Monoclonal antibodies that recognize 8-oxo-adenine in nucleic acids and their use in diagnostic and quality control assays

Abstract

The present invention relates to antigen-binding molecules, preferably antibodies or antigen-binding fragments thereof, that selectively bind to 8-oxo-adenine. The antibodies disclosed here recognize 8-oxo-adenine within a variety of nucleic acid substrates, including single-strand and double-strand RNA and DNA and 8-oxo-adenine-nucleotides. The invention further relates to immunocohjugates comprising the antigen-binding molecules. The antigen-binding molecules and immunoconjugates of the invention may be employed alone, or in combination for use in 8-oxo-adenine detection and quantification assays. The present invention also relates to nucleotide sequences encoding anti-8-oxo-adenine antibodies, and host cells, cell-free transcription-translation systems and cell-free translation systems containing the nucleotide sequences, and methods of making anti-8-oxo-adenine-antibodies. The antigen-binding molecules, antibodies and compositions of the invention are useful in DNA or RNA quality control assays and diagnostic or monitoring applications for diseases in which levels of 8-oxo-adenine are altered. The present invention also obviates the requirement for binding substrate denaturation.

Description

[0001] MONOCLONAL ANTIBODIES THAT RECOGNIZE 8-OXO-ADENINE IN NUCLEIC ACIDS AND THEIR USE IN DIAGNOSTIC AND QUALITY CONTROL ASSAYS

[0002] TECHNICAL FIELD

[0003] The present invention relates generally to antigen-binding molecules, and more specifically antibodies or antigen-binding fragments thereof, that selectively bind to the modified nucleobase of RNA or DNA, 8-oxo-adenine. The present invention further relates to novel monoclonal antibodies that recognize 8-oxo-adenine within a variety of nucleic acid substrates including single-strand and double-strand RNA and DNA and their precursors and decay fragments as well as 8-oxo-adenine-nucleotides. The invention further relates to immunoconjugates comprising the antigen-binding molecules, in particular the anti-8-oxo- adenine antibodies, or antigen-binding fragments thereof. The present invention also relates to nucleotide sequences encoding anti-8-oxo-adenine antibodies, and host cells, cell-free transcription-translation systems and cell-free translation systems containing the nucleotide sequences, and methods of making anti-8-oxo-adenine-antibodies. The antigenbinding molecules, antibodies and compositions of the invention are useful in DNA or RNA quality control assays and diagnostic or monitoring applications for diseases in which levels of 8-oxo-adenine are altered, in particular in which 8-oxo-adenine levels are increased, such as in autoimmune disease, cancer, cardiovascular disease, metabolic syndrome, and neurodegenerative disease.

[0004] BACKGROUND OF THE INVENTION

[0005] Oxidative damage to nucleic acids, and more generally oxidative stress, is a seminal feature of cancer, aging, neurodegenerative diseases, and metabolic syndrome (Olinski et al. 1992; Beckman and Ames, 1998; Shigenaga et al. 1994; Sies 2015; Hayes et al. 2020). The hydroxyl radical (*OH), formed in cells by ionizing radiation, chemical toxins, and rogue mitochondrial electrons, is the most potent nucleic acid oxidizer, generating a dosedependent assortment of nucleic acid modifications and strand breaks (Kang et al. 1998; Dizdaroglu and Jaruga, 2012; Raha and Robinson, 2000). The *OH modification of guanine at C8 (yielding 8-oxo-guanine) is often considered the most frequent oxidative lesion in DNA due to the low redox potential of the nucleobase and is specifically targeted for removal by DNA repair glycosylases (Steenken and Jovanovic, 1997; Dizdaroglu and Jaruga, 2012; Shigdel et al. 2020). Nevertheless, oxidized and potentially mutagenic species of all purine and pyrimidine nucleobases have been observed in both DNA and RNA (Loft and Poulsen, 1996; Liu et al. 1996; Yanagawa et al. 1992; Wallace 2022). Like its purine counterpart, adenine readily oxidizes at C8 (yielding 8-oxo-adenine; Steenken and Jovanovic, 1997; Dizdaroglu and Jaruga, 2012). 8-oxo-adenine predominantly exists as a keto tautomer distinguished from adenine by a C8 carbonyl and an N7 proton (Cho and Evans, 1991 ; Guschlbauer et al. 1991 ; Choi et al. 2017). Adenine is chemically considered the nucleobase second most prone to oxidation, with levels of 8- oxo-adenine in irradiated cells or purified DNA 3-6 fold less than 8-oxo-guanine (Ide et al. 1997; Jaruga and Dizdaroglu, 1996; Malins and Haimanot, 1990; Frelon et al. 2000; Cadet et al. 2003). However, in some instances 8-oxo-adenine frequency in DNA may be at near parity with 8-oxo-guanine, as observed in a rat and fish liver cancer and aging model (Wang et al. 1995). 8-oxo-adenine, along with the ring cleaved FapyA, an 8-oxo-adenine oxidation product, accumulate in a variety of cancers and in the DNA extracted from cells exposed to ionizing radiation, ischemia-reperfusion, and other sources of oxidative (*OH) stress (Jackson et al. 1989; Olinski et al. 1992; Jaruga and Dizdaroglu, 1996; Liu et al. 1996; Cui et al. 1999; Tuo et al. 2002). This increase arises either by direct oxidation of DNA or vis-a- vis DNA polymerase misincorporation of 8-oxo-dATP (Koag et al. 2019; Koag et al. 2020). The mutagenic potential of 8-oxo-adenine is driven by its structural propensity to form mispairs, especially when encountered by an error prone DNA polymerase (Kamiya et al. 1995; Koag et al. 2020; Jung and Lee, 2020), its influence on the excision rate of tandem abasic sites (Lomax et al. 2005), and the tendency of C2 to form protein cross-links (Nilov et al. 2013).

[0006] The 8-oxo-adenine nucleobase is also important in RNA function and RNA metabolism (Choi et al. 2017). Xanthine oxidase produces 8-oxo-adenine as an intermediate in purine catabolism and irradiation of polyadenylic acid (poly-A) generates 8-oxo-adenine and other adenine adducts (Wyngaarden and Dunn, 1957; Alexander et al. 1987). In highly damaged regions of the late-stage Alzheimer’s brain, mRNA accumulated 8-oxo-adenine moieties at levels greater than 8-oxo-guanine, hinting at a role for the poly-A tail as a redox sink (Weinder et al. 2011 ). Unlike 8-oxo-guanine, when 8-oxo-adenine arises in the DNA template, elongation by the RNA polymerase 11 -transcription factor IIS complex is inhibited (Kuraoka et al. 2007). When 8-oxo-adenine is localized to coding regions of mRNA, translation arrest produces truncated proteins and mRNA endonucleases are recruited to the ribosome to initiate no-go decay (NGD) (Calabretta et al. 2015; Simms et al. 2017; Yan and Zaher, 2019). Similarly, translation is attenuated when a catalytic adenosine (A2451) in 23S ribosomal RNA is C8 oxidized (Willi et al. 2018). Interestingly, 8-oxo-adenine derivatives are potent site 1 agonists of Toll-like receptor 7 (TLR7), a central innate immune sensor (Hirota et al. 2002; Isobe et al. 2006; Kurimoto et al. 2010; Koga-Yamakawa et al. 2013; Nakamura et al. 2013; Evans et al. 2019; Gential et al. 2019; Tojo et al. 2020), raising the prospect that lysosomal processing of damaged RNA or its precursors activate the innate immune system. Monoclonal antibodies capable of selectively recognizing specific nucleobase modifications have found diverse scientific applications, which include, among other methods, immunoaffinity chromatography, enzyme-linked immunosorbent assay (ELISA), Western blotting, and high-resolution immunohistochemistry. More recently, immunoprecipitation by modified nucleobase-recognizing monoclonal antibodies has been coupled with nextgeneration DNA / RNA sequencing to locate, quantify, and characterize nucleobase modifications across the genome and the transcriptome (Feederle and Schepers, 2017). While these technologies hold great promise for fundamental discoveries and clinical applications, their utility is dependent on the discovery of optimized monoclonal antibodies. Given the widespread involvement of the 8-oxo-adenine modification in nucleic acid function, metabolism, and the pathology of a diverse scope of diseases, 8-oxo-adenine is one such important epitope for research, diagnostic and therapeutic purposes. In the prior art, mouse monoclonal antibodies that bind to 8-oxo-adenine have been described (US6187551 B1 , US6900291 B2; Holmes and Greene, 2001 and 2005). The antibodies are able to discriminate 8-oxo-adenine from other nucleobase types in the context of ribonucleosides and are able to bind to (detect) 8-oxo-adenine in physiological DNA samples that have been denatured. However, there remains need for monoclonal anti-8- oxo-adenine antibodies having high on-target affinity and low cross-reactivity in combination with an ability to bind to and detect 8-oxo-adenine in the context of singlestranded RNA or double-helical RNA and double helical DNA molecules. By obviating the need to denature nucleic acid samples, such antibodies moreover minimize the introduction of oxidation artifacts (Bruskov et al. 2002). These technical problems are solved by the embodiments as defined in the claims and summary of the invention.

[0007] SUMMARY OF THE INVENTION

[0008] The prior art (US6187551 B1 , US6900291 B2) encompasses mouse monoclonal antibodies that bind to 8-oxo-adenine in the context of ribonucleosides and ribonucleotides and are able to bind to (detect) 8-oxo-adenine in physiological DNA samples that have been denatured. However, there remains need for monoclonal anti-8-oxo-adenine antibodies having high on-target affinity and low cross-reactivity in combination with an ability to bind to and detect 8-oxo-adenine in the context of single-stranded or double-helical RNA and DNA molecules, and especially in chemically oxidized DNA or RNA. This technical problem has been solved by the present invention, as outlined below.

[0009] The invention specifically relates to:

[0010] 1. An antigen-binding molecule that specifically binds to 8-oxo-adenine, wherein the antigen-binding molecule interacts with the hydrogen atom at position N7 of 8-oxo- adenine, either directly or via an ordered H2O molecule bridge, and / or with the oxygen atom at position 08 of 8-oxo-adenine. The antigen-binding molecule according to item 1 , wherein the antigen binding molecule further interacts with at least one of: a) the N atom at position 1 of 8-oxo-adenine; b) the N atom at position 3 of 8-oxo-adenine; c) the amino group at position 6 of 8-oxo-adenine; and / or d) the apical or basal faces of the purine ring of 8-oxo-adenine. The antigen-binding molecule according to item 1 or 2, wherein the 8-oxo-adenine is comprised in 8-oxo-adenosine or 8-oxo-deoxyadenosine. The antigen-binding molecule according to item 3, wherein the 8-oxo-adenosine is comprised in a ribonucleoside or a ribonucleotide or an RNA molecule or the 8- oxo-deoxyadenosine is comprised in a deoxyribonucleoside or a deoxyribonucleotide or a DNA molecule or the 8-oxo-adenosine is comprised in an adenine nucleotide derivative or an adenine derivative, including but not limited to, nicotinamide adenine dinucleotide (NAD) or flavin adenine dinucleotide (FAD) or 2’3’-cyclic adenosine monophosphate (2’3’-cAMP) or 3’5’-cyclic adenosine monophosphate (3’5’-cAMP) or 2’3’-cyclic guanosine monophosphate-adenosine monophosphate (2’3’-cGAMP) or 3’-phosphoadenosine 5’-monophosphate (pAp). The antigen-binding molecule according to item 3, wherein the 8-oxo-adenosine is comprised in a ribonucleoside or a ribonucleotide or a ribonucleotide triphosphate or an RNA molecule, including in vitro transcribed messenger RNA, or the 8-oxo- deoxyadenosine is comprised in a deoxyribonucleoside or a deoxyribonucleotide or a deoxyribonucleotide triphosphate or a DNA molecule or the 8-oxo-adenosine is comprised in an adenine nucleotide derivative or an adenine derivative, including but not limited to, nicotinamide adenine dinucleotide (NAD) or flavin adenine dinucleotide (FAD) or 2’3’ -cyclic adenosine monophosphate (2’3’-cAMP) or 3’5’- cyclic adenosine monophosphate (3’5’-cAMP) or 2’3’-cyclic guanosine monophosphate-adenosine monophosphate (2’3’-cGAMP) or 3’- phosphoadenosine 5’-monophosphate (pAp). The antigen-binding molecule according to any one of items 1 to 4, wherein the antigen-binding molecule is an antibody, an antigen-binding fragment thereof, an ankyrin repeat protein, an aptamer or another antibody mimetic. The antigen-binding molecule according to item 5, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a multi-specific antibody, or an antibody displayed upon the surface of a phage or displayed upon the surface of a chimeric antigen receptor (CAR) T cell. The antigen-binding molecule according to item 6, wherein the antibody is an I gG 1 , lgG2a or I gG2b, lgG3 or lgG4 antibody. The antigen-binding molecule of item 5, wherein the antigen-binding fragment is a Fab fragment, a F(ab')2 fragment or a Fv fragment. The antigen-binding molecule according to any one of items 5 to 8, wherein binding of 8-oxo-adenine is achieved through the variable heavy (VH) chain of the antibody. The antigen-binding molecule according to any one of items 5 to 9, wherein the antibody comprises a variable heavy (VH) chain comprising a CDR3 having the amino acid sequence SIWATRLWYFDV (SEQ ID NO: 5). The antigen-binding molecule according to any one of items 5 to 10, wherein the antibody comprises a variable heavy (VH) chain comprising a CDR1 having the amino acid sequence GYKFTTYWIN (SEQ ID NO: 3), a CDR2 having the amino acid sequence DFYPGNGSNNFNDKFRN (SEQ ID NO: 4) and a CDR3 having the amino acid sequence SIWATRLWYFDV (SEQ ID NO: 5). The antigen-binding molecule according to any one of items 5 to 11 , wherein the antibody comprises a variable heavy (VH) chain sequence comprising the amino acid sequence

[0011] QVQLQQPGAEWKPGTSVKLSCKASGYKFTTYWINWLKLRPGQGLEWIGDFYP GNGSNNFNDKFRNKATLTVDTSSNTAYMQLSSLASEDSALYYCARSIWATRLWY FDVWGAGTTVTVSS (SEQ ID NO: 1) or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 1 . The antigen-binding molecule according to any one of items 10 to 12, wherein the antibody comprises a variable light (VL) chain comprising a CDR3 having the amino acid sequence QQHYRLPFT (SEQ ID NO: 8). The antigen-binding molecule according to any one of items 10 to 13, wherein the antibody comprises a variable light (VL) chain comprising a CDR1 having the amino acid sequence RASQDVNTAVA (SEQ ID NO: 6), a CDR2 having the amino acid sequence WASTRHT (SEQ ID NO: 7) and a CDR3 having the amino acid sequence QQHYRLPFT (SEQ ID NO: 8). The antigen-binding molecule according to any one of items 10 to 14, wherein the antibody comprises a variable light (VL) chain sequence comprising the amino acid sequence DIVMTQSHKFMSTSVGDRISIPCRASQDVNTAVAWYQQKPGQSPKLLIYWASTR HTGVPDRFTGSGSGTDYALTISGVQAEDLTLYYCQQHYRLPFTFGGGTKLEIK (SEQ ID NO: 2) or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 2. The antigen-binding molecule according to any one of items 5 to 9, wherein the antibody comprises a variable heavy (VH) chain comprising a CDR3 having the amino acid sequence SADYLAMDY (SEQ ID NO: 13). The antigen-binding molecule according to any one of items 5-9 or 16, wherein the antibody comprises a variable heavy (VH) chain comprising a CDR1 having the amino acid sequence GYIFTSYWIN (SEQ ID NO: 11 ), a CDR2 having the amino acid sequence DFHPGRGITNNNEKFKT (SEQ ID NO: 12) and a CDR3 having the amino acid sequence SADYLAMDY (SEQ ID NO: 13). The antigen-binding molecule according to any one of items 5-9 or 16-17, wherein the antibody comprises a variable heavy (VH) chain sequence comprising the amino acid sequence

[0012] QVQLQQPGAELVKPGASVKMSCKASGYIFTSYWINWLRQRPGQGLEWIGDFH PGRGITNNNEKFKTKVKLTLETSSSTAYMQLSSLTSEDSAVYYCSRSADYLAMD YWGQGTSVTVSS (SEQ ID NO: 9) or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 9. The antigen-binding molecule according to any one of items 16 to 18, wherein the antibody comprises a variable light (VL) chain comprising a CDR3 having the amino acid sequence QNGHSFPPT (SEQ ID NO: 16). The antigen-binding molecule according to any one of items 16 to 19, wherein the antibody comprises a variable light (VL) chain comprising a CDR1 having the amino acid sequence RASQSISDYLH (SEQ ID NO: 14), a CDR2 having the amino acid sequence YASQPIS (SEQ ID NO: 15) and a CDR3 having the amino acid sequence QNGHSFPPT (SEQ ID NO: 16). The antigen-binding molecule according to any one of items 16 to 20, wherein the antibody comprises a variable light (VL) chain sequence comprising the amino acid sequence DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIRYASQPI SGIPSRFTGSGSGSDFTLTINSVEPEDVGVYYCQNGHSFPPTFGGGTKLEIK (SEQ ID NO: 10) or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 10. An antibody, or an antigen-binding fragment thereof, binding to the same epitope as an antibody, or antigen-binding fragment thereof, of any one of items 10 to 21 . A polynucleotide encoding the antibody, or the antigen-binding fragment thereof, or a polynucleotide sequence capable of hybridizing to said polynucleotide encoding the antibody or the antigen-binding fragment thereof, according to any one of items 5 to 22. A host cell comprising the polynucleotide of item 23. A method for producing an antibody, or an antigen-binding fragment thereof, comprising culturing the host cell according to item 24. A cell-free transcription-translation system or cell-free transcription system or cell- free translation system comprising the polynucleotide of item 23. A method for producing an antibody, or an antigen-binding fragment thereof, the method comprising a step of transcribing and / or translating the polynucleotide of item 23 in vitro', in particular wherein the antibody, or the antigen-binding fragment thereof, is produced with the cell-free transcription-translation system or the cell- free transcription system or the cell free translation system according to item 26. An immunoconjugate comprising the antibody, or the antigen-binding fragment thereof, according to any one of items 5 to 22. The immunoconjugate according to item 28, wherein the immunoconjugate comprises radioisotope or label. The immunoconjugate according to item 29, wherein the label is selected from the group of: an affinity tag, an enzyme, and a fluorescent tag. A method of detecting 8-oxo-adenine in a biological sample, the method comprising a step of contacting the biological sample with the antigen-binding molecule according to any one of items 1 to 22 or the immunoconjugate according to item 28 to 30 under conditions permissive for binding of the antigen-binding molecule to 8-oxo-adenine, and detecting whether a complex is formed between the antigen-binding molecule and the 8-oxo-adenine in the biological sample. The method according to item 31 further comprising a step of quantifying the amount of complex formed. A method for diagnosing or monitoring the status of a disease associated with elevated levels of 8-oxo-adenine in a human or animal comprising the steps of: a) providing a biological sample comprising nucleic acids that has been obtained and / or derived from the human or animal; b) contacting the biological sample with the antigen-binding molecule according to any one of items 1 to 22 or the immunoconjugate according to item 28 to 30 to form a complex with 8-oxo-adenine comprised in the biological sample; and c) determining the amount of complex formed as a measure of the presence or amount of 8-oxo-adenine in the biological sample, wherein the amount of complex determined is indicative of a disease state associated with elevated levels of 8-oxo-adenine or is correlated with the status of a disease associated with elevated levels of 8-oxo-adenine. The method according to item 33, wherein the disease associated with elevated levels of 8-oxo-adenine is cancer, autoimmune disease, aging-related diseases, cardiovascular disease, acute radiation syndrome, chronic inflammatory disease, neurodegenerative disease, metabolic syndrome, rheumatoid arthritis, systemic lupus erythematosus, or sickle cell anemia. A method for detecting or quantifying levels of 8-oxo-adenine in DNA or RNA comprising the steps of: a) providing a DNA or RNA sample that has been synthesized or produced in vitro or has been obtained and / or derived from a eukaryote, bacteria, archaea, virus, or bacteriophage. This includes, but is not limited to, DNA or RNA obtained and / or derived from human, plant, animal, fungi, protozoa, and other single-celled prokaryotes and their viruses. b) contacting the biological sample with the antigen-binding molecule according to any one of items 1 to 22 or the immunoconjugate according to item 28 to 30 to form a complex with 8-oxo-adenine comprised in the DNA or RNA sample; and c) determining the amount of complex formed as a measure of the presence or amount of 8-oxo-adenine in the sample, wherein the amount of complex determined is indicative of the oxidation extent or degradation level associated with the DNA or RNA sample or is correlated with a measure of sample quality control.

[0013] 36. The method according to any one of items 31 to 35, wherein the detection and / or quantification of the complex comprises one or more steps of immunohistochemistry, immunofluorescence imaging, immunoprecipitation, DNA immunoprecipitation, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin or DNA or RNA immunoprecipitation-based nucleic acid sequencing, enzyme-linked immunosorbent assay (ELISA), immunoblotting, ultra-sensitive immunoassay, Western blotting, radioimmunoassay, flow cytometry, fluorescence- activated cell sorting (FACS) or radiographic imaging.

[0014] 37. A kit for detecting the presence and / or quantifying the levels of 8-oxo-adenine in a sample; the kit comprising the antigen-binding molecule according to any one of items 1 to 22 or the immunoconjugate according to item 28 to 30.

[0015] 38. A kit for diagnosing or monitoring the status of a disease associated with elevated levels of 8-oxo-adenine in a eukaryote, bacteria, archaea, virus, or bacteriophage. This includes, but is not limited to, RNA obtained and / or derived from human, plant, animal, fungi, protozoa, and other single-celled prokaryotes and their viruses; the kit comprising the antigen-binding molecule according to any one of items 1 to 22 or the immunoconjugate according to item 28 to 30.

[0016] Accordingly, the present invention relates to antigen-binding molecules, preferably antibodies or antigen-binding fragments thereof, that selectively bind to 8-oxo-adenine in the context of the isolated base, deoxyribonucleoside, deoxyribonucleotide, ribonucleoside, ribonucleotide or when the binding substrate is a double-helical RNA or a DNA molecule.

[0017] That is, the present invention is based, at least in part, on the surprising discovery that antibodies can recognize 8-oxo-adenine within a variety of nucleic acid substrates, which include single-strand and double-strand RNA and DNA, including their precursors and decay fragments, and 8-oxo-adenine-nucleotides.

[0018] Accordingly, the invention relates to antigen-binding molecules that specifically bind to 8- oxo-adenine by interacting with the hydrogen atom at position 7 of 8-oxo-adenine and / or with the oxygen atom at position 8 of 8-oxo-adenine.

[0019] In the past, antibodies that bind to 8-oxo-adenine have been described in US6187551 B1 and US6900291 B2. In particular, LIS6187551 B1 / US6900291 B2 describes antibodies that are able to discriminate 8-oxo-adenine from other nucleobase types in the context of ribonucleosides and ribonucleotides and are able to bind to (detect) 8-oxo-adenine in physiological DNA samples that have been denatured through heat-treatment.

[0020] Accordingly, the antibodies of the present invention are advantageous over those known in the art since they bind to 8-oxo-adenine in folded or double-helical RNA or DNA. Therefore, the anti-8-oxo-adenine antibodies of the present invention also obviate the requirement for binding substrate denaturation, that is denaturation of the RNA or DNA samples by heattreatment prior to antibody binding. This allows for greater versatility and accuracy in the applications of the antibodies of the present invention. More specifically, this invention eliminates the need for heat-treatment of RNA or DNA samples, which is known to introduce oxidation artefacts into nucleic acid samples.

[0021] In some embodiments, the antigen-binding molecule of the invention is an antibody, or an antigen-binding portion thereof, a bispecific antibody, or an antigen-binding portion thereof, a designed ankyrin repeat protein (DARPIN), an aptamer or another antibody mimetic, such as affibody molecules, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, fynomers, kunitzdomain peptides, monobodies.

[0022] The antibody of the invention can be, inter alia, a monoclonal antibody, a chimeric antibody, a recombinant antibody, an antigen-binding fragment of a recombinant antibody, a humanized antibody, or an antibody displayed upon the surface of a phage or displayed upon the surface of a chimeric antigen receptor (CAR) T cell. The antibody of the invention can furthermore be an lgG1 , lgG2a, lgG2b, lgG3 or lgG4 antibody.

[0023] The invention also relates to an immunoconjugate comprising the antigen-binding molecules, in particular the anti-8-oxo-adenine antibodies, or antigen-binding fragments thereof.

[0024] In some embodiments, the antigen-binding molecules and immunoconjugates of the invention may be employed alone, or in combination with other antibodies, immunoconjugates or other diagnostic agents, for use in 8-oxo-adenine detection and quantification assays.

[0025] The present invention also relates to nucleotide sequences encoding anti-8-oxo-adenine antibodies, and host cells, cell-free transcription-translation systems and cell-free translation systems containing the nucleotide sequences, and methods of making anti-8- oxo-adenine-antibodies.

[0026] In some embodiments, methods are provided where the antigen-binding molecules, antibodies and compositions of the invention are employed in RNA quality control assays and diagnostic or monitoring applications for diseases in which levels of 8-oxo-adenine are altered, in particular in which 8-oxo-adenine levels are increased, such as in autoimmune disease, cancer, cardiovascular disease, aging-related diseases, metabolic syndrome, and neurodegenerative disease.

[0027] In some embodiments, methods for the quantification of 8-oxo-adenine in a nucleic acid (DNA or RNA) molecule are provided.

[0028] BRIEF DESCRIPTION OF THE FIGURES

[0029] FIG. 1 Design of the 8-oxo-adenosine (8oxoA) ELISA. Experimental overview of the competitive ELISA assay used to measure binding to 8oxoA. Wells are coated with goat anti-mouse IgG Fc domain and BSA as a blocking agent. A mixture of free 8oxoA, AChE conjugated to 8oxoA, and the indicated antibody are added to the well, followed by washing with buffer to remove unbound material. Ellman’s reagent and acetylthiocholine are added as substrate for bound AChE-8oxoA. The emission of yellow light is then monitored at 414 nm on a BioTek Epoch microplate spectrophotometer and absorbance plotted as % B / Bo.

[0030] FIG. 2A Antibody purification and titration. SDS-PAGE of 5 pg each of non-reduced (NR) and reduced (R) purified mAb 6E4 is shown after staining by Coomassie Blue. A control, mouse IgGkl mAb, is displayed for comparison. The location and molecular weight (kDa) of protein standards are exhibited on the left.

[0031] FIG. 2B Antibody purification and titration. Titration of purified mAb 6E4 binding to AChE- 8oxoA in the absence of competitor. The absorbance (A) at 414 nm is plotted as a function of the mass (ng) of purified mAb 6E4. The binding assay was performed as described in Materials and Methods.

[0032] FIG. 3A Antibody binding specificity for 8oxoA variants. The structure of 8oxoA; the 08 and H7 atoms that distinguish 8oxoA from A are shaded grey. The light grey box demarcates the ribose (R).

[0033] FIG. 3B Antibody binding specificity for 8oxoA variants. Specificity for 8oxoA determined by an AChE-8oxoA competitive ELISA. The binding assay was performed as described in Materials and Methods. The %B / Bo is plotted as a function of the concentration (nM) of the indicated competitor: 8-oxoadenosine (8oxoA); 8-oxoadenosine 5’ -triphosphate (8oxoATP); 8-oxo-2’-deoxyadenosine (8oxodA); 8-oxo-2’-deoxyadenosine 5’-triphosphate (8oxodATP); 8-oxoadenine (8oxoAde); Fapyadenine (FapyAde); adenosine (A). Each datum is the average of at least 2 independent experiments + / - SEM. %B / Bo is the ratio of A414 nm for a specific well divided by the maximum absorbance, Bo, of a competitor-free well. 100% B / Bo indicates no inhibition by the indicated competitor. FIG. 4A Antibody binding to pyrimidines, purines, and methylations. The %B / Bo is plotted as a function of the concentration (nM) of free 8oxoA or the indicated competing pyrimidine nucleoside: Uridine (U); 5-hydroxyuridine (5hoU); cytidine (C); 5-hydroxycytidine (5hoC).

[0034] FIG. 4B Antibody binding to pyrimidines, purines, and methylations. The %B / Bo is plotted as a function of the concentration (nM) of the indicated competing pyrimidine nucleoside: Adenosine (A); 8-oxoguanosine (8oxoG); guanosine (G); 2-oxoadenosine (2oxoA); inosine (I). 8oxoA is used as a control. Each datum is the average of at least 2 independent experiments + / - SEM.

[0035] FIG. 4C Antibody binding to pyrimidines, purines, and methylations. The %B / Bo is plotted as a function of the concentration (nM) of the indicated competing modified adenosine: N1- methyladenosine (m1A); N6-methyladenosine (m6A); and 8-aminoadenosine (8aminoA). 8oxoA is used as a control. Each datum is the average of at least 2 independent experiments + / - SEM.

[0036] FIG. 5 Detection of 8oxoA in single- and double-strand DNA. The structure of the 21 nt 8oxoA DNA oligonucleotide duplex is shown at top. The 8oxoA base is located at position 11 in the sense strand (ssDNA-8oxodA) and pairs with T in the antisense strand (dsDNA- 8oxodA). The ssDNAand dsDNA contain an unmodified A at position 21 of the sense strand. The ELISA binding assay was performed as described in Materials and Methods. The %B / Bo is plotted as a function of the concentration (nM) of the indicated competing DNA oligonucleotide. 8oxoA is used as a control.

[0037] FIG. 6 Detection of 8oxoA in mismatched DNA oligonucleotides. The structure of the 21 nt 8oxoA DNA oligonucleotide duplex is shown at top. The 8oxoA base, at position 11 in the sense strand, is variably paired with X in the antisense strand. The ssDNA-8oxodA is the unpaired sense strand. The ELISA binding assay was performed as described in Materials and Methods. The %B / Bo is plotted as a function of the concentration (nM) of the indicated competing DNA oligonucleotide. 8oxoA is used as a control.

[0038] FIG. 7 Detection of 8oxoA in single- and double-strand RNA. The structure of the 21 nt 8oxoA RNA oligonucleotide duplex is shown at top (dsRNA-8oxoA). The 8oxoA base is located at position 11 in the sense strand and pairs with U in the antisense strand. The ssRNA-8oxoA is the unpaired sense strand. The ssRNA and dsRNA contain an unmodified A at position 21 of the sense strand. The ELISA binding assay was performed as described in Materials and Methods. The %B / Bo is plotted as a function of the concentration (nM) of the indicated competing RNA oligonucleotide. 8oxoA is used as control.

[0039] FIG. 8A Detection of 8oxoA in mismatched RNA oligonucleotides. The structure of the 21 nt 8oxoA RNA oligonucleotide duplex is shown at top. The 8oxoA base, at position 11 in the sense strand, is variably paired with X in the antisense strand. The ssRNA-8oxoA is the unpaired sense strand. The ELISA binding assay was performed as described in Materials and Methods. The %B / Bo is plotted as a function of the concentration (nM) of the indicated competing RNA oligonucleotide. 8oxoA is used as control.

[0040] FIG. 8B Detection of 8oxoA in mismatched RNA oligonucleotides. Image of an 8oxoA:G mispair. 8oxoA is shown in the syn conformation forming two H-bonds with G in the anti- conformation. The two unique atoms that define 8oxoA, (C8oxo and N7 hydrogen) are shaded grey.

[0041] FIG. 9A Primary structure of 8oxoA antibodies. Sequence alignment of the variable heavy domains of mouse anti-8oxoA monoclonal antibody clone 6E4 in comparison to the sequence of anti-8oxoA clone 5G7. The complementary determining regions (CDR) are colored blue. Residues are numbered according to Kabat.

[0042] FIG. 9B Primary structure of 8oxoA antibodies. Sequence alignment of the variable light kappa domain of mouse anti-8oxoA monoclonal antibody clone 6E4 in comparison to the sequence of the mouse anti-8oxoA clone 5G7. The complementary determining regions (CDR) are shown bold. Residues are numbered according to Kabat.

[0043] FIG. 10 Homology model of mAb 6E4 bound to 8oxoAMP. scFv homology model of mouse 6E4 with a docked 8oxoAMP was created in SabPred ABodyBuilder-ML using the PDB 1JV5 model as a template. Figure was created in Pymol (Schrodinger, Inc, New York, NY, USA).

[0044] FIG. 11A 8oxoA sensing in polyadenylated mRNA. The architecture of a 996 nt in vitro transcribed messenger RNA encoding the enhanced green fluorescent protein (eGFP- mRNA) is depicted with a m7G cap 1 structure comprised of a 7-methyl guanosine inverted 5’-5’ triphosphate fused to a 2’-O-methyl adenosine (m7GpppAm) and a 120 nt 3’-polyadenylate tail. The eGFP-mRNA is shown before (top) and after (bottom) chemical oxidation with a Fenton reaction mixture of Fe2+(1 mM), HsOs G O mM), and ascorbic acid (1 mM). 8oxoA is rendered as a circle, while other possible oxidative modifications are depicted with varied shapes. The region between the rightward arrow and the stop codon indicates the 717 nt eGFP open reading frame.

[0045] FIG. 11 B 8oxoA sensing in polyadenylated mRNA. Competitive AChE-8oxoA ELISA utilizing the anti-8oxoA monoclonal antibody 6E4. The %B / Bo is plotted as a function of the concentration (nM) of the indicated competing mRNA: eGFP-mRNA (square) or oxidized eGFP-mRNA (hexagon). 8oxoA is used as a control. Each datum is the average of at least 2 independent experiments + / - SEM. FIG. 12 Detection of 8oxoA in Total Human Universal Reference RNA. A sample of total RNA extracted from a mixture of primary human tissues was oxidized by the Fenton reaction described above and analyzed for 8oxoA content by competitive ELISA. A non-oxidized sample of the total human RNA was used as a control. The % B / Bo is plotted as a function of RNA concentration (pg / ml). Untreated, total human RNA (square) or in vitro oxidized total human RNA (circle) were employed as competitive antigens. Each datum is the average of at least 2 independent experiments + / - SEM.

[0046] FIG. 13 Determining 8-oxoA Sensitivity of Clones 5G7 and 6E4. The %B / Bo is plotted as a function of the concentration (nM) of 8-oxoA. The indicated curves are as follows: Clone 5G7 supernatant at 1 :3000 dilution (circles); clone 6E4 supernatant at 1 :1200 dilution (squares); clone 6E4 at 70 ng / ml after purified by protein G (triangles), and clone 6E4 at 500 ng / ml purified by HiTrap Blue (diamonds).

[0047] DETAILED DESCRIPTION

[0048] An “antigen-binding molecule,” as used herein, is any molecule that can specifically or selectively bind to an antigen. A binding molecule may include or be an antibody or a fragment thereof. An anti-8-oxo-adenine binding molecule is a molecule that binds to the 8- oxo-adenine antigen, such as an anti-8-oxo-adenine antibody or fragment thereof, at a specific recognition site, epitope as detailed further above. That is, antigen-binding molecules of the invention bind to an epitope within deoxyribonucleoside or deoxyribonucleotide or ribonucleoside or ribonucleotide or a polynucleotide sequence of an RNA or DNA molecule. Other anti-8-oxo-adenine binding molecules may also include multivalent molecules, multi-specific molecules (e.g., diabodies), fusion molecules, aptamers, avimers, or other naturally occurring or recombinantly created molecules. Illustrative antigen-binding molecules useful in the present invention include antibody-like molecules. An antibody-like molecule is a molecule that can exhibit functions by binding to a target molecule (See, e.g., Current Opinion in Biotechnology 2006, 17:653-658; Current Opinion in Biotechnology 2007, 18:1-10; Current Opinion in Structural Biology 1997, 7:463- 469; Protein Science 2006, 15:14-27), and includes, for example, DARPins (WO 2002 / 020565), Affi body (WO 1995 / 001937), Avimer (WO 2004 / 044011 ; WO 2005 / 040229), Adnectin (WO 2002 / 032925) and fynomers (WO 2013 / 135588).

[0049] The terms "anti-8-oxo-adenine antibody" and "antigen-binding molecule that specifically binds to 8-oxo-adenine" as used herein refer to an antibody that is capable of binding 8- oxo-adenine with sufficient affinity and selectivity such that the antibody is useful in quality control assays and diagnostic or monitoring applications in detecting 8-oxo-adenine. In general, the term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), fully mouse antibodies and antibody fragments so long as they exhibit the desired antigen-binding activity. Antibodies within the present invention may also be chimeric antibodies, recombinant antibodies, antigen-binding fragments of recombinant antibodies, humanized antibodies or antibodies displayed upon the surface of a phage or displayed upon the surface of a chimeric antigen receptor (CAR) T cell.

[0050] An "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab' -SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); single-domain antibody molecules (nanobodies); and multispecific antibodies formed from antibody fragments.

[0051] The term “specific binding” or “specifically binds” as used in accordance with the present invention means that the antibody or antigen-binding fragment thereof of the invention does not or does not essentially cross-react with nucleobases of similar structure as 8- oxo-adenine. Accordingly, the antibody or antigen-binding fragment thereof of the invention specifically binds to / interacts with 8-oxo-adenine in the context of the isolated base or deoxyribonucleoside or deoxyribonucleotide or ribonucleoside or ribonucleotide or when the binding substrate is a single-stranded or double-helical RNA or DNA molecule. Specific examples of such molecules are provided herein.

[0052] The term “monoclonal antibody” 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 mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are advantageous in that they may be synthesized by a hybridoma culture, essentially uncontaminated by other immunoglobulins. The modified "monoclonal" indicates the character of the antibody as being amongst a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. As mentioned above, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method described by Kohler and Milstein, 1975.

[0053] The term “polyclonal antibody” as used herein, refers to an antibody which was produced among or in the presence of one or more other, non-identical antibodies. In general, polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B-lymphocytes which produced non-identical antibodies. Usually, polyclonal antibodies are obtained directly from an immunized animal.

[0054] The term “chimeric antibodies”, refers to an antibody which comprises a variable region of the present invention fused or chimerized with an antibody region (e.g., constant region) from another, human or non-human species (e.g., mouse, horse, rabbit, dog, cow, chicken).

[0055] The term "recombinant antibody" includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (other than mouse) that is transgenic for mouse immunoglobulin genes; antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial mouse antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of mouse immunoglobulin gene sequences to other DNA sequences. Such recombinant mouse antibodies have variable and constant regions (if present) derived from mouse germline immunoglobulin sequences. Such antibodies can, however, be subjected to in vitro mutagenesis (or, when an animal transgenic for mouse Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to mouse germline VH and VL sequences, may not naturally exist within the mouse antibody germline repertoire in vivo.

[0056] The term antibody also relates to humanized antibodies. "Humanized" forms of nonhuman (e.g. murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Often, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibody may comprise residues, which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see: Jones Nature 321 (1986), 522-525; Reichmann Nature 332 (1998), 323-327 and Presta Curr Op Struct Biol 2 (1992), 593-596.

[0057] A popular method for humanization of antibodies involves CDR grafting, where a functional antigen-binding site from a non-human ‘donor’ antibody is grafted onto a human ‘acceptor’ antibody. CDR grafting methods are known in the art and described, for example, in US 5,225,539, US 5,693,761 and US 6,407,213. Another related method is the production of humanized antibodies from transgenic animals that are genetically engineered to contain one or more humanized immunoglobulin loci which are capable of undergoing gene rearrangement and gene conversion (see, for example, US 7,129,084).

[0058] Accordingly, in context of the present invention, the term “antibody” relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules (i.e., “antigen-binding fragment thereof”). Furthermore, the term relates, as discussed above, to modified and / or altered antibody molecules. The term also relates to recombinantly or synthetically generated / synthesized antibodies. The term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fv, Fab’, Fab’-SH, F(ab’)2. The term antibody also comprises but is not limited to fully mouse antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies and antibody constructs, like single-chain variable fragments (scFvs), single-domain antibodies (nanobodies), or antibody-fusion proteins.

[0059] “Single-chain variable fragment” “single-chain Fv” or “scFv” antibody fragments have, in the context of the invention, the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. Techniques described for the production of single chain antibodies are described, e.g., in Pluckthun in The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore eds. Springer-Verlag, N.Y. (1994), 269- 315.

[0060] A “Fab fragment” as used herein is comprised of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. An "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0061] A "Fab1fragment" contains one light chain and a portion of one heavy chain that contains the VH domain and the C H1 domain and also the region between the CH1 and C H2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0062] A "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

[0063] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0064] A ’’single-domain antibody” comprises the variable heavy chain or variable light chain only. A “nanobody” comprises a single antigen binding fragment derived from the camelid VHH domain or engineered by camelization of a mammalian VH domain. A single-domain antibody or nanobody thus is composed of a single monomeric variable antibody domain.

[0065] Antibodies, antibody constructs, antibody fragments, antibody derivatives (all being Ig- derived) to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and / or recombination(s) and / or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook (1989), loc. cit. The term “Ig- derived domain” particularly relates to (poly) peptide constructs comprising at least one CDR. Fragments or derivatives of the recited Ig-derived domains define (poly) peptides which are parts of the above antibody molecules and / or which are modified by chemical / biochemical or molecular biological methods. Corresponding methods are known in the art and described inter alia in laboratory manuals (see Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition (1989) and 3rd edition (2001 ); Gerhardt et al., Methods for General and Molecular Bacteriology ASM Press (1994); Lefkovits, Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press (1997); Golemis, Protein- Protein Interactions: A Molecular Cloning Manual Cold Spring Harbor Laboratory Press (2002)).

[0066] The term “CDR” as employed herein relates to “complementary determining region”, which is well known in the art. The CDRs are parts of immunoglobulins that determine the specificity of said molecules and make contact with a specific ligand. The CDRs are the most variable part of the molecule and contribute to the diversity of these molecules. There are three CDR regions CDR1 , CDR2 and CDR3 in each V domain. CDR-H depicts a CDR region of a variable heavy chain and CDR-L relates to a CDR region of a variable light chain. VH means the variable heavy chain and VL means the variable light chain. The CDR regions of an Ig-derived region may be determined as described in Kabat “Sequences of Proteins of Immunological Interest”, 5th edit. NIH Publication no. 91-3242 U.S. Department of Health and Human Services (1991 ); Chothia J. Mol. Biol. 196 (1987), 901-917 or Chothia Nature 342 (1989), 877-883.

[0067] Accordingly, in the context of the present invention, the antibody molecule described herein above is selected from the group consisting of a full antibody (immunoglobulin, like an lgG1 , an lgG2, an lgG2a, an lgG2b, an lgA1 , an lgGA2, an lgG3, an lgG4, an IgA, an IgM, an IgD or an IgE), F(ab)-, Fab’-SH-, Fv-, Fab’-, F(ab’)2- fragment, a chimeric antibody, a CDR-grafted antibody, a fully mouse antibody, a nanobody, a bivalent antibody-construct, an antibody-fusion protein, a synthetic antibody, bivalent single chain antibody, a trivalent single chain antibody and a multivalent single chain antibody.

[0068] “Humanization approaches” are well known in the art and in particular described for antibody molecules, e.g. Ig-derived molecules. The term “humanized” refers to humanized forms of non-human (e.g., murine) antibodies or fragments thereof (such as Fv, Fab, Fab’, F(ab’), scFvs, nanobodies, or other antigen-binding partial sequences of antibodies) which contain some portion of the sequence derived from non-human antibody. Humanized antibodies include human immunoglobulins in which residues from a complementary determining region (CDR) of the human immunoglobulin are replaced by residues from a CDR of a non-human species such as mouse, rat or rabbit having the desired binding specificity, affinity and capacity. In general, the humanized antibody will comprise substantially all of at least one, and generally two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin ; see, inter alia, Jones et al., Nature 321 (1986), 522-525, Presta, Curr. Op. Struct. Biol. 2 (1992), 593-596. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acids introduced into it from a source which is non-human still retain the original binding activity of the antibody. Methods for humanization of antibodies / antibody molecules are further detailed in Jones et al., Nature 321 (1986), 522-525; Reichmann et al., Nature 332 (1988), 323-327; and Verhoeyen et al., Science 239 (1988), 1534-1536. Specific examples of humanized antibodies, e.g. antibodies directed against EpCAM, are known in the art, see e.g. (LoBuglio, Proceedings of the American Society of Clinical Oncology Abstract (1997), 1562 and Khor, Proceedings of the American Society of Clinical Oncology Abstract (1997), 847).

[0069] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, E, y, and p, respectively.

[0070] The terms “nucleic acid,” “nucleic acid molecule” and “polynucleotide” may be used interchangeably and include both single-strand and double-strand RNA, DNA and RNA:DNA hybrids. These terms are intended to include, but are not limited to, a polymeric form of nucleotides that may have various lengths, including deoxyribonucleotides and / or ribonucleotides, or analogs or modifications thereof. A nucleic acid molecule may encode a full-length polypeptide or RNA or a fragment of any length thereof, or may be non-coding. Nucleic acids can be naturally-occurring, modified, or synthetic polymeric forms of nucleotides.

[0071] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0072] The term “cell-free system" may include a combination of cytoplasmic and / or nuclear components from cells. The components may include extracts, purified components, or combinations thereof. The extracts, purified components, or combinations thereof include reactants for protein synthesis, transcription, translation, DNA replication and / or additional biological reactions occurring in a cellular environment identifiable by a person skilled in the art.

[0073] The terms “cell-free transcription-translation system”, “cell-free transcription system” and “cell-free translation system” refer to a system that is able to conduct transcription and / or translation outside of the context of a cell. In the art, such systems are also referred to as “cell-free system”, “cell-free transcription and translation”, “cell-free transcription”, “cell-free translation”, “TX-TL”, “TXTL”, “TX / TL”, “extract systems”, “in vitro system”, “ITT”, or “artificial cells.” Exemplary in vitro transcription and translation systems include purified or partially purified protein systems that are made from hosts, purified or partially purified protein systems that are not made from hosts, and protein systems made from a host strain that is formed as an “extract”. In an embodiment, extracts include whole-cell extracts, nuclear extracts, cytoplasmic extracts, combinations thereof, and the like. Whole-cell extracts are also termed lysates. Lysates, and lysate systems, are intended to be non-limiting examples of extracts; where lysate is described, it is contemplated that other extracts, or extracts and protein combinations, may be used.

[0074] An "immunoconjugate" is an antibody conjugated with or coupled to or linked to or associated with or covalently bound to one or more heterologous molecule(s), including but not limited to an affinity tag, an enzyme, and a fluorescent tag.

[0075] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0076] All publications, patent applications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference(s) should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document is authoritative. EXAMPLES

[0077] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

[0078] Example 1: Design, Construction, and Selection of an Anti-8-0xo-Adenine Monoclonal Antibody

[0079] An 8-oxoadenosine (8oxoA or 8-oxoA) antigen was constructed by fusing periodate- oxidized 8oxoA (8oxoA-dialdehyde) to keyhole limpet hemocyanin (KLH-8oxoA) and then immunizing three BALB / c female mice. Serum was screened for 8oxoA affinity and specificity by a competitive ELISA assay whereby free antigens are used to disrupt antibody binding to an 8oxoA acetylcholinesterase conjugate (AchE-8oxoA) (Figure 1 ). Binding to 8oxoA is measured by monitoring hydrolysis of acetylthiocholine by the 8oxoA-fused AChE and monitoring absorbance at 414 nm after addition of Ellman’s reagent.

[0080] Serum from the mouse exhibiting the strongest 8oxoA-specific titer was sacrificed and prepared for splenocyte fusion with the plasmacytoma line P3X63Ag8.653. Of the ~90 stable hybridoma clones, we proceeded to characterize clone 6E4 due to its affinity for both 8oxoA and a 21 mer RNA oligonucleotide harboring a central 8oxoA (ssRNA-8oxoA) and its low cross-reactivity with nucleosides A, G, and 8-oxo-guanine (8oxoG; > 1000-fold competitor concentration required relative to 8oxoA). A mouse-specific, lateral flow assay was used to confirm the isotype encoded in clone 6E4 is IgGkl . We also confirmed an IgGkl isotype for a second hybridoma, clone 5G7, with robust specificity for 8oxoA (Fig. 13).

[0081] A. Construction of the Antigen and Tracer

[0082] An ~10 mg / mL solution of 8-oxoadenosine (8oxoA) (TCI America) in ddFLO was treated with 2 equivalents of sodium periodate (NalO4) overnight at room temperature (RT) and protected from light to convert the ribose 2',3'-vicinal diol into a dialdehyde. The reaction was quenched by addition of 50 pl of 100% ethylene glycol.

[0083] 4 mg of 8oxoA dialdehyde intermediate was mixed with 4 mg of keyhole limpet hemocyanin (KLH) (Thermo-Fisher) in a total volume of 838 pl, followed by the addition of 400 pl of 100 mM sodium borate pH 8.5. The reaction mix was incubated for 2 h at RT, and then 4 mg of sodium cyanoborohydride (NaBHsCN) was added and further incubated overnight at RT and protected from light. The 8oxoA-KLH conjugation reaction was dialyzed twice into 4L of phosphate buffered saline (PBS) to remove unreacted 8oxoA and reaction components. Protein concentration in the dialyzed 8oxoA-KLH was quantified by the bicinchoninic acid assay (Pierce™ BCA assay; Thermo-Fisher). Presence of 8oxoA in both antigen and tracer was monitored by a wavelength scan with max of 271 nm. 8oxoA-KLH was frozen and stored at -20°C until further use. The activated 8oxoA dialdehyde (1 mg) was similarly fused to 500 II of E. electricus acetylcholinesterase (AChE) (Cayman Chemical) vis-a-vis incubation in 25 mM borate buffer pH 9.5 for ~2 h at room temperature, followed by treatment with NaBHsCN as described.

[0084] B. Immunization

[0085] 3 BALB / c female mice (Charles River) were immunized intraperitoneally (i.p.), when 28 days old, with 50 pg of KLH-8oxoA solubilized in equimolar Freund’s complete adjuvant in PBS, followed by i.p. boosters of 50 pg KLH-8oxoA resuspended in Freund’s incomplete adjuvant 14 and 35 days later. Mice were i.p. injected ~21 days later with 100 pl of KLH- 8oxoA without adjuvant. Serum was harvested from the submandibular vein ~24 and 44 days after initial immunization for titer determination by competitive 8oxoA ELISA. Serum from the mouse with the highest 8oxoA-specific titer was selected for fusion.

[0086] C. Hybridoma Production

[0087] Mice were euthanized by CO2 inhalation followed by cervical dislocation. Spleens were harvested and prepared for fusion. Hybridoma’s were generated by electrofusion of Dulbecco’s Modified Eagle Medium (DMEM)-washed splenocytes with the P3X63Ag8.653 (ATCC® CRL-1580™) plasmacytoma line in the presence of polyethylene glycol 1450 at 37°C and 5% CO2. Fusions were cultured in hypoxanthine-aminopterin- thymidine (HAT) medium. Culture media from HAT-resistant hybridomas was assayed for 8oxoA specificity by competitive ELISA. 8oxoA-specific parental hybridomas were then cloned by limiting dilution at 1 cell per well. Clones were then cultured in RPMI 1640, 2 mM glutamine, 10% fetal bovine serum (FBS), and prepared by centrifugation at 1 ,000 rpm for 10 min at 4°C. Cell pellets were resuspended at ~107cells / ml in RPMI 1640 with 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide. Aliquots of 1 ml were distributed into cryovials, stored overnight at -80°C, and then transferred the into liquid N2.

[0088] Example 2: Monoclonal Antibody Purification and Determination of 8-Oxo-Adenine Binding and Selectivity

[0089] To discern its nucleobase specificity, we purified monoclonal antibody (mAb) 6E4 by protein G chromatography from the supernatant of hybridoma 6E4 cultured in ultra-low IgG fetal bovine serum (Figure 2A). We then titrated increasing mass of purified 6E4 and measured its ability to bind the 8oxoA-AChE conjugate in a non-competitive ELISA assay (Figure 2B). 6E4 exhibited a dose-dependent ability to capture 8oxoA-AChE. All subsequent assays were then carried out using an antibody concentration extrapolated from the linear range of the titration curve.

[0090] Purified 6E4 was then assayed by competitive ELISA on a series of 8oxoA derivatives to determine the requirement of the exocyclic 8oxo moiety for antibody binding and how nucleobase specificity is affected by the presence of a ribose, 2’-deoxyribose, or 5’- triphosphate (Figure 3). The structure of 8oxoA is depicted in Figure 3A, highlighting the exocyclic 8oxo and the additional H-atom at N7. The ability of increasing concentrations of competitive 8oxoA variants (nM) to dislodge 6E4 from 8oxoA-AChE is depicted in Figure 3. The relative affinities, as gauged by ICso (Table 1), indicate a preference for the 8-oxo- adenine (8oxoAde) nucleobase = 20 nM)

[0091] (8oxoAde>8oxodA>8oxodATP>8oxoA>8oxoAT . The presence of a ribose 2’-OH is slightly repulsive (2.4-fold decrease odA vs. 8oxoA) and a 5’- triphosphate is well tolerated when fused to 8o nucleosides (ICso = 91 nM and 29 nM, respectively). The 8oxoAde oxidative cleavage product, FapyAde, is an ineffective competitor.

[0092] A. Antibody Purification, Characterization, and Isotyping

[0093] The anti-8oxoA hybridoma clone 6E4 was sequentially adapted to an ultra-low IgG FBS media (Avantor Seradigm). Hybridoma cell culture supernatant was then applied to a protein G chromatography column in the presence of Gentle Ag / Ab Binding Buffer (pH 8.0) (Thermo Scientific). Bound antibodies were eluted with Thermo Gentle Ag / Ab Elution Buffer (pH 6.6) dialyzed first into Tris-buffered saline (TBS) and then PBS. Fractions were then either stored at -20°C or mixed 1 :1 with ELISA buffer and stored at 4°C. The purified antibody isotype was determined with the mouse-specific Pierce™ Rapid Antibody Isotyping Kit (Thermo Scientific).

[0094] B. Competitive ELISA

[0095] A goat polyclonal antibody targeting the Fc domain of mouse IgG was coated on a 96 well surface, blocked in the presence of bovine serum albumin (BSA). Binding assays (150 pl) were carried out in ELISA buffer (100 mM phosphate, 400 mM NaCI, 1 mM EDTA, 0.1 % BSA, 0.01 % sodium azide) containing AChE-8oxoA conjugate (3.75 mU), competitive antigen at the indicated concentration, and either dilutions of immunized serum or protein G purified mAb 6E4 (5 ng). Binding was initiated by addition of the immunized serum or mAb and then incubated for 2 h at RT. Antibody titration experiments against AChE-8oxoA were performed in the absence of competitor.

[0096] Table 1

[0097] Summary of IC50and Cross Reactivities for mAb 6E4

[0098] Competitor IC50(nM) Fold Change*

[0099] 8oxoAde 20 0.3

[0100] 8oxodA 27 0.4

[0101] 8oxodATP 29 0.5 8oxoA 65 1.0

[0102] 8oxoATP 91 1.4 dsRNA-8oxoA:C 632 10 dsRNA-8oxoA:A 671 10 ssRNA-8oxoA 692 11 ssDNA-8oxoA 774 12 dsRNA-8oxoA:U 861 13 dsDNA-8oxodA:dA 875 13 dsDNA-8oxodA:dG 1151 18 dsDNA-8oxodA:dC 1206 18 dsDNA-8oxodA:dT 1298 20 dsRNA-8oxoA:G 4597 70 ssDNA nc** / dsDNA nc /

[0103] 8aminoA nc /

[0104] 2oxoA nc / ssRNA nc / dsRNA nc /

[0105] 8oxoG nc /

[0106] FapyAde nc /

[0107] C nc /

[0108] 5hoU nc /

[0109] G nc /

[0110] U nc /

[0111] A nc /

[0112] I nc / m1A nc / m6A nc /

[0113] * ( IC50Competitor) / (IC508oxoA)

[0114] ** Not calculated

[0115] To visualize binding, Ellman’s reagent (5,5’-dithio-b / s-2 nitrobenzoic acid) and the AChE substrate acetylthiocholine were added and then plates were developed in the dark for at least 2 h (Bo>1 .0). The emission of yellow light was monitored at 414 nm on a BioTek Epoch microplate spectrophotometer and absorbance plotted as % bound (B / Bo) versus log competitor concentration using a 4-parameter logistic fit. ICso values were calculated from the 4-paramater logistic fit. Plots and renderings displayed in figures were created in Prism 9 (GraphPad).

[0116] Nucleoside and nucleobase competitors were purchased from Carbosynth-Biosynth, Cayman Chemical, Medchem, LGC Biosearch, and Sigma. 8oxoATP and 8oxodATP were purchased from TriLink.

[0117] Example 3: Characterization of Anti-8-Oxo-Adenine Antibody Binding of and Selectivity against Different Pyrimidine, Purine, and Methylated Base Substrates

[0118] Selectivity of an 8oxoA mAb must be robust in the presence of other pyrimidine and purine nucleosides to be effective in complex mixtures of nucleic acids that include precursors, degradation products, and polynucleotide chains. Furthermore, oxidized variants of the other nucleosides can also be elevated by oxidative stress and display exocyclic oxygen atoms that could serve as 6E4 epitopes. We thus expanded our investigation of the binding properties of mAb 6E4 to the native pyrimidine ribonucleosides, uridine (U) and cytidine (C), along with their most common oxidation products, 5-hydroxyuridine (5hoU) and 5- hydroxycytidine (5hoC), both of which occur as native modifications in cells and are enriched after oxidative stress (Loft and Poulsen, 1996; Liu et al. 1996; Yanagawa et al. 1992; Wallace 2022) (Figure 4A). The native pyrimidine nucleosides U and C were unable to compete off mAb 6E4 bound to AChE-8oxoA. Similarly, the exocyclic oxidation products, 5hoU and 5hoC were also poor competitors to the AChE-8oxoA antigen. These findings indicate neither native nor oxidized pyrimidine nucleosides are targeted by mAb-6E4, and that the antibody specifically detects 8oxoA in the context of native or commonly oxidized nucleosides.

[0119] We next surveyed native purine nucleosides, adenosine (A) and guanosine (G), along with important oxidized, purine variants: 2-oxoadenosine (2oxoA), a cytotoxic, oxidation product of adenosine (Asada et al. 2017) and 8-oxoguanosine (8oxoG), the most ubiquitous oxidized base, which like 8oxoA harbors an N7 proton and a C8 oxo moiety (Shigdel et al. 2020). Inosine (I), an N6 deaminated adenosine with essential biological roles in both DNA and RNA, was also studied. The nucleosides A, G, and I were not competitors for mAb- 6E4, even as concentrations were increased to 100 pM, indicating N6 / O6 variants or an exocyclic amine at C2 do not induce cross-reactivity. 8oxoG, a purine nucleoside harboring the same exocyclic oxygen atom at C8 as 8oxoA, was similarly ineffective in displacing mAb 6E4 from AChE-8oxoA. Finally, 2oxoA (isoguanosine) was also a poor competitor out to 1 M (B / Bo = 100%), with modest antibody binding at doses >10 pM (IC50 = 73 pM). These outcomes further substantiate the 8oxoA specificity of mAb 6E4.

[0120] We proceeded to measure cross-reactivity with three distinct, modified adenosine analogues. Binding by 6E4 to either N1-methyladenosine (m1A), a modification originally discovered in non-coding RNA and important for RNA structure and stability (Jin etal. 2022), or N6-methyladenosine (m6A), a pervasive, dynamic modification in eukaryotic mRNA with roles in splicing, stability, and translation (Zaccara et al. 2019; Matsuzawa et al. 2019) was undetectable by competitive ELISA (Figure 4C). We then examined 8-aminoadenosine (8aminoA), a chemotherapeutic agent that inhibits multiple steps in RNA synthesis and processing including polyadenylation (Frey and Gandhi, 2010). Unlike m1A and m6A, 8aminoA is structurally comparable to 8oxoA in that it harbors a C8 modification (though one with H-bond donors). Nevertheless, mAb 6E4 is able to effectively discriminate 8aminoA from 8oxoA (>1000-fold increase in competitor required to reach 50% B / Bo), though 8aminoA is a superior antigen to the described methylated adenosines.

[0121] Example 4: Antibody Recognition of 8-Oxo-Deoxyadenosine in Single- and DoubleStrand DNA

[0122] The most prevalent adenine lesion in DNA damaged by ionizing radiation is 8-oxo- deoxyadenosine (8oxodA; Bonicel et al. 1980). A single 8oxodA in single-strand DNA (ssDNA) alters adjacent base-stacking and phosphodiester conformation (Malins et al. 2000). When correctly paired with dT, 8oxodA does not alter the structural properties of the DNA duplex by adopting the anf / -orientation (Guschlbauer ef al. 1991). The structures of an 8oxodA:G base pair in double-strand DNA (dsDNA) shows a syn.anti Hoogsteen arrangement that involves H-bonds between the 8oxo moiety and the N1 of guanine (Leonard et al. 1992). To determine the ability of mAb-6E4 to identify 8oxodA embedded in single-strand and correctly paired double-strand DNA, we synthesized a 21 nt DNA oligonucleotide containing a central 8oxodA residue at position 11 and then annealed the ssDNA-8oxodA to its complementary anti-sense strand to create a dsDNA with a central 8oxodA (Figure 5). Versions of the ssDNA and dsDNA antigens in which position 11 is an unmodified dA were used as controls. mAb 6E4 effectively binds both ssDNA-8oxodA and dsDNA-8oxodA, though the affinity is inferior to the nucleoside 8oxoA (Table 1 & Figure 5). Recognition of 8oxoA is efficient in the ssDNA and dsDNA (ICso = 774 nM and 1.3 pM, respectively). The corresponding unmodified ssDNA and dsDNA oligonucleotides are both poor antigens, with a 6.2-fold and 4.3-fold decrement, respectively, at 6.25 pM. These findings highlight that mAb 6E4 is able to detect a site-specific, individual 8oxodA in ssDNA and dsDNA.

[0123] A. Deoxyoligonucleotide Binding Substrates

[0124] DNA oligonucleotides were chemically synthesized at Integrated DNA Technologies (IDT). DNA duplex was annealed by mixing sense and anti-sense strands at a 1 :1 .2 molar ratio in 10 mM Tris-HCI (pH 7.5), 50 mM NaCI, and 1 mM EDTA in sterile, nuclease free ddH?O and then heating to 95°C. The annealing reactions were then slowly cooled to room temperature (~22°C) over 45 minutes, followed by centrifugation at -10,000 rpm. described methylated adenosines. Example 5: Effect of Base Mispairs with 8-Oxo-Deoxyadenosine Embedded in Double-Strand DNA on Antibody Recognition of the 8-Oxo-Adenine

[0125] We created 4 distinct duplex DNA oligonucleotides in which we varied the base (X) opposite 8oxodA to better understand the properties of mAb-6E4 when encountering potential mutagenic 8oxodA mispairs (Table 1 & Figure 6). The results of the competitive ELISA shown in Figure 6 show clear dose-dependent binding to 8oxodA in all DNA oligonucleotides tested. Varying the base (T,C,A,G) that pairs with 8oxodA only modestly alters 6E4’s ability to detect the lesion in dsDNA. Additionally, 8oxodA is distinguished with similar facility in single- and double-strand DNA contexts. The comparable ICso magnitudes suggest that 8oxodA is predominantly present in the ant / -conformation in all 4 duplex DNA antigens, rendering the 8oxo epitope available for antibody binding. Amongst the mispairs, 6E4 marginally prefers 8oxodA pairing in the order A>G>C>T. Interestingly, 6E4’s affinity for the 8oxodA:dA pair (ICso = 875 nM) is nearly equivalent to that of ssDNA-8oxodA (ICso - 775 nM) and moderately superior to the other 3 mispairs. We conclude that recognition of 8oxodA in DNA is largely driven by nucleobase specificity and epitope accessibility, a property in duplex DNA enhanced by the 8oxodA:dA mispair.

[0126] Example 6: Antibody Recognition of 8-0xo-Adenosine in Single- and Double-Strand RNA

[0127] We interrogated the aptitude of mAb-6E4 for recognition of a single 8oxoA inserted in RNA (Table 1 & Figure 7). A ssRNA version of the ssDNA-8oxodA oligonucleotide described above was synthesized and used as an antigen (ssRNA-8oxoA) in the competitive ELISA assay. Similarly, the ssRNA-8oxoA oligonucleotide was hybridized to its RNA complement to form dsRNA-8oxoA (Figure 7). Control ssRNA and dsRNA competitors in which an unmodified A replaces 8oxoA at position 11 were also examined. The ssRNA-8oxoA antigen is well-recognized by mAb 6E4 (ICso = 692 nM), exhibiting nM affinity, though reduced affinity compared to the 8oxoA nucleoside (an 11-fold decrement). The unmodified ssRNA counterpart is not effectively bound by mAb-6E4, with a >300-fold concentration necessary relative to ssRNA-8oxoA to initiate displacement from AchE-8oxoA. The binding behavior of 6E4 to dsRNA-8oxoA antigens was reasonably robust. The dsRNA-8oxoA:U pair is well detected by 6E4 (ICso = 861 nM), though not with the same affinity as ssRNA-8oxoA (1.2- fold decrement). The dsRNA control lacking 8oxoA is feebly recognized, exhibiting a competitor titration curve resembling the unmodified ssRNA. Consistent with its capacity for 8oxoAde detection in nucleosides and DNA oligonucleotides, mAb 6E4 adeptly senses 8oxoA damage in RNA.

[0128] A. Oligonucleotide Binding Substrates

[0129] Unmodified RNA oligonucleotides were chemically synthesized at Integrated DNA Technologies (IDT). The 8oxoA RNA oligonucleotide was custom synthesized by ChemGenes. RNA duplex was annealed by mixing sense and anti-sense strands at a 1 : 1 .2 molar ratio in 10 mM Tris-HCI (pH 7.5), 50 mM NaCI, and 1 mM EDTA in sterile, nuclease free ddH2O and then heating to 95°C. The annealing reactions were then slowly cooled to room temperature (~22°C) over 45 minutes, followed by centrifugation at -10,000 rpm. described methylated adenosines.

[0130] Example 7 Effect of 8oxoA Base Mispairs in Double-Strand RNA on Antibody Recognition of the 8-oxo-Adenine Nucleobase

[0131] Double-strand RNA was generated, in which 8oxoA is individually paired with each one of the four standard RNA bases. Single-strand RNA and double-strand RNA, in which 8oxoA is paired with II, C, or A, are all roughly equivalent antigens for mAb-6E4, with ICso values of 861 , 632, and 671 nM, respectively (Table 1 & Figure 8A). However, affinity for dsRNA with a central 8oxoA:G mispair is relatively poor, with 4.6 pM of the competitor required for 50% inhibition - a 5.3-fold decrement in affinity relative to the dsRNA-8oxoA:ll pair and a 6.6-fold decrement relative to ssRNA-8oxoA. The structure of a syn 8oxoA.anti G base pair with 2 H-bonds is shown in Figure 8B. The 2 atomic additions of 8oxoA- a proton at N7 and an O atom at C8 (both shaded grey) - are fundamental to its pairing scheme with guanine. The N7 proton of 8oxoA forms H-bonds with the guanine 06, while the 08 adduct Hoogsteen pairs with the guanine N1 proton. These findings indicate that 8oxoA:G pairs in the dsRNA tested here, renders the 8-oxo-adenine epitope less accessible to antibody recognition.

[0132] Example 8: Anti-8-0xo-Adenine Antibody Sequencing and Homology Modeling of 8- Oxo-Adenine Binding

[0133] \Ne carried out hybridoma antibody sequencing and homology modeling, which suggests that 8-oxo-adenine specificity resides exclusively in the VH domain. Homology modeling of 8oxoATP into the VH domain of mAb-6E4 was achieved by first structurally aligning the apo mAb-6E4 8oxoA scFv model on our unpublished X-ray structure of scFv-8oxoG bound to 8oxoGTP (Cymba X, LLC, a Delaware limited liability company). An 8oxoA ligand model was then superimposed onto the 8oxoGTP, replacing it. In the model shown here (Figure 10), the beta and gamma phosphates were deleted to generate 8oxoAMP. However, CDR2 contacts to 5’-phosphates are also observed. The minimal and broader 8-oxo-adenine epitope was deduced from our biochemical data, in combination with comparisons to the apo-homology model (SAbDab) and the modeling of 8oxoAMP and 8oxoATP into the mAb- 6E4 heavy chain. Although this collectively indicates that 8-oxo-adenine specificity resides exclusively in the VH domain, larger nucleic acid substrates, such as longer single-strand 8-oxo-adenine-RNA molecules, may have non-specific elements that are contacted by residues from both the VH and VL domains. Key features of the data-informed model are outlined below. Summary of mAb-6E4 VH Groups Predicted to Contact 8oxoAMP

[0134] CDR1 W33 and N35

[0135] CDR2 D50, Y52, N55 and N59

[0136] CDR3 1100, W101 , A102, T103, R104, L105 and W106, whereby I100-L105 comprise the 80x0 recognition loop

[0137] 8oxoAMP Contacts (bold) with mAb-6E4 VH Residues

[0138] N1 contacted by CDR2 D50, N59, and main chain of CDR1 W33

[0139] C2 contacted as part of the base stacking with CDR1 W33

[0140] N3 contacted by CDR2 N59

[0141] N6 contacted by CDR2 D50 side chain

[0142] N7-H contacted by CDR3 groups via a bridging H2O: the CDR3 S99 hydroxyl group, 1100 main chain carbonyl oxygen atom, and W101 main chain amide hydrogen atom form H-bonds with an ordered H2O molecule, which is in turn H-bonded to the N7 hydrogen atom; alternatively, the N7 hydrogen atom forms a H-bond with the CDR3 L105 main chain carbonyl oxygen atom

[0143] C8-oxo moiety is the key epitope element, contacted by multiple CDR3 groups: the W101 NE1 group and the A102 and T103 main chain amide groups all serve as donors in hydrogen bonding with the C8-oxo oxygen atom

[0144] Purine ring contacted by CDR1 W33 (apical side) and CDR3 W106 (basal side) via stacking of the aromatic groups

[0145] Alpha phosphate group contacted by CDR2 N55; it is not an essential epitope element based on -equal affinity for the 8oxoA nucleosides vs. nucleoside triphosphates

[0146] Ribose / Deoxyribose is not part of the epitope; this is consistent with periodate oxidation during antigen linkage to KLH, making the sugar unavailable; also supported by the biochemical data, including the highest affinity associated with the 8-oxo-adenine nucleobase alone

[0147] A. Hybridoma Antibody Sequencing

[0148] Total RNA was purified from clone 6E4 and 5G7 hybridoma cell pellets (Zymogen Quick- RNA) and then reverse transcribed using a modified Moloney murine leukemia virus reverse transcriptase (SMARTScribe™) and an oligo dT primer and a universal template switching oligonucleotide (TSO) to incorporate 5’-terminal transcript sequences. Rapid amplification of cDNA ends (RACE) was then performed to amplify the VH and VL domains using a high-fidelity DNA polymerase, the universal TSO as forward primer and an lgG1 k constant domain as reverse primer. Products were inserted into the pCR-Blunt-TOPO and then transformed into E. coli TOP10. Colony PCR was performed to accurately size select clones. At least 5 colonies harboring accurate VH and VL insert lengths were Sanger sequenced. A consensus sequence for the VH and VL chains of each hybridoma 6E4 and 5G7 were then determined from nucleotide multiple sequence alignments.

[0149] Example 8: Sensing 8-oxoA in Oxidized, Polyadenyiated mRNA

[0150] Further, the inventors determined if mAb 6E4’s observed ssRNA-8-oxoA affinity (Figure 7) extends to full-length RNA species such as polyadenylated, messenger RNA (mRNA). Unmodified mRNA encoding enhanced green fluorescent protein (eGFP) was transcribed in vitro with T7 RNA polymerase (RNAP) and then chemically oxidized via the Fenton reaction utilizing Fe(ll), ascorbic acid, and H2O2 (Figure 11 ). The linear architectures of both the untreated (top) and oxidized (bottom) eGFP mRNAs are depicted in Figure 11 A, highlighting the terminal 5’-m7G cap1 structure, 3’-polyadenylate tail, and assorted nucleobase damage. The native and ox-mRNAs were then compared by means of the anti- 8-oxoA, competitive ELISA using free 8-oxoA as control (Figure 11 B). The ox-mRNA effectively disrupted 6E4 binding from the AChE-8-oxoA conjugate, generating a doseresponse curve that mirrors the free 8-oxoA competitor profile. Interestingly, the affinity of 6E4 for ox-mRNA is greater than a singly modified ssRNA oligonucleotide (Figure 7) and comparable to the free nucleobase, implying multiple C8-oxo lesions are induced by the mRNA oxidation conditions. In contrast, the untreated eGFP mRNA is not bound by 6E4. These results indicate Fenton oxidation of an IVT mRNA, harboring a 5’-m7G cap and a 3’- polyadenylate tail, generates antigens specifically sensed by the anti-8-oxoA monoclonal antibody 6E4.

[0151] Example 9: Oxidation and Lesion Recognition in Total Human RNA

[0152] To further understand the facility of 6E4 to identify 8-oxoA in complex RNA mixtures, the inventors treated total RNA extracted from assorted, healthy primary human tissues (including brain, heart, lung) with the Fe(ll), ascorbic acid, and H2O2 regimen to elicit nucleobase oxidation. The RNA samples were highly pure (A260 / 280 1.9) and included intact ribosomal RNA, a broad mRNA population, and microRNAs. The inventors then employed both the treated and untreated, RNA samples as competitors in the 6E4 powered, 8-oxoA ELISA (Figure 12). The data demonstrate that 6E4 effectively binds increasing concentrations of oxidized, total human RNA. Conversely, the untreated RNA control is an ineffective 6E4 antigen, evidenced by a 2.7-fold decrease in relative affinity at 15 pg / ml of competitor RNA. These findings demonstrate the ability of mAb 6E4 to distinguish 8-oxoA lesions amidst the complex structural and nucleobase modification inventory present in chemically oxidized, total human RNA. CITATIONS

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[0208] Yanagawa H, Ogawa Y, and Ueno M. 1992. Redox ribonucleosides. Isolation and characterization of 5-hydroxyuridine, 8-hydroxyguanosine, and 8-hydroxyadenosine from Torula yeast RNA. J. Biol. Chem. 267: 13320-13326.

[0209] Zaccara S, Ries RJ, and Jaffrey SR. 2019. Reading, writing and erasing mRNA methylation. Nature Reviews Molecular Cell Biology, 20 608-624.

Claims

CLAIMS1 . An antigen-binding molecule that specifically binds to 8-oxo-adenine, wherein the antigen-binding molecule interacts with the hydrogen atom at position N7 of 8-oxo- adenine, either directly or via an ordered H2O molecule bridge, and / or with the oxygen atom at position 08 of 8-oxo-adenine.

2. The antigen-binding molecule according to claim 1 , wherein the antigen binding molecule further interacts with at least one of: a) the N atom at position 1 of 8-oxo-adenine; b) the N atom at position 3 of 8-oxo-adenine; c) the amino group at position 6 of 8-oxo-adenine; and / or d) the apical or basal faces of the purine ring of 8-oxo-adenine.

3. The antigen-binding molecule according to claim 1 or 2, wherein the 8-oxo-adenine is comprised in 8-oxo-adenosine or 8-oxo-deoxyadenosine.

4. The antigen-binding molecule according to claim 3, wherein the 8-oxo-adenosine is comprised in a ribonucleoside or a ribonucleotide or a ribonucleotide triphosphate or an RNA molecule or the 8-oxo-deoxyadenosine is comprised in a deoxyribonucleoside or a deoxyribonucleotide or a deoxyribonucleotide triphosphate or a DNA molecule or the 8-oxo-adenosine is comprised in an adenine nucleotide derivative or an adenine derivative, including but not limited to, nicotinamide adenine dinucleotide (NAD) or flavin adenine dinucleotide (FAD) or 2’3’-cyclic adenosine monophosphate (2’3’-cAMP) or 3’5’-cyclic adenosine monophosphate (3’5’-cAMP) or 2’3’-cyclic guanosine monophosphate-adenosine monophosphate (2’3’-cGAMP) or 3’-phosphoadenosine 5’-monophosphate (pAp).

5. The antigen-binding molecule according to any one of claims 1 to 4, wherein the antigen-binding molecule is an antibody, an antigen-binding fragment thereof, an ankyrin repeat protein, an aptamer or another antibody mimetic.

6. The antigen-binding molecule according to claim 5, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a multispecific antibody, or an antibody displayed upon the surface of a phage or displayed upon the surface of a chimeric antigen receptor (CAR) T cell.

7. The antigen-binding molecule according to claim 6, wherein the antibody is an I gG 1 , lgG2a or lgG2b, lgG3 or lgG4 antibody.

8. The antigen-binding molecule of claim 5, wherein the antigen-binding fragment isa Fab fragment, a F(ab')2 fragment or a Fv fragment.

9. The antigen-binding molecule according to any one of claims 5 to 8, wherein binding of 8-oxo-adenine is achieved through the variable heavy (VH) chain of the antibody.

10. The antigen-binding molecule according to any one of claims 5 to 9, wherein the antibody comprises a variable heavy (VH) chain comprising a CDR3 having the amino acid sequence SIWATRLWYFDV (SEQ ID NO: 5).11 . The antigen-binding molecule according to any one of claims 5 to 10, wherein the antibody comprises a variable heavy (VH) chain comprising a CDR1 having the amino acid sequence GYKFTTYWIN (SEQ ID NO: 3), a CDR2 having the amino acid sequence DFYPGNGSNNFNDKFRN (SEQ ID NO: 4) and a CDR3 having the amino acid sequence SIWATRLWYFDV (SEQ ID NO: 5).

12. The antigen-binding molecule according to any one of claims 5 to 11 , wherein the antibody comprises a variable heavy (VH) chain sequence comprising the amino acid sequenceQVQLQQPGAEWKPGTSVKLSCKASGYKFTTYWINWLKLRPGQGLEWIGDFYP GNGSNNFNDKFRNKATLTVDTSSNTAYMQLSSLASEDSALYYCARSIWATRLWY FDVWGAGTTVTVSS (SEQ ID NO: 1 ) or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 1 .

13. The antigen-binding molecule according to any one of claims 10 to 12, wherein the antibody comprises a variable light (VL) chain comprising a CDR3 having the amino acid sequence QQHYRLPFT (SEQ ID NO: 8).

14. The antigen-binding molecule according to any one of claims 10 to 13, wherein the antibody comprises a variable light (VL) chain comprising a CDR1 having the amino acid sequence RASQDVNTAVA (SEQ ID NO: 6), a CDR2 having the amino acid sequence WASTRHT (SEQ ID NO: 7) and a CDR3 having the amino acid sequence QQHYRLPFT (SEQ ID NO: 8).

15. The antigen-binding molecule according to any one of claims 10 to 14, wherein the antibody comprises a variable light (VL) chain sequence comprising the amino acid sequence DIVMTQSHKFMSTSVGDRISIPCRASQDVNTAVAWYQQKPGQSPKLLIYWASTR HTGVPDRFTGSGSGTDYALTISGVQAEDLTLYYCQQHYRLPFTFGGGTKLEIK (SEQ ID NO: 2) or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 2.

16. The antigen-binding molecule according to any one of claims 5 to 9, wherein the antibody comprises a variable heavy (VH) chain comprising a CDR3 having the amino acid sequence SADYLAMDY (SEQ ID NO: 13).

17. The antigen-binding molecule according to any one of claims 5-9 or 16, wherein the antibody comprises a variable heavy (VH) chain comprising a CDR1 having the amino acid sequence GYIFTSYWIN (SEQ ID NO: 11 ), a CDR2 having the amino acid sequence DFHPGRGITNNNEKFKT (SEQ ID NO: 12) and a CDR3 having the amino acid sequence SADYI_AMDY (SEQ ID NO: 13).

18. The antigen-binding molecule according to any one of claims 5-9 or 16-17, wherein the antibody comprises a variable heavy (VH) chain sequence comprising the amino acid sequenceQVQLQQPGAELVKPGASVKMSCKASGYIFTSYWINWLRQRPGQGLEWIGDFH PGRGITNNNEKFKTKVKLTLETSSSTAYMQLSSLTSEDSAVYYCSRSADYLAMD YWGQGTSVTVSS (SEQ ID NO: 9) or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 9.

19. The antigen-binding molecule according to any one of claims 16 to 18, wherein the antibody comprises a variable light (VL) chain comprising a CDR3 having the amino acid sequence QNGHSFPPT (SEQ ID NO: 16).

20. The antigen-binding molecule according to any one of claims 16 to 19, wherein the antibody comprises a variable light (VL) chain comprising a CDR1 having the amino acid sequence RASQSISDYLH (SEQ ID NO: 14), a CDR2 having the amino acid sequence YASQPIS (SEQ ID NO: 15) and a CDR3 having the amino acid sequence QNGHSFPPT (SEQ ID NO: 16).

21. The antigen-binding molecule according to any one of claims 16 to 20, wherein the antibody comprises a variable light (VL) chain sequence comprising the amino acid sequence DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIRYASQPI SGIPSRFTGSGSGSDFTLTINSVEPEDVGVYYCQNGHSFPPTFGGGTKLEIK (SEQ ID NO: 10) or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 10.

22. An antibody, or an antigen-binding fragment thereof, binding to the same epitope as an antibody, or antigen-binding fragment thereof, of any one of claims 10 to 21.

23. A polynucleotide encoding the antibody, or the antigen-binding fragment thereof, or a polynucleotide sequence capable of hybridizing to said polynucleotideencoding the antibody or the antigen-binding fragment thereof, according to any one of claims 5 to 22.

24. A host cell comprising the polynucleotide of claim 23.

25. A method for producing an antibody, or an antigen-binding fragment thereof, comprising the culturing the host cell according to claim 24.

26. A cell-free transcription-translation system or cell-free transcription system or cell- free translation system comprising the polynucleotide of claim 23.

27. A method for producing an antibody, or an antigen-binding fragment thereof, the method comprising a step of transcribing and / or translating the polynucleotide of claim 23 in vitro, in particular wherein the antibody, or the antigen-binding fragment thereof, is produced with the cell-free transcription-translation system or the cell- free transcription system or the cell free translation system according to claim 26.

28. An immunoconjugate comprising the antibody, or the antigen-binding fragment thereof, according to any one of claims 5 to 22.

29. The immunoconjugate according to claim 28, wherein the immunoconjugate comprises radioisotope or label.

30. The immunoconjugate according to claim 29, wherein the label is selected from the group of: an affinity tag, an enzyme, and a fluorescent tag.

31. A method of detecting 8-oxo-adenine in a biological sample, the method comprising a step of contacting the biological sample with the antigen-binding molecule according to any one of claims 1 to 22 or the immunoconjugate according to claim 28 to 30 under conditions permissive for binding of the antigen-binding molecule to 8-oxo-adenine, and detecting whether a complex is formed between the antigen-binding molecule and the 8-oxo-adenine in the biological sample.

32. The method according to claim 31 further comprising a step of quantifying the amount of complex formed.

33. A method for diagnosing or monitoring the status of a disease associated with elevated levels of 8-oxo-adenine in a human or animal comprising the steps of: a) providing a biological sample comprising nucleic acids that has been obtained and / or derived from the human or animal;b) contacting the biological sample with the antigen-binding molecule according to any one of claims 1 to 22 or the immunoconjugate according to claim 28 to 30 to form a complex with 8-oxo-adenine comprised in the biological sample; and c) determining the amount of complex formed as a measure of the presence or amount of 8-oxo-adenine in the biological sample, wherein the amount of complex determined is indicative of a disease state associated with elevated levels of 8-oxo-adenine or is correlated with the status of a disease associated with elevated levels of 8-oxo-adenine.

34. The method according to claim 33, wherein the disease associated with elevated levels of 8-oxo-adenine is cancer, autoimmune disease, aging-related diseases, cardiovascular disease, chronic inflammatory disease, neurodegenerative disease, metabolic syndrome, rheumatoid arthritis, systemic lupus erythematosus, or sickle cell anemia.

35. A method for detecting or quantifying levels of 8-oxo-adenine in DNA or RNA comprising the steps of: a) providing a DNA or RNA sample that has been synthesized or produced in vitro or has been obtained and / or derived from a eukaryote, bacteria, archaea, virus, or bacteriophage. This includes, but is not limited to, DNA or RNA obtained and / or derived from human, plant, animal, fungi, protozoa, and other single-celled prokaryotes and their viruses. b) contacting the biological sample with the antigen-binding molecule according to any one of claims 1 to 22 or the immunoconjugate according to claim 28 to 30 to form a complex with 8-oxo-adenine comprised in the DNA or RNA sample; and c) determining the amount of complex formed as a measure of the presence or amount of 8-oxo-adenine in the sample, wherein the amount of complex determined is indicative of the oxidation extent or degradation level associated with the DNA or RNA sample or is correlated with a measure of sample quality control.

36. The method according to any one of claims 31 to 35, wherein the detection and / or quantification of the complex comprises one or more steps of immunohistochemistry, immunofluorescence imaging, immunoprecipitation, DNA immunoprecipitation, RNA immunoprecipitation, chromatin immunoprecipitation,chromatin or DNA or RNA immunoprecipitation followed by nucleic acid sequencing, enzyme-linked immunosorbent assay (ELISA), immunoblotting, ultrasensitive immunoassay, Western blotting, radioimmunoassay, flow cytometry, fluorescence-activated cell sorting (FACS) or radiographic imaging.

37. A kit for detecting the presence and / or quantifying the levels of 8-oxo-adenine in a sample; the kit comprising the antigen-binding molecule according to any one of claims 1 to 22 or the immunoconjugate according to claim 28 to 30.

38. A kit for diagnosing or monitoring the status of a disease associated with elevated levels of 8-oxo-adenine in a eukaryote, bacteria, archaea, virus, or bacteriophage. This includes, but is not limited to, DNA or RNA obtained and / or derived from human, plant, animal, fungi, protozoa, and other single-celled prokaryotes and their viruses; the kit comprising the antigen-binding molecule according to any one of claims 1 to 22 or the immunoconjugate according to claim 28 to 30.

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