucleotides (ASOs), microRNAs (miRNAs), aptamers, and small interfering RNAs (siRNAs), contain short fragments of nucleic acids complementary to a specific mRNA that can suppress or amplify the expression of a gene, for instance by inhibiting or promoting transcription and translation of the resulting protein. Oligonucleotide drugs and mRNA vaccines have been researched for decades. The rate of oligonucleotide drug approvals is increasing, resulting in burgeoning pipelines for new drug development. Challenges in getting oligonucleotide drugs to market include: (1) poor stability and sensitivity to endo and exonucleases in cells and serum, (2) poor uptake into target cells or tissues, (3) off-target effects due to partial complementarity to unintended targets, and (4) immunogenicity or immuno-stimulation. Other challenges include targeting extrahepatic tissue via a systemic administration of drugs, and the lack of analytical tools to demonstrate absorption, distribution, metabolism, excretion, and toxicity (ADMET). Analytical tools are the primary limiting factor in the collection of analytical data needed to satisfy regulatory agencies who require such data to ensure efficacy and safety.

[0049] Many of the central barriers to the use of oligonucleotide drugs and mRNA have been at least partially addressed over the last several decades of development, especially concerning strategies for drug delivery such as the use of GalNAc for hepatic receptor targeting, or the use of lipid nanoparticles for drug encapsulation. One such development, chemical modification, has significantly improved the stability of oligonucleotide drugs without modifying the nucleobase sequence. Chemical modifications have also been shown to decrease toxicity while improving resistance to degradation. Often drug developers use different chemical modifications in combinations by embracing a chemical platform strategy, such as the use of a gapmer to recruit RNaseH for degradation of the target RNA. Gapmers employ locked nucleic acid (LNA) or other modified RNA (e.g., 2'-0Me or 2'- MOE) flanking a central “gap” of antisense DNA. Consequently, several clinical trials are underway for oligonucleotide drugs to treat diseases including cancer, HIV / AIDS, Duchenne muscular dystrophy, Cytomegalovirus retinitis, and many others including numerous rare diseases.

[0050] According to this disclosure, an approach to develop monoclonal antibodies as analytical tools to be used as ONT detection reagents comprises: (1) design of an immunogen; (2) design of screening assays and controls used during the development of the antibodies; (3) design of strategies to isolate hybridomas with desired sensitivity and specificity to the target; and (4) strategies for production and purification of selected monoclonal antibodies. In embodiments, the process can further comprise: (5) design of final release criteria; (6) design of elements to enhance stability and shelf life of monoclonal antibodies; (7) determination of additional physical parameters to broaden the application of these tools, such as determination of amino acid sequences, characterizing epitope, and quantifying binding affinities.

[0051] In embodiments, the process comprises assessing drug structure. Oligonucleotide and nucleic acid therapies often contain one or more structural modifications that increase their stability, uptake and effectiveness, but may increase the likelihood of toxicity. Different modifications may be incorporated, in varying degrees, to the nitrogenous bases or to the sugar-phosphate backbone. An exemplary chemical modifications is phosphorothioate (PS) linked backbone, where the naturally occurring phosphodiester (PO) is replaced by the phosphorothioate backbone (where phosphorus is bound to sulfur). Other backbone modifications, such as phosphorodi thioate, morpholino (as well as thiomorpholino), and peptide nucleic acid (PNA), are known in the art and may also be used. Alternatively or in addition, the oligonucleotide may contain sugar modifications, such as locked nucleic acid (LNA) and other bicyclic structures such as constrained ethyl (cEt) and ENA. Other sugar modifications include 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (MOE), 2 '-Methyl, 2 -F, and 2'-ethyl, among others. In still other embodiments, the nucleic acid target comprises nucleobase modifications, such as pseudouridine or N1 -methylpseudouridine. Other nucleobase modifications are known in the art. See US Patent No. 10,064,959, which is hereby incorporated by reference in its entirety.

[0052] By assessing drug structure, it was concluded that only a monoclonal antibody would suffice to recognize a single atom change in structure. To be functional as a broad detection reagent the monoclonal antibody would need binding properties such that the chemical modification could be detected regardless of nucleic acid composition or sequence, structure (e.g., ASO, siRNA, or mRNA), strandedness (e.g., single stranded or double stranded), configuration (e.g., RNA:RNA, DNA:DNA or RNA:DNA), or platform (e.g., gapmer).

[0053] In embodiments, an immunogen is constructed comprising the selected chemical modification(s). For example, in embodiments, the immunogen comprises a single-stranded nucleic acid configured as an antisense oligonucleotide (ASO), where inter-nucleotide linkages are modified with PS backbone (e.g., at least 50%, such as 100%, of the inter- nucleotide linkages are modified with PS backbone). In embodiments, the immunogen comprises a single-stranded RNA configured as an antisense oligonucleotide (ASO), where sugars are converted to 2'-0Me and / or 2 -MOE (at least 50%, such as 100%, of the sugars are modified with 2'-0Me or 2 -MOE). In embodiments, oligonucleotides contain from 10 to 50 nucleotides, such as from 10 to 30 nucleotides, or from 12 to 25 nucleotides. In embodiments, the immunogen involves a conjugated nucleotide (e.g., a single nucleotide) with the desired (e.g., target) chemical modification. For example, the immunogen may comprise a nucleotide with a target 2' sugar modification, such as those already described.

[0054] The immunogen further comprises a carrier protein, that is, the immunogen is a nucleotide-carrier protein or oligonucleotide-carrier protein conjugate, with selected conjugation chemistry and ratio of nucleotide / oligonucleotide-to-protein deemed sufficient to illicit an immune response. The dosage used for the initial immunization of mice and subsequent booster injections, as well as the route of administration are selected. In embodiments, the carrier protein is keyhole limpet hemocyanin (KLH), where the heterobifunctional crosslinking agent MBS (or comparable bivalent reagents) can be selected to couple a derivatized modified nucleotide / oligonucleotide to a free primary amine (-NH2) on KLH via a free reduced sulfhydryl group (-SH). In embodiments, modified nucleotide or modified oligonucleotide is conjugated to the carrier protein at a ratio of from about 20: 1 to about 1: 1, such as from about 12: 1 to about 2: 1, or from about 10:1 to about 4: 1.

[0055] Monoclonal antibodies generated are screened for activity. A surrogate assay was conceptualized as a screening assay that would mimic the desired properties of anti-PS antibodies (for example) useful for both in vitro and in vivo assays including those assays intended to assess the biological effects of therapeutic drugs. An exemplary assay for screening of prospective clones are ELISA and immunofluorescence microscopy. Control oligonucleotides are also synthesized and used to assess both the sensitivity and specificity of the antibody clones during the development phase. Oligonucleotide controls would possess the same specific nucleotide sequence as the (e.g., fully) PS, 2'-0Me and / or 2 -MOE modified oligonucleotide (for example) used as a positive control. However, unlike the modified oligonucleotide used for immunization, oligonucleotide controls for use in ELISA can be conjugated to bovine serum albumin (BSA) as a carrier protein to facilitate binding to the ELISA plate. Alternatively, oligonucleotide controls can be bound to ELISA plates with the candidate antibody (capture antibody), and detected with the same antibody carrying a detectable label or ligand (detection antibody).

[0056] Modified oligonucleotides as controls can be synthesized with varying levels of the selected modification, e.g., from 100% to 0%. For example, the following percentage of PS modification, relative to naturally occurring PO backbone, can be employed, such as from 1% to about 100% (e.g., from about 5% to about 100%). In exemplary embodiments, one or more oligonucleotides are screened having about 100%, about 31%, about 10%, about 3% or 0% PS modification. Additional oligonucleotides can be synthesized without PS modification but with 2’-0-M0E or 2’-O-methyl modifications to be used as negative controls. As another example, the following percentage of 2’-0-M0E and / or 2’-O-methyl modifications, or relative to naturally occurring ribose or deoxyribose sugar, can be used for screening: from 1% to about 100% (e.g., from about 5% to about 100%). In exemplary embodiments, one or more oligonucleotides are screened having about 100%, about 31%, about 10%, about 3% or 0% of 2 -0 modification. Additional oligonucleotides can be synthesized without 2’-0-M0E or 2’-O-methyl modifications but, e.g., with PS modification to be used as negative controls. Various other positive control oligonucleotides comprising the chemical modification, optionally with other modifications (such as gapmers), and with varying nucleotide sequence, can be synthesized and used for screening assays.

[0057] Hybridomas are created for screening clones with the desired sensitivity and specificity to the target, and desired results against positive and negative controls. After splenocyte-myeloma fusion, hybridoma cells secreting antibody reactive to the relevant chemical modification are screened by iterative limiting dilution steps. In embodiments, the amount of BSA-conjugated oligonucleotide bound to the well of the plates is modified to vary from 0.1 pg to 0.5 pg so that a dilution series of tissue culture supernatant fluid can be applied, to not only determine the specificity of the antibody during clonal selection, but also the amount and robustness of antibody secreted by the hybridoma cells. Only those wells containing clones reactive to the positive control oligonucleotides and non-reactive to the negative control oligonucleotides are considered for further screening. In addition, only clones showing a sufficient absorbance value at a determined dilution point are selected for hybridoma development, as we concluded that these clones were secreting sufficient quantities of antibodies that demonstrated adequate binding properties for use in the diverse array of immunoassays intended for use with these antibodies.

[0058] The likelihood that a single hybridoma would secrete an antibody capable of detecting all of the possible configurations of PS containing oligonucleotide drugs, e.g., ASO, siRNA, mRNA, aptamer, and in all of the possible immunoassays considered to be valuable to support the collection of analytical data, was believed to be low. Thus, the approach according to this disclosure was to select multiple clones from multiple splenocyte- myeloma fusions, to prepare a panel of monoclonal antibodies that demonstrate differential sensitivity and specificity to the target (e.g., PS target). Surprisingly, however, it was found that some clones were able to detect single PS, 2'-0Me or 2 -MOE modifications independent of sequence or format (e.g., ASO or gapmer) of the oligonucleotide.

[0059] By developing panels of highly reactive and specific clones that each possess subtle differences in binding the epitope increases the likelihood that at least one clone would be useful for any one drug developed for use as a therapeutic. Prospective clones were further selected for chromosomal stability by screening over an extended period of time. In embodiments, IgG class clones are selected that show an IgGl, IgG2a or IgG2b subclass chain configuration for long term stability and usage.

[0060] Selected hybridoma cells are grown in larger quantities in both stationary flasks and roller bottle flasks. During each passage and scale up cells are screened by ELISA to ensure the integrity of the antibody produced by the cells. Cells are frozen in liquid nitrogen for the production of working banks of cells for long term use once a sufficient quantity of cells is obtained. This process ensures that the cells are viable after the freezing process. Thawed cells can be grown in larger volumes of culture fluid for the purification of antibody using protein A chromatography, for example. Once purified, resultant antibodies can be again screened by ELISA using the full complement of positive and negative control oligonucleotides described above.

[0061] In some embodiments, the candidate monoclonal antibodies recognize a single chemical modification in a substantially sequence-non-specific manner. In some embodiments, the candidate monoclonal antibodies recognize a single chemical modification in a manner that is substantially independent of structure or platform (e.g., ASO, gapmer, siRNA, or mRNA). In some embodiments, the candidate monoclonal antibody recognizes a single chemical modification in a manner that is substantially independent of strandedness (e.g., single stranded or double stranded). In some embodiments, the candidate monoclonal antibody recognizes a single chemical modification in a manner that is substantially independent of configuration (e.g., RNA:RNA, DNA:DNA or RNA:DNA).

[0062] In some embodiments, the single chemical modification that the candidate monoclonal antibody recognizes is a nucleotide backbone modification, optionally selected from phosphor othioate, phosphoacetate linkage (PACE), methylphosphonate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, phosphoramidite, phosphorodi ami date, phosphorodithioate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, thiomorpholino, peptide nucleic acid, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, or sulfone internucleoside linkages. In some embodiments, the candidate monoclonal antibody recognizes a phosphorothioate linkage. In some embodiments, the candidate monoclonal antibody specifically recognizes the right-handed (R) enantiomers of phosphorothioate linkage. In some embodiments, the candidate monoclonal antibody specifically recognizes the left-handed (S) enantiomers of phosphorothioate linkage.

[0063] In some embodiments, the single chemical modification that the candidate monoclonal antibody recognizes is a 2' sugar modification of DNA or RNA nucleotides. In some embodiments, the candidate monoclonal antibody recognizes a 2' sugar modification, optionally selected from 2’-0 methoxyethyl (MOE), 2'-0 methyl (2'-methoxy), 2'-0 ethyl, 2'-Fluoro, 2'-Methyl, and 2'-Ethyl. In some embodiments, the candidate monoclonal antibody recognizes a 2'-0 methoxyethyl (MOE) modification. In some embodiments, the candidate monoclonal antibody recognizes a 2'-0 methyl (2'-methoxy) modification.

[0064] In some embodiments, the single chemical modification that the candidate monoclonal antibody recognizes is a bridged nucleotide, such as Locked Nucleic Acid (LNA). LNA or “locked nucleotides” are described, for example, in U.S. Patent Nos. 6,268,490; 6,316,198; 6,403,566; 6,770,748; 6,998,484; 6,670,461; and 7,034,133, all of which are hereby incorporated by reference in their entireties. LNAs are modified nucleotides that contain a bridge between the 2' and 4' carbons of the sugar moiety resulting in a “locked” conformation, and / or bicyclic structure. Other suitable locked nucleotides that can be used according to this disclosure include those described in U.S. Pat. Nos. 6,403,566 and 6,833,361, both of which are hereby incorporated by reference in their entireties. In exemplary embodiments, the locked nucleotides are independently selected from a 2' to 4' methylene or ethylene bridge and a constrained ethyl (cEt) bridge (see, US Patent Nos. 7,399,845 and 7,569,686, which are hereby incorporated by reference in their entireties).

[0065] In still other embodiments, the candidate monoclonal antibody recognizes a morpholino and / or a thiomorpholino backbone.

[0066] Selected antibodies can be cloned, and produced in any desired format, including monoclonal antibody, single chain variable fragment (ScFv), heavy chain only antibodies, as well as antigen binding fragments (e g., Fab or F(ab')2).

[0067] Antibodies produced should show purity of the antibody from other proteins as determined by SDS-PAGE, the accepted subclass of immunoglobulin as determined by RID isotyping using commercially available kits, freedom from mycoplasma, bacterial or fungal contamination in cultures, the predicted positive or negative pattern of reactivity by ELISA against positive and negative oligonucleotide controls used during the screening process, positive reactivity by ELISA against the chemically modified target (e.g., ‘gapmer’ drug), and a minimum level of acceptable binding by ELISA as determined by titer, enzyme kinetic rate, or acceptable IC50 and / or EC50 concentrations as determined by ELISA. Furthermore, biotin and fluorochrome conjugated antibodies should show acceptable levels of reactivity and specificity by ELISA against the target and controls and show a degree of labeling (DOL) for biotin and fluorochrome conjugations.

[0068] Accordingly, this disclosure utilizes monoclonal antibodies to recognize a chemical modification on oligonucleotide drugs, substantially independent of nucleic acid sequence, drug format, structure, or platform in various embodiments. Screening strategies can constitute an important component of certain embodiments, leading to the development of select clones that demonstrate the desired properties needed for immunoassays.

[0069] A panel approach rather than a single antibody can be an important consideration for certain embodiments, because the likelihood of a single hybridoma secreting an antibody capable of detecting all of the possible configurations of a modified oligonucleotide drug in all of the possible immunoassays considered to be valuable to support the collection of analytical data, was considered to be unlikely. The panel approach, where we developed multiple clones from multiple splenocyte-myeloma fusions, was conceived to be more likely to generate monoclonal antibodies with differential sensitivity and specificity to the target modification and was considered to be more likely to generate analytical tools capable to satisfying the needs for analytical data in diverse permutations of in vitro and in vivo assays.

[0070] In some aspects, the disclosure provides a method for detecting a chemically- modified oligonucleotide. The method comprises providing a sample suspected of comprising the chemically-modified oligonucleotide (e.g., comprising a chemical modification described herein). In some embodiments, the chemically-modified oligonucleotide comprises one or more phosphorothioate linkages. In some embodiments, the chemically-modified oligonucleotide comprises one or more 2 -0 modifications as described herein, such as 2'-0 methoxyethyl (MOE), 2'-0 methyl (2'-methoxy), or bridged nucleotide modifications. In some embodiments, the method comprises detecting the presence or absence or amount of the chemically modified oligonucleotide in the sample by contacting the sample with the antibody.

[0071] In some embodiments, the presence, absence, or amount of the chemically-modified oligonucleotide is detected using an assay selected from immunohistochemistry, immunofluorescence microscopy, ELISA, Western blot, dot blot, flow cytometry / FACS analysis, and immunoprecipitation. In some embodiments, the ELISA is a quantitative ELISA.

[0072] In some embodiments, the sample is a test sample from manufactured batch of the oligonucleotide therapeutic. In some embodiments, the method is used for qualitative or quantitative analysis of the manufactured batch. In some embodiments, the method is used for quality control of the manufactured batch. That is, a manufactured batch has a predetermined binding characteristic with an antibody of this disclosure or a panel of such antibodies.

[0073] In some embodiments, the sample is a test sample from a subject that was administered the oligonucleotide therapeutic. Illustrative samples comprise a fluid e.g., a bodily fluid such as blood, serum, urine, or saliva) and / or at least one cell or tissue (e.g., one or a collection of organs or tissues derived from the subject). In some embodiments, the method is used for the determination of pharmacokinetics or biodistribution of the oligonucleotide therapeutic. In some embodiments, the method is used for the determination of delivery of the chemically-modified oligonucleotide to a tissue or a cell, including to determine a subcellular compartment. In some embodiments, the subcellular compartment is selected from nucleus, endosomes, and cytoplasm.

[0074] In some embodiments, the sample comprises cells that were contacted in vitro with the oligonucleotide. In some embodiments, the method is used for the determination of delivery of the chemically-modified oligonucleotide to a subcellular compartment. In some embodiments, the subcellular compartment is selected from nucleus, endosomes and cytoplasm.

[0075] In some aspects, the disclosure provides a method of quantifying phosphorothioate (PS) stereochemistry in a PS-modified oligonucleotide. In some embodiments, the method comprises contacting the PS-modified oligonucleotide with a monoclonal antibody that binds to right-handed (R) enantiomers of phosphorothioate linkage. Additionally or alternatively, the method comprises contacting the PS-modified oligonucleotide with a monoclonal antibody that binds to left-handed (S) enantiomers of phosphorothioate linkage. In some embodiments, the binding of the PS-modified oligonucleotide to the monoclonal antibodies is quantified. In some embodiments, the binding of the PS-modified oligonucleotide to the antibody is quantified using an assay selected from immunohistochemistry, immunofluorescence microscopy, ELISA, northern blot, Southern blot, flow cytometry / FACS analysis, and immunoprecipitation.

[0076] In some embodiments, e.g., involving ELISA, the monoclonal antibody may be conjugated with a detectable label (e.g., a fluorescent label, a detectable ligand such a biotin, or an enzyme). Exemplary detectable labels include a fluorescent or fluorogenic marker, a luminescent or luminogenic marker e.g., luciferase), an enzyme (e.g., alkaline phosphatase), and biotin. Exemplary fluorescent detectable labels include 6-carboxyfluorescein, tetrachlorofluorescin, green fluorescent protein, and red fluorescent protein.

[0077] In some aspects, the present disclosure provides a method of enriching or purifying an oligonucleotide comprising an enantiomer of phosphorothioate (PS) linkage. In some embodiments, the method comprises contacting a sample comprising the oligonucleotide with a monoclonal antibody specific for PS linkages to produce a complex of the monoclonal antibody and the oligonucleotide. In some embodiments, the monoclonal antibody binds to right-handed (R) enantiomers of phosphorothioate linkage. Additionally or alternatively, the monoclonal antibody binds to left-handed (S) enantiomers of phosphorothioate linkage. In some embodiments, the method further comprises eluting the oligonucleotide bound to the monoclonal antibody, or sequestering (e.g., removing) oligonucleotide bound to the monoclonal antibody.

[0078] In some embodiments, the monoclonal antibody is bound to a solid support, optionally wherein the solid support is a bead, optionally a magnetic bead, or a chromatography column. Any other support can be used. In some embodiments, the method further comprises contacting the complex with a solid support capable of binding the monoclonal antibody directly or indirectly, optionally wherein the solid support is a bead, optionally a magnetic bead, or a chromatography column. In some embodiments, the method further comprises washing the complex with a buffer.

[0079] In some embodiments, the method produces an oligonucleotide comprising at least 60% or at least 70% or at least 80% or at least 90% or at least 95% R enantiomers of phosphorothioate linkage (or essentially 100% R enantiomers). Alternatively, the method produces an oligonucleotide comprising at least 60% or at least 70% or at least 80% or at least 90% or at least 95% S enantiomers of phosphorothioate linkage (or essentially 100% S enantiomers).

[0080] In embodiments, the method allows for stereospecific oligonucleotide synthesis with respect to phosphorothioate (PS) linkage. In some embodiments, the method comprises contacting a synthetic intermediate of the oligonucleotide with a monoclonal antibody of this disclosure that recognizes a specific enantiomer of PS linkage, to produce a complex of the monoclonal antibody and the intermediate comprising a specific enantiomer of PS linkage. In some embodiments, the monoclonal antibody binds to right-handed (R) enantiomers of phosphorothioate linkage. Alternatively, the monoclonal antibody binds to left-handed (S) enantiomers of phosphorothioate linkage. In some embodiments, the method comprises eluting the intermediate bound to the monoclonal antibody or sequestering or removing the intermediate bound to the monoclonal antibody.

[0081] In some embodiments, the monoclonal antibody is bound to a solid support, optionally wherein the solid support is a bead, optionally a magnetic bead, or a chromatography column. In some embodiments, the method further comprises contacting the complex with a solid support capable of binding the monoclonal antibody directly or indirectly, optionally wherein the solid support is a bead, optionally a magnetic bead, or a chromatography column. In some embodiments, the method further comprises washing the complex with a buffer. In some embodiments, the method further comprises subjecting the eluted intermediate to further synthesis, or subjecting the intermediate after sequestering an undesired enantiomer to further synthesis. In some embodiments, the method further comprises contacting a subsequent intermediate with the monoclonal antibody, and repeating the process. For example, such a process can be used for each step of nucleotide addition.

[0082] In some embodiments, the oligonucleotide synthesis is chemical synthesis (e.g., solid phase synthesis), biochemical synthesis or a combination thereof.

[0083] As used herein, unless the context requires otherwise, the term “about” means a value that is ±10% of a reference value.

[0084] Various aspects and embodiments of this disclosure will be apparent from the following non-limiting examples.

[0085] As used herein, unless the context requires otherwise, the term “oligonucleotide” includes dinucleotides, trinucleotides, and an oligomer of more than three nucleotides. In embodiments, the term “oligonucleotides” includes those containing from 10 to 50 nucleotides, such as from 10 to 30 nucleotides, or from 12 to 25 nucleotides.

[0086] EXAMPLES

[0087] Oligonucleotide therapeutics (ONTs) have increased in development since their inception. ONTs, such as antisense oligonucleotides (ASO) and small interfering RNA (siRNA), include short nucleic acids complementary to a specific mRNA target, and upon administration can modulate protein synthesis in patients. ONT therapeutic platforms are depicted in FIG. 2. The “drug-likeness” of ONTs is facilitated by chemical modifications to the sugar-phosphate backbone and / or nitrogenous bases. Modifications are designed to increase ONT drug stability, uptake, and efficacy, but may increase the likelihood of toxicity. First generation chemical modifications include phosphorothioate (PS) modification of the phosphate portion of the backbone, whereas second generation modifications include adding substituent groups to the 2’ position of the ribose portion of the backbone, such as 2’-O- methyl (OMe), 2 ’-O-m ethoxy-ethyl (MOE), and 2’-fluoro modifications. Third generation modifications include peptide nucleic acids (PNA), locked nucleic acids (LNA) and other bridged nucleotides, morpholino phosphoroamidate (PMO) modifications, and others (FIG. 1). Analytical tools and methods for detecting these chemical modifications significantly independent of nucleotide sequence would be a significant advancement for the development and / or commercialization of these drugs.

[0088] Example 1: Generation of Monoclonal Antibodies that Recognize Chemical Modifications According to this example, modified ONTs were conjugated to keyhole limpet hemocyanin (KLH) to construct immunogens for inducing antibody production in mice. Antibodies were screened for those that detect the desired chemical modification, independent of other features, such as nucleotide sequence.

[0089] Immunogens used to produce monoclonal antibodies to chemical modifications within ONTs included (a) antisense oligonucleotides (ASOs), and (b) individual nucleotides with chemical modification, and representing each of the nitrogenous bases naturally occurring within DNA and RNA. Internucleotide linkages were fully chemically modified. The chemically modified ASOs or nucleotides were then conjugated to a carrier protein such as KLH and used to immunize mice by either injecting individual ASOs or cocktails of mixtures of chemically modified nucleotides. The dosages administered for primary immunization and subsequent booster injections were bracketed at 0.5x, 1 x and 2* standard dosages to increase the likelihood of maximizing the intended immune response. Animal immunizations were performed using multiple cohorts to assess the immune response based on variables used for antigen presentation. Booster injections were given in 21 -day intervals followed by collection of whole blood 10 days after the booster was given. Whole blood was processed to obtain sera which was then applied to an ELISA screening assay. The screening assay included a panel of controls including the ASOs and / or individual nucleotides used for immunization.

[0090] For screening purposes, the controls were conjugated to BSA rather than KLH (positive control). The corresponding negative controls for ASOs contain PO rather than PS linkages. Free BSA was used as a negative control. Other controls include known ONT drugs containing either the intended chemical modification (positive control) or other chemical modifications (negative control). Other controls include nucleotides containing chemical modifications on the nitrogenous bases (negative control).

[0091] The cycles of booster injection, blood sample collection and screening were repeated over the course of several months until a suitable immune response was achieved. The duration of the immunization protocol was typically greater than that used for the routine production of monoclonal antibodies against peptides or proteins or most other types of immunogens. An acceptable immune response was correlated to an absorbance readout on an ELISA microtiter plate reader of 3.0 or greater using test serum diluted to 1:800 in PBS for positive controls with a minimal response to negative controls. Once this level of immune response was obtained a penultimate booster injection was given, the ELISA screen was repeated, and if the level of immune response remained high, a final booster injection was given prior to splenocyte harvest and myeloma fusion to produce stable parental hybridomas. Parental clones were then subcloned to ensure stability and obtain individual subclones. Subclones were screened initially by ELISA against a similar panel of positive and negative controls and for isotype composition. Clones of desirable class and isotype showing robust signal by ELISA against positive controls and negligible or minimal signal against negative controls were selected for production, purification and further analysis.

[0092] A generalized workflow for the development of monoclonal antibodies is shown in FIG. 12. Once purified, monoclonal antibodies were further characterized for reactivity by assays including ELISA, quantitative capture ELISA, immunohistochemistry, immunofluorescence microscopy, biolayer interferometry, peptide sequence analysis, reactivity in 3D cell culture and other analytical assays. An expanded set of ONTs, including drugs approved by the United Stated Food and Drug Administration configured as gapmers, were used an additional means of characterizing monoclonal antibody sensitivity and specificity.

[0093] Using this process, panels of monoclonal antibodies specific for PS (clones PS01, PS02, PS03, PS04, PS05, PS06, PS07, PS08, and PS09), 2'-M0E (M0E1, M0E3, M0E4, M0E9 and MOEC) or 2'-OMe (clones 0ME1, 0ME2, 0ME3, 0ME4, and 0ME5) were generated. Example 2: Specificity of Monoclonal Antibodies

[0094] A panel of monoclonal antibodies specific for PS was studied using ELISA. Briefly, an ASO having 0, 3, 33, or 100% PS modification or a gapmer having PS modifications were assayed using an ELISA assay using monoclonal antibody clones PS01, PS02, PS03, PS04, PS05, PS06, PS07, PS08, and PS09. Binding affinity was quantified using Sartorius Octet

[0095] R4 with SA Biosensor and biotinylated ASO or gapmer, which contains PS and MOE modifications. The results are shown in the Table 1 below: aAffinity Constant;bAssociation Rate;cDissociation Rate; (-) negative; (+ / -) weak; (+) positive; (++) strong; (+++) very strong; (ND) not determined. These results demonstrate that the antibodies are specific to PS modification. A complete lack of binding ( / .<?., not even weak binding) to the ASO having 0% modification, indicates a lack of sequence-specificity.

[0096] A panel of monoclonal antibodies specific for 2'-M0E was studied using ELISA. Briefly, a gapmer having MOE modifications was assayed by ELISA using monoclonal antibody clones M0E1, M0E3, M0E4, M0E9 and MOEC. Affinity binding was determined using Sartorius Octet R4 with SA Biosensor and biotinylated gapmer containing

[0097] PS and MOE modifications. The results are shown in Table 2 below:

[0098] Key: (+) positive; (++) strong; (+++) very strong.

[0099] FIG. 3 is a radar chart showing the aggregate nucleotide base specificity of anti-2'- MOE clones by relative ELISA titer.

[0100] A panel of monoclonal antibodies specific for 2'-0Me was studied using ELISA. Briefly, an ASO having 2'-0Me modifications was assayed by ELISA using monoclonal antibody clones 0ME1, 0ME2, 0ME3, 0ME4, and 0ME5. The results are shown in the Table 3 below: Key: (+) positive; (++) strong; (+++) very strong.

[0101] These results demonstrate that the antibodies are specific to 2’0ME modification.

[0102] Collectively, these results demonstrate that the method disclosed herein produces antibodies that recognize a single chemical modification, and which can be in a sequence- non-specific manner. Example 3: Detection of ONT containing PS modifications by Immunofluorescence

[0103] To evaluate an anti-phosphorothioate (PS) monoclonal antibody in immunofluorescence assay, HeLa cells were cultured and treated with 100 nM of a fully-PS modified 16-mer ASO by gymnosis for 72 hours. After fixation with paraformaldehyde, cells were incubated with a 1 :1,000 dilution of anti-PS monoclonal antibody clone PS03 in PBS and an anti-alpha tubulin antibody, followed by counterstaining of nuclei with DAPI. As shown in FIG. 9, the fully-PS modified ASO showed sequestration inside the cytoplasmic compartment.

[0104] In another such experiment, mouse glioma cells (GL261) derived from C57 black mice were cultured and treated with an ASO drug comprising PS modifications. After fixation with paraformaldehyde, cells were stained with DAPI and anti-PS antibody clone PS05 (1 :2000 dilution) either with (FIG. 4, left panel) or without (FIG. 4, right panel) staining with anti-alpha tubulin clone DMA1. The observed punctate cytoplasmic staining is consistent with endosomal storage of ASO within the cell, as expected for this ONT drug (FIG. 4, right panel).

[0105] To explore whether the drug colocalizes with endosomes within the cell, HeLa cells were cultured, fixed with paraformaldehyde and reacted with anti-PS antibody clone PS03 at a 1 : 1000 dilution. Nuclei were stained with DAPI and endosomes were identified using a RAB9A marker. As shown in FIG. 5, the ONT showed a co-localization with the endosomal marker.

[0106] These results demonstrate, inter alia, that the antibodies developed according to the present disclosure may be used to understand localization inside tissues or cells, including the cytosolic and nuclear penetration of ONTs.

[0107] Example 4: Use of Monoclonal Antibodies to Quantify ONT Drugs

[0108] To evaluate the use of an anti-phosphorothioate (PS) monoclonal antibody in quantification of ONT drugs, a quantitative ELISA assay was performed with fully PS modified ASO using anti-PS monoclonal antibody (clone PS04). Non-modified PO oligonucleotide of the same sequence was used as a negative control (PO Oligo). FIG. 6 shows quantification of fully PS modified ASO. The curve was generated using PS-modified ASO diluted in buffer from 55 pM to 21 nM in concentration, and subsequently detected using anti-PS monoclonal antibody. A standard curve was plotted as the average OD result versus the log of ASO concentration in pM using a 4PL best fit formula. The lower limit of quantitation (LLOQ) was defined as the lowest standard detected (55 pM). The limit of detection (LOD) was determined to be <1 IpM based on standard definition of the term. The upper limit of quantitation (ULOQ) was determined to be 21 nM. It is anticipated that further optimization and sensitivity enhancement will increase the LLOQ by one log unit.

[0109] These results demonstrate, inter alia, that the antibodies according to the disclosure may be used to quantify the ONTs. Accordingly, the antibodies of this disclosure may be used to determine pharmacokinetics of ONTs.

[0110] Example 5: The Sequence, Chemistry Reactivity of the ant i -PS Monoclonal Antibodies

[0111] To evaluate the sequence / chemistry reactivity of the anti-PS antibodies, of six candidate ONT drugs with different configurations (ASO, DNA, and a gapmer) were assayed using ELISA, using anti-PS antibody clone PS04, clone PS05, and clone PS03. Reactivity was assayed for PS modified drug and unmodified PO negative control. FIG. 7A shows sequence / chemistry reactivity of anti-PS antibody clone PS04, clone PS05, and clone PS03 against the panel. As shown in FIG. 7A, irrespective of ONT platform used, each of the assayed antibodies showed higher specificity to PS modified drug compared to unmodified PO negative control.

[0112] The amount of PS linkages in a PS modified ONT drug was varied from 100% to 95%, 75%, 50%, 25% and 5%, and resultant molecules were assayed using ELISA using anti-PS antibody clone PS04, clone PS05, and clone PS03. Anti-PS antibodies were assayed at a 1: 10,000 dilution followed by fluorescein conjugated anti-Mouse IgG secondary antibody. As shown in FIG. 7B, while the signal strength trended downward with the degree of modification, all antibodies tested in this series reacted against an ONT drug with as few as only 5% modified bases, e.g., 1 modified base out of a total of 20 bases, relative to baseline.

[0113] These results indicate that the antibodies produced according to the disclosure can recognize a single modified PS linkage, and in a sequence-nonspecific manner.

[0114] These results also indicate that immunodetection by the antibodies according to the disclosure may be used for detecting even low levels of chemical modification, and for correlating the biological activity or stability of the drugs with modifications and / or for drug ranking. Example 6: Detection of a Fully PS-Modified A SO by ELISA in Biological Samples

[0115] To explore the use of the anti-PS antibodies for assays with biological samples, a fully PS modified ASO was detected by ELISA using anti-PS monoclonal antibody (clone PS04) (FIG. 10). The curve was generated using PS-modified ASO diluted in RIPA lysis buffer, HeLa whole cell lysate (0.3 pg / mL), or mouse serum (2.5 mg / mL) as indicated over a concentration range from 44 pM to 11 nM (FIG. 10). Non-modified PO oligonucleotide of the same sequence was used as a negative control (PO Oligo) diluted in HeLa whole cell lysate. A standard curve was plotted as the average OD result versus the log of ASO concentration in pM using a 4PL best fit formula. The lower limit of quantitation (LLOQ) was defined as the lowest standard detected (44 pM). The limit of detection (LOD) was determined to be <33pM based on standard definitions of the term. The upper limit of quantitation (ULOQ) was 11 nM. Further optimization and sensitivity enhancement will likely increase the LLOQ by one log unit (FIG. 10).

[0116] These results indicate that the antibodies according to the present disclosure are useful for applications such as pharmacokinetic studies.

[0117] Example 7: Use of the anti-PS Monoclonal Antibodies for Evaluating Biodistribution

[0118] Human hepatocytes in tissue were treated with either 100% modified ASO, 80% modified ASO, or unmodified ASO to allow uptake, and then stained by with DAPI, phalloidin, and anti-PS antibody, or with secondary antibody only as a negative control and analyzed by immunofluorescence microscopy. Anti-PS antibodies were used at a dilution range of 1 :200 to 1 : 1000. FIG. 8 top panels show the images representing staining with clone PS03, FIG. 8 middle panels show the images representing staining with clone PS04, and FIG. 8 bottom panels show the images representing staining using clone PS05. As shown in FIG. 8, the tissue treated with 100% and 80% PS-modified drug showed clear staining of cells

[0119] These results indicate that the anti-PS monoclonal antibodies according to the present disclosure can be used for studying the biodistribution of an ONT drug.

[0120] Example 8: Use of the Anti-PS Monoclonal Antibodies to Distinguish between Right- Handed (R) and Left-Handed (S) Enantiomers of Phosphorothioate linkages

[0121] To evaluate the use of an anti-PS monoclonal antibody according to the present disclosure to distinguish between right-handed (R) and left-handed (S) enantiomers of phosphorothioate linkage, an ELISA assay is performed with substrates comprising only R enantiomers of phosphorothioate, only S enantiomers of phosphorothioate, or a mixture of R and S enantiomers of phosphorothioate. An unmodified substrate lacking PS linkages can be used as a control. The substrates may be assayed using the anti-PS monoclonal antibody clones PS01, PS02, PS03, PS04, PS05, PS06, PS07, PS08, and PS09, or other clones. It is expected that some of the antibodies will recognize the substrates comprising only R enantiomers of phosphorothioate and the substrate comprising a mixture of R and S enantiomers of phosphorothioate, while others recognize the substrates comprising only R enantiomers of phosphorothioate and the substrate comprising a mixture of R and S enantiomers of phosphorothioate. As shown above, none of the clones are expected to recognize the substrate lacking PS linkages (see Example 2).

[0122] FIG. 11 shows the results of an assay to characterize selectivity for the stereochemistry of the PS bond recognized by the panel of anti-PS antibodies. Antibodies PS03 to PS05 are shown to preferentially recognize the (R) form as well as the T I PS ASO blend. PS09 is shown to preferentially recognize the (S) form, as well as recognition of the blend.

[0123] Example 9: Use of the Anti-PS Monoclonal Antibodies to Enrich and or Purify Oligonucleotide Therapeutics Comprising Right-Handed (R) or Left-Handed (S) Enantiomers of Phosphorothioate linkages

[0124] To evaluate the use of the anti-PS monoclonal antibody according to the present disclosure to enrich and / or purify oligonucleotides comprising right-handed (R) and lefthanded (S) enantiomers of phosphorothioate linkage, the anti-PS monoclonal antibody clones PS01, PS02, PS03, PS04, PS05, PS06, PS07, PS08, and PS09 (or other clones) may be immobilized on a solid support (e.g., a magnetic bead or a column). To study enrichment or purification, a mixture of substrates comprising only R enantiomers of phosphorothioate, only S enantiomers of phosphorothioate, a mixture of R and S enantiomers of phosphorothioate, and optionally an unmodified substrate lacking PS linkages is used. The mixture is contacted with the immobilized antibodies, washed and eluted. The eluate is then assayed for the presence of the different substrates.

[0125] It is expected that some of the antibodies will enrich and / or purify the substrates comprising only R enantiomers of phosphorothioate and the substrate comprising a mixture of R and S enantiomers of phosphorothioate, while others enrich and / or purify the substrates comprising only R enantiomers of phosphorothioate and the substrate comprising a mixture of R and S enantiomers of phosphorothioate.

Claims

CLAIMS1. A method for making a monoclonal antibody specifically recognizing a nucleic acid chemical modification, comprising: providing an immunogen comprising an oligonucleotide or nucleotide containing the chemical modification, the oligonucleotide or nucleotide conjugated to a carrier protein to illicit an immune response in a mouse; immunizing a mouse with the immunogen; screening antibody-producing B lymphocytes isolated from the mouse and / or hybridomas generated therefrom against one or more positive and negative control oligonucleotides; and selecting one or more monoclonal antibodies that display binding specificity for the positive control oligonucleotide(s) over the negative control oligonucleotides.

2. The method of claim 1, wherein the chemical modification comprises a nucleotide backbone modification.

3. The method of claim 2, wherein the backbone modification is phosphorothioate intemucleotide linkages.

4. The method of claim 2, wherein the backbone modification is phosphoacetate linkage (PACE), methylphosphonate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, phosphoramidite, phosphorodiamidate, phosphorothioate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, thiomorpholino, peptide nucleic acid, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, or sulfone internucleoside linkages.

5. The method of any one of claims 1 to 4, wherein the chemical modification comprises a 2' sugar modification of DNA or RNA nucleotides.

6. The method of claim 5, wherein the 2' sugar modification is selected from 2'-0 methoxyethyl (MOE), 2'-0 methyl (2'-methoxy), 2'-0 ethyl, 2'-Fluoro, 2'-Methyl, and 2'- Ethyl.

7. The method of claim 5, wherein the 2' sugar modification is a bridged nucleotide.

8. The method of claim 7, wherein the bridged nucleotide has a 2' to 4' bridge.

9. The method of claim 8, wherein the bridged nucleotide has a methylene bridge (LNA), 2'-O,4'-C -ethylene-bridged nucleic acid (ENA), or a constrained ethyl bridge (cEt).

10. The method of any one of claims 5 to 9, wherein the immunogen comprises a nucleotide with the 2' sugar modification conjugated to the carrier protein.

11. The method of any one of claims 1 to 10, wherein the chemical modification comprises a nucleobase modification.

12. The method of claim 11, wherein the chemical modification is 5-methylcytosine, pseudouridine, or N1 -methylpseudouridine.

13. The method of any one of claims 1 to 12, wherein the oligonucleotide is an antisense oligonucleotide, and is optionally a gapmer, or is a PNA or morpholino, or thiomorpholino oligonucleotide.

14. The method of any one of claims 1 to 12, wherein the oligonucleotide is an siRNA, miRNA, or aptamer.

15. The method of any one of claims 1 to 12, wherein the oligonucleotide is an mRNA.

16. The method of any one of claims 1 to 12, wherein the oligonucleotide is single stranded or double stranded, and optionally comprises one or more of RNA:RNA, RNA-DNA, and DNA:DNA hydrogen-bonded nucleotides, which are optionally Watson-Crick base-pairing.

17. The method of any one of claims 1 to 16, wherein the carrier protein is keyhole limpet hemocyanin (KLH).

18. The method of any one of claims 1 to 17, wherein the oligonucleotide is conjugated to the carrier protein using a linking reagent, which is optionally m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS).

19. The method of claim 18, wherein the linking reagent conjugates to free primary amines of the carrier protein.

20. The method of claim 19, wherein the reagent conjugates to a reduced sulfhydryl group on the oligonucleotide, which is optionally on a terminus of the oligonucleotide.

21. The method of any one of claims 1 to 20, wherein the oligonucleotide is conjugated to the carrier protein at a ratio of from about 20: 1 to about 1: 1, such as from about 12: 1 to about 2: 1, or from about 10: 1 to about 4: 1.

22. The method of any one of claims 1 to 21, wherein the positive control oligonucleotides comprise a panel with varying amount of the chemical modification, and the negative control(s) lack the chemical modification.

23. The method of claim 22, wherein the positive control oligonucleotides comprise different nucleotide sequences, and / or contain the chemical modification at different positions.

24. The method of claim 22 or 23, wherein the negative control oligonucleotide(s) comprise other chemical modifications not included in the immunogen or no chemicalmodification, and wherein at least one negative control has the same nucleobase sequence as the oligonucleotide used for the immunogen.

25. The method of any one of claims 1 to 24, wherein candidate monoclonal antibodies are screened using ELISA.

26. The method of any one of claims 1 to 25, comprising selecting antibodies with the desired specificity for positive controls over negative controls, and characterizing the antibodies by one or more of: amino acid sequence analysis, epitope characterization, binding affinity for positive controls, and binding affinity for negative controls.

27. The method of any one of claims 1 to 26, wherein the candidate monoclonal antibodies recognize a single chemical modification in a substantially sequence-non-specific manner.

28. The method of claim 27, wherein the single chemical modification is a nucleotide backbone modification.

29. The method of claim 28, wherein the backbone modification is a phosphorothioate linkage.

30. The method of claim 29, wherein the candidate monoclonal antibodies specifically recognize right-handed (R) enantiomers of phosphorothioate linkage.

31. The method of claim 29, wherein the candidate monoclonal antibodies specifically recognize left-handed (S) enantiomers of phosphorothioate linkage.

32. The method of claim 27, wherein the single chemical modification comprises a sugar modification of DNA or RNA nucleotides.

33. The method of claim 32, wherein the 2' sugar modification is 2'-0 methoxyethyl (MOE).

34. The method of claim 32, wherein the 2' sugar modification is 2’-0 methyl (21- methoxy).

35. The method of claim 32, wherein the 2' sugar modification comprises a 2' to 4' bridge.

36. The method of any one of claims 1 to 35, comprising selecting antibodies with the desired specificity for positive controls over negative controls, and characterizing the antibodies by one or more of: amino acid sequence analysis, epitope characterization, binding affinity for positive controls, and binding affinity for negative controls.

37. A monoclonal antibody prepared by the method of any one of claims 1 to 36.

38. A method for detecting a chemically-modified oligonucleotide, comprising providing a sample suspected of comprising the chemically-modified oligonucleotide, and detecting the presence or absence or amount of the chemically modified oligonucleotide in the sample by contacting the sample with the antibody prepared according to any one of claims 1 to 36.

39. The method of claim 38, wherein the presence, absence, or amount of the chemically- modified oligonucleotide is detected using an assay selected from immunohistochemistry, immunofluorescence microscopy, ELISA, Western blot, dot blot, flow cytometry / FACS analysis, and immunoprecipitation.

40. The method of claim 39, wherein the ELISA is a quantitative ELISA.

41. The method of any one of claims 38 to 40, wherein the sample is a test sample from a subject.

42. The method of claim 41, wherein the sample comprises fluid, tissue or cells from an animal that was administered the chemically-modified oligonucleotide.

43. The method of claim 42, wherein the method is used for the determination of pharmacokinetics or biodistribution.

44. The method of any one of claims 38 to 40, wherein the sample is a manufactured batch of the oligonucleotide therapeutic, and where the method is optionally used for quality control.

45. A method of quantifying phosphorothioate (PS) stereochemistry in a PS-modified oligonucleotide, the method comprising contacting the oligonucleotide with a monoclonal antibody prepared by the method of claim 30 or claim 31, and quantifying the binding of the oligonucleotide to the candidate monoclonal antibody.

45. The method of claim 44, wherein the chemically-modified oligonucleotide is quantified using an assay selected from immunohistochemistry, immunofluorescence microscopy, ELISA, southern blot, northern blot, flow cytometry / FACS analysis, and immunoprecipitation.

46. A method of enriching or purifying an oligonucleotide comprising an enantiomer of phosphorothioate (PS) linkage, the method comprising contacting a sample comprising the oligonucleotide with a candidate monoclonal antibody prepared by the method of claim 30 or claim 31 to produce a complex of the candidate monoclonal antibody and the oligonucleotide, and eluting or sequestering the oligonucleotide bound to the candidate monoclonal antibody.

47. The method of claim 46, wherein the candidate monoclonal antibody is bound to a solid support, optionally wherein the solid support is a bead, optionally a magnetic bead, or a chromatography column.

48. The method of claim 46, wherein the method further comprises contacting the complex with a solid support capable of binding the candidate monoclonal antibody directly or indirectly, optionally wherein the solid support is a bead, optionally a magnetic bead, or a chromatography column.

49. A method of stereospecific oligonucleotide synthesis with respect to phosphorothioate (PS) linkage, the method comprising:(i) contacting a synthetic intermediate of the oligonucleotide with a candidate monoclonal antibody prepared by the method of claim 30 or claim 31 to produce a complex of the candidate monoclonal antibody and the intermediate comprising a specific enantiomer of PS linkage, and(ii) eluting the intermediate bound to the candidate monoclonal antibody or sequestering the intermediate bound to the candidate monoclonal antibody.

50. The method of claim 49, wherein the monoclonal antibody or the complex is bound to a solid support, optionally wherein the solid support is a bead, optionally a magnetic bead, or a chromatography column.

51. The method of claim 49 or 50, wherein the method further comprises (iii) subjecting the eluted intermediate to further synthesis, or subjecting the intermediate after sequestration of a specific enantiomer of PS linkage to further synthesis.

52. The method of claim 51, wherein the method further comprises repeating steps (i) and (ii).

53. The method of any one of claims 49 to 52, wherein the oligonucleotide synthesis is chemical synthesis, biochemical synthesis or a combination thereof.

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