WO2012110824A1

WO2012110824A1 - Binding agents with specificity for a nucleic acid modification

Info

Publication number: WO2012110824A1
Application number: PCT/GB2012/050357
Authority: WO
Inventors: Clive Graham Copley; Catherine Jane MARSDEN
Original assignee: Astrazeneca Ab; Dynavax Technologies Corporation; Astrazeneca Uk Limited
Priority date: 2011-02-18
Filing date: 2012-02-16
Publication date: 2012-08-23

Abstract

A method for identifying binding agents having binding specificity for a nucleic acid modification, the binding agents comprising an antigen binding site and including for example, antibodies or fragments or derivatives thereof. Binding agents having binding specificity for a nucleic acid modification, particularly of the backbone or sugar component, the binding agents comprising an antigen binding site, and including for example, antibodies or fragments or derivatives thereof. Uses of the binding agents and associated compositions in diagnostics and therapy.

Description

BINDING AGENTS WITH SPECIFICITY FOR A NUCLEIC ACID MODIFICATION

Field of the invention

The invention relates to binding agents which can discriminate between naturally occurring and modified nucleic acids, and to the use of such agents in therapy and diagnostics, in particular as an adjunct to oligonucleotide therapy. The invention further relates to methods for identifying and isolating such agents.

Background of the invention

Oligonucleotides offer promise as pharmacophores through a variety of modes of action including: antisense (Crooke Annu. Rev. Med., 55:61-95, 2004; Rubenstein et al, Drugs Fut., 29:893-909, 2004); antigene (Stull et al. Pharmaceut. Res., 12:465-483, 1995);

ribozyme (Pyle Science, 261 :709-14, 1993; Narlikar et al., Annu. Rev. Biochem., 66: 19- 59, 1997); immunostimulatory sequences (Hyashi Am. J. Med. 119:897el-897e6, 2006) and as aptamers (Kaur et al., Expert Opin. Investig. Drugs, 17:47-60, 2008).

As part of the development process there is a need to measure and monitor pharmacophore levels in samples during pre-clinical and clinical trials. In the case of oligonucleotides this is usually carried out by a sandwich hybridisation assay, or a variation of this method, which uses capture and detection sequences that are complementary to part of the sequence that requires measurement (Tremblay et.al, Bioanalysis, 1 :595-609, 2009). However, these methods are problematic for a number of reasons.

Firstly, the pharmacophore can often be relatively short with a significant degree of self- complementarity, meaning the consecutive bases available to complement capture or detection probes are few. Secondly, degraded forms of the pharmacophore can

demonstrate significant biological effect (Fearon et al., Eur. J. Immunol., 33:2114-2122, 2003) reducing the number of bases available for detection even further. Finally, circulating DNA (van der Vaart Clin. Chem., 53:2215, 2007; Fatouros et al., Clin. Chem., 52:1820-1824, 2006) in patients' serum has the propensity to interfere with the

hybridization assay by binding to either the detection and capture probes or to the pharmacophore itself thus increasing the background and decreasing the sensitivity of any hybridization assay.

Thus there is a need in the art for a more effective means of monitoring and measuring oligonucleotide pharmacaphore levels.

Summary of the invention

The inventors have addressed this need by providing a means for isolating binding agents which specifically bind to a nucleic acid pharmacaphore molecule (or a fragment thereof) but which do not bind to the corresponding naturally occurring nucleic acid and can therefore be used to assay pharmacaphore levels.

Detection methods using the present binding agents do not display the disadvantages of the prior art methods. Thus for example, binding agents are provided which bind to

oligonucleotides with a significant degree of self-complementarity and secondary structure, so that such pharmacaphores can be detected. Binding agents may be prepared which bind to degraded or partially degraded (but potentially still active) pharmacaphore, allowing for example monitoring of degradation and clearance of a pharmacaphore. The binding agents do not bind to the naturally occurring or background nucleic acid in the assay sample, thus addressing the problem of background interference.

The binding agents identified by the inventors comprise antigen binding sites, such as antibody antigen binding sites.

Antibodies that bind DNA are known, particularly as components of the pathology of conditions such as Systemic Lupus Erythromatous (Koffler et ah, J. Exp. Med. 134:294- 312, 1971; Fournie et ah, Adv. Nephrol. 6:47-61, 1976). Studies of antibodies derived from this condition, both in man and in a mouse model, have demonstrated specificities for: ssDNA, both ds DNA and ss DNA or preferences for certain base composition or sequence (Eivazova et ah, Immunology, 101 :371-377, 2000; Stevens et ah, Biochem., 38:560-568, 1999; DiPietro et al, Biochem., 42:6218-6227, 2003).

However, deliberate attempts to raise anti-oligonucleotide antibodies by conventional immunisation using oligonucleotide conjugated to a carrier protein has had only limited success. Results have been slightly better with the use of an immunogen that comprises an oligonucleotide in association with a specific DNA binding protein (Ramussen et al. , J. Immunol. Methods, 189:47-58, 1996; Cerutti et al, J. Biol. Chem. 276: 12769-12773, 2008).

The inventors applied phage naive antibody libraries technology (de Haard et al., J. Biol. Chem., 274: 18218-3, 1999) to the problem of isolating DNA-specific binding agents . This approach has the added advantage that it is possible to select for binding agents that have particular specificities by carrying out specific absorption during the isolation procedure (de Haard et al., 1999 ibid).

The inventors noted that, in order to improve their properties, many oligonucleotide pharmacophores have modified backbone groups (Micklefield Curr. Med. Chem. 8: 1157- 1179, 2001). Such modifications include replacement of the phosphodiester linkers with more esterase-resistant analogues such as phosphorothioate. The inventors realised that these modifications might present an opportunity for the isolation of antibodies that bind to the modified backbone regardless of the DNA sequence, and that this would be of use in detecting modified oligonucleotide pharmacophores in human serum since patient samples are, at present at least, unlikely to accidentally contain other such modified entities.

Phage naive antibody libraries technology have been used to isolate single chain antibodies against DNA aptamers for use as adapter molecules in nanoscale DNA arrays (Li et al. , Org. Biomol. Chem., 4:3420-3426, 2006). However, the inventors have for the first time developed a method which includes both a step of screening such display libraries with a target modified nucleic acid, and also a subtractive screening step with the corresponding unmodified nucleic acid, to allow isolation of binding agents with specificity for the modification in the modified nucleic acid.

Accordingly, in one aspect the invention provides a method for identifying a binding agent having binding specificity for a nucleic acid modification, the method comprising:

(a) contacting a nucleic acid having the modification (modified nucleic acid) with a display library of binding agents, wherein each binding agent in the library comprises an antigen binding site; (b) selecting one or more binding agents having an antigen binding site which is able to bind to the modified nucleic acid;

(c) contacting the binding agents selected in (b) with nucleic acid which does not have the modification (unmodified nucleic acid); and

(d) selecting one or more binding agents having an antigen binding site which is not able to bind to the unmodified nucleic acid.

The invention additionally provides a binding agent identified using the present methods.

Further, the invention provides a binding agent having binding specificity for a nucleic acid modification, wherein the binding agent comprises an antigen binding site which binds to a nucleic acid having the modification (modified nucleic acid), and wherein the nucleic acid modification comprises a modified backbone, or a modified sugar component.

In a further aspect, the invention provides a pharmaceutical composition comprising a binding agent of the invention and a pharmaceutically acceptable diluent, excipient or carrier.

Also provided herein is the use of a binding agent of the invention in an in vitro assay to determine the presence and/or concentration of a pharmacaphore in a sample, wherein the pharmacaphore comprises a modified nucleic acid and wherein the binding agent binds specifically to the modified nucleic acid.

Further provided is a method for detecting or quantifying a pharmacaphore in a human or animal subject to which the pharmacaphore has been administered, the method comprising

(e) providing a sample taken from the subject;

(f) contacting the sample with binding agent of the invention; and

(g) detecting pharmacaphore-bound binding agent; wherein the pharmacaphore comprises a modified nucleic acid and wherein the binding agent binds specifically to the modified nucleic acid. The invention also provides a binding agent or a pharmaceutical composition of the invention, for use in medicine. In one aspect the invention provides a binding agent or a pharmaceutical composition of the invention for use in a method of detecting a

pharmacaphore in a human or animal subject, wherein the pharmacaphore comprises a modified nucleic acid and wherein the binding agent binds specifically to the modified nucleic acid. In another aspect the invention provides a binding agent or a pharmaceutical composition of the invention for use in a method of oligonucleotide therapy in a human or animal subject

Also provided herein is a method for detecting a pharmacaphore in a human or animal subject to which has been administered a pharmacaphore and detectably labelled binding agent of the invention, wherein the pharmacaphore comprises a modified nucleic acid and wherein the binding agent binds specifically to the modified nucleic acid, the method comprising detecting the position of the binding agent in the patient body by means of the detectable label.

In a further aspect the invention provides a method of treating a human or animal subject with a therapeutic oligonucleotide comprising administration of a binding agent or a pharmaceutical composition according to the invention, wherein the therapeutic oligonucleotide comprises a modified nucleic acid and wherein the binding agent binds specifically to the modified nucleic acid.

Also provided herein is a kit for determining the presence and/or concentration of a pharmacaphore in a sample, the kit comprising a binding agent according to the invention, wherein the pharmacaphore comprises a modified nucleic acid and wherein the binding agent binds specifically to the modified nucleic acid.

Brief description of the drawings

Figure 1 shows a comparison of binding specificities of binding agent clones 3749.03 and 3749.22 as described in the present Examples. Figure 2 shows the effect of temperature on the binding of biotinylated C274

oligonucleotide having a phosphorothioate backbone (5 'bC274PS) to immobilised binding agent, for binding agent clones 3742.30, 3745.58 and 3749.93, as in the present Examples.

Figure 3 shows the results of a competition assay to determine sensitivity of selected binding agent (clones 3749.93, 3749.22 and 3749.03) for binding C274PS.

Figure 4 shows Table 1 , listing the names and sequences of the oligonucleotides used in the present Examples.

Figure 5 shows Table 2, listing the number of positive isolates for each library/nucleic acid antigen combination in primary screening in the present Examples.

Figure 6 shows Table 3, listing the percentage inhibition of binding of individual binding agents to 5'bC274PS by C274PS as in the present Examples. Only binding agent clones showing greater than 40% signal knockdown in the presence of C274PS are reported.

Figure 7 shows Table 4, listing the percentage inhibition of binding of 5'bC274PS to selected binding agents, by various oligonucleotides, in a competition assay carried out as in the present Examples to determine binding specificity of the binding agents.

Figure 8 shows Table 5, listing the results of chessboard titrations carried out as in the present Examples for selected binding agents.

Figure 9 shows Table 6, listing the percentage inhibition of binding agent binding to 5'bC274PS by unlabelled C274PS in a competition assay to determine IC50 values for binding agents as in the present Examples.

Brief description of the sequences

Sequences described and used herein are listed below. SEQ ID NOs 1-68 are included in the appended sequence listing. HEX1 - HEX6 are shown in Table 1 (Figure 4).

SEQ ID NO: 1 shows the amino acid sequence of HCDRl of binding agent clone 3749.03. SEQ ID NO: 2 shows the amino acid sequence of HCDR2 of binding agent clone 3749.03.

SEQ ID NO: 3 shows the amino acid sequence of HCDR3 of binding agent clone 3749.03.

SEQ ID NO: 4 shows the amino acid sequence of LCDR1 of binding agent clone 3749.03.

SEQ ID NO: 5 shows the amino acid sequence of LCDR2 of binding agent clone 3749.03.

SEQ ID NO: 6 shows the amino acid sequence of LCDR3 of binding agent clone 3749.03.

SEQ ID NO: 7 shows the amino acid sequence of HFR1 of binding agent clone 3749.03.

SEQ ID NO: 8 shows the amino acid sequence of HFR2 of binding agent clone 3749.03.

SEQ ID NO: 9 shows the amino acid sequence of HFR3 of binding agent clone 3749.03.

SEQ ID NO: 10 shows the amino acid sequence of HFR4 of binding agent clone 3749.03.

SEQ ID NO: 1 1 shows the amino acid sequence of LFR1 of binding agent clone 3749.03.

SEQ ID NO: 12 shows the amino acid sequence of LFR2 of binding agent clone 3749.03.

SEQ ID NO: 13 shows the amino acid sequence of LFR3 of binding agent clone 3749.03.

SEQ ID NO: 14 shows the amino acid sequence of LFR4 of binding agent clone 3749.03.

SEQ ID NO: 15 shows the amino acid sequence of the VH domain of binding agent clone 3749.03.

SEQ ID NO: 16 shows the amino acid sequence of the VL domain of binding agent clone 3749.03.

SEQ ID NO: 17 shows the amino acid sequence of HCDR1 of binding agent clone 3742.30.

SEQ ID NO: 18 shows the amino acid sequence of HCDR2 of binding agent clone 3742.30. SEQ ID NO: 19 shows the amino acid sequence of HCDR3 of binding agent clone 3742.30.

SEQ ID NO: 20 shows the amino acid sequence of LCDR1 of binding agent clone 3742.30.

SEQ ID NO: 21 shows the amino acid sequence of LCDR2 of binding agent clone 3742.30.

SEQ ID NO: 22 shows the amino acid sequence of LCDR3 of binding agent clone 3742.30.

SEQ ID NO: 23 shows the amino acid sequence of HFR1 of binding agent clone 3742.30.

SEQ ID NO: 24 shows the amino acid sequence of HFR2 of binding agent clone 3742.30.

SEQ ID NO: 25 shows the amino acid sequence of HFR3 of binding agent clone 3742.30.

SEQ ID NO: 26 shows the amino acid sequence of HFR4 of binding agent clone 3742.30.

SEQ ID NO: 27 shows the amino acid sequence of LFR1 of binding agent clone 3742.30

SEQ ID NO: 28 shows the amino acid sequence of LFR2 of binding agent clone 3742.30.

SEQ ID NO: 29 shows the amino acid sequence of LFR3 of binding agent clone 3742.30.

SEQ ID NO: 30 shows the amino acid sequence of LFR4 of binding agent clone 3742.30.

SEQ ID NO: 31 shows the amino acid sequence of the VH domain of binding agent clone 3742.30.

SEQ ID NO: 32 shows the amino acid sequence of the VL domain of binding agent clone 3742.30.

SEQ ID NO: 33 shows the amino acid sequence of HCDR1 of binding agent clone 3745.58. SEQ ID NO: 34 shows the amino acid sequence of HCDR2 of binding agent clone 3745.58.

SEQ ID NO: 35 shows the amino acid sequence of HCDR3 of binding agent clone 3745.58.

SEQ ID NO: 36 shows the amino acid sequence of LCDR1 of binding agent clone 3745.58.

SEQ ID NO: 37 shows the amino acid sequence of LCDR2 of binding agent clone 3745.58.

SEQ ID NO: 38 shows the amino acid sequence of LCDR3 of binding agent clone 3745.58.

SEQ ID NO: 39 shows the amino acid sequence of HFR1 of binding agent clone 3745.58.

SEQ ID NO: 40 shows the amino acid sequence of HFR2 of binding agent clone 3745.58.

SEQ ID NO: 41 shows the amino acid sequence of HFR3 of binding agent clone 3745.58.

SEQ ID NO: 42 shows the amino acid sequence of HFR4 of binding agent clone 3745.58.

SEQ ID NO: 43 shows the amino acid sequence of LFR1 of binding agent clone 3745.58

SEQ ID NO: 44 shows the amino acid sequence of LFR2 of binding agent clone 3745.58.

SEQ ID NO: 45 shows the amino acid sequence of LFR3 of binding agent clone 3745.58.

SEQ ID NO: 46 shows the amino acid sequence of LFR4 of binding agent clone 3745.58.

SEQ ID NO: 47 shows the amino acid sequence of the VH domain of binding agent clone 3745.58.

SEQ ID NO: 48 shows the amino acid sequence of the VL domain of binding agent clone 3745.58. SEQ ID NO: 49 shows the amino acid sequence of HCDR1 of binding agent clone 3749.93.

SEQ ID NO: 50 shows the amino acid sequence of HCDR2 of binding agent clone 3749.93.

SEQ ID NO: 51 shows the amino acid sequence of HCDR3 of binding agent clone 3749.93.

SEQ ID NO: 52 shows the amino acid sequence of LCDR1 of binding agent clone 3749.93.

SEQ ID NO: 53 shows the amino acid sequence of LCDR2 of binding agent clone 3749.93.

SEQ ID NO: 54 shows the amino acid sequence of LCDR3 of binding agent clone 3749.93.

SEQ ID NO: 55 shows the amino acid sequence of HFR1 of binding agent clone 3749.93.

SEQ ID NO: 56 shows the amino acid sequence of HFR2 of binding agent clone 3749.93.

SEQ ID NO: 57 shows the amino acid sequence of HFR3 of binding agent clone 3749.93.

SEQ ID NO: 58 shows the amino acid sequence of HFR4 of binding agent clone 3749.93.

SEQ ID NO: 59 shows the amino acid sequence of LFR1 of binding agent clone 3749.93

SEQ ID NO: 60 shows the amino acid sequence of LFR2 of binding agent clone 3749.93.

SEQ ID NO: 61 shows the amino acid sequence of LFR3 of binding agent clone 3749.93.

SEQ ID NO: 62 shows the amino acid sequence of LFR4 of binding agent clone 3749.93.

SEQ ID NO: 63 shows the amino acid sequence of the VH domain of binding agent clone 3749.93. SEQ ID NO: 64 shows the amino acid sequence of the VL domain of binding agent clone 3749.93.

SEQ ID NO: 65 shows the nucleotide sequence of oligonucleotide C274PO. SEQ ID NO: 66 shows the nucleotide sequence of oligonucleotide C274PS SEQ ID NO: 67 shows the nucleotide sequence of oligonucleotide C274 core. SEQ ID NO: 68 shows nucleotide sequence of oligonucleotide irrelevant core.

Detailed description of the invention

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd.,

1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed. ), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc.,

1995 (ISBN 1-56081-569-8). Definitions and additional information known to one of skill in the art in immunology can be found, for example, in Fundamental Immunology, W. E. Paul, ed., fourth edition, Lippincott-Raven Publishers, 1999. Description of conventional techniques of molecular biology (including recombinant techniques), are explained in for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) and Current Protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987, including supplements through 2001).

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference in to the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. The inventors have devised a method for identifying binding agents which will bind specifically to nucleic acids which are modified in some way compared to naturally occurring nucleic acid, for example, nucleic acids having a modified backbone. The method is based upon a two-stage panning of a displayed library of potential binding agents, e.g. antibodies, or antibody fragments, with nucleic acid bait. In the first stage, the library is panned with a nucleic acid bearing the modification of interest (modified nucleic acid), and binding agents which bind the modified nucleic acid are collected. In the second stage, these collected binding agents are panned with the equivalent nucleic acid but this time lacking the modification. This time, non-binding binding agents are collected. Thus the panning specifically selects for binding agents which bind the modified but not the unmodified nucleic acid. These binding agents thus have binding specificity for the particular nucleic acid modification used in panning.

Some of the binding agents selected in library panning will have binding specificity dependent only upon the particular nucleic acid modification used in panning. However, for some agents, binding specificity will additionally depend upon the base sequence and/or secondary structure of the modified nucleic acid used in panning. Thus, in one embodiment the present methods identify a binding agent for which binding is specific for the nucleic acid modification alone (irrespective of base sequence or secondary structure). In another embodiment the present methods identify a binding agent for which binding is specific for the nucleic acid modification and additionally for a particular base sequence and/or a particular secondary structure. Binding agents selected in library panning can be subjected to additional screening to find out more about the type of binding specificity that the binding agent has. Additional screening steps can also be carried out to determine properties of the binding agents, such as IC50 value for binding to the modified nucleic acid. The methods devised by the inventors, and the binding agents provided are particularly useful because they can be used to reliably detect, or target quantify nucleic acid pharmacaphores (which typically include modified nucleic acids) without the risk of cross- reactivity with naturally occurring nucleic acid that may be present, for example, in a clinical sample or in the body of a patient.

Library panning

Accordingly, in one aspect the invention provides a method for identifying a binding agent having binding specificity for a nucleic acid modification. In general the method comprises:

(a) contacting a nucleic acid having the modification (modified nucleic acid) with a display library of binding agents, wherein each binding agent in the library comprises an antigen binding site;

(b) selecting one or more binding agents having an antigen binding site which is able to bind to the modified nucleic acid;

By "binding agent" is meant a molecule having an antigen binding site, such as an antibody antigen binding site. A binding agent may comprise an antibody or a fragment or derivative thereof. Binding agents for use in the present methods typically comprise an antigen binding site which is able to bind to a nucleic acid antigen. Structural and functional properties of binding agents are further described herein.

By "having binding specificity for a nucleic acid modification" is meant that the ability of the binding agent to bind a nucleic acid molecule is determined in whole or in part by the presence or absence of a particular nucleic acid modification. Thus, for example, the binding agent binds to a nucleic acid having the modification (modified nucleic acid) but does not bind to an equivalent nucleic acid which does not have the modification

(unmodified nucleic acid), within the limits of detection of the particular assay. Put another way the binding agent is able to discriminate between modified and unmodified nucleic acid. Binding specificity of binding agents is described further herein.

A "nucleic acid modification" as used herein refers to an alteration, typically a chemical alteration, which is present in the modified nucleic acid molecule compared to the commonly or naturally occurring nucleic acid molecule. A modification may have been made to any component of the nucleic acid, for example, to the backbone, sugar component, or base. By way of example, a modified nucleic acid may have a

phosphorothioate backbone, instead of the naturally occurring phosphodiester backbone. Modifications of this kind are often made to nucleic acids which are to be used in therapy. Modifications and modified nucleic acids are described further herein.

A library for use in the panning method comprises a display library in which binding agents are displayed on the surface of suitable structures such as particles or cells. For example, the library may be displayed on yeast, bacteria, bacteriophage or phagemid, viruses, cells, ribosomes, or other in vitro display systems.

Any suitable display library may be used. For example, a cell-free display system is described in WO 01/05808. A ribosome display library is described in Groves et al 2006, Journal of Immunological Methods 313: 129-139. Phage display technology is well known in the art (see, for example, W091/17271 or WO92/001047, or de Haard et al supra). Phage used in such methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with binding agent protein recombinantly fused to either the phage gene III or gene VIII protein.

As above, a binding agent encoded in the display library generally comprises an antigen binding site, and may comprise an antibody, or a fragment or derivative thereof, or a non- antibody molecule having an antigen binding site, such as an Affibody or DARPin. The structures of binding agents are described in more detail herein. Typically each particle or cell in the library comprises nucleic acid encoding an antigen binding site for example, e.g, an antibody heavy chain variable (VH) domain, an antibody VH and light chain variable (VL) domain, a single chain fragment variable (scFv) antibody fragment, or a Fab fragment. A display library may comprise for example, antigen binding domains from a repertoire of combinatorial antibody library, e.g. human or murine or any other suitable species A library may be derived from a species in which a binding agent is to be used.

Libraries which can be used in the invention are known in the art.

For example, Vaughan et al. Nature Biotechnol. 14:309-314, 1996 describes the Bone Marrow Vaughan (BMV) library, a nonimmunised library of 1.4 x 10¹⁰ scFv fragments. Lloyd et al., Protein Eng. Des. Sel. 22: 159-168, 2009 describes a combined spleen (CS) library of 1.29 x 10¹¹ scFv fragments. Groves et al., J. Immunol. Meth. 313, 129-139, 2006 describes the DP47 library, a naive human library of 1 x 10¹⁰ scFv fragments, de Haard et al., J. Biol. Chem., 274: 18218-3, 1999 describes a Fab-phage library, pCES-1 from Dyax Corp, of >37 billion Fab fragments. All of these libraries are phagemid-based and require rescue with helper phage M13K07 for the production of scFv (or Fab) bearing phage. The BMV, CS and DP47 libraries are available from Medimmune Cambridge (formerly Cambridge Antibody Technology). The pCES-1 library is available from Dyax Corporation. Another suitable library of >10⁷ scFv molecules is described in Marks et al (J Mol Biol (1991) 222: 581-597).

Display libraries of non-antibody binding agents are also available as described herein.

In general in steps (a) and (b), of the method, the display library is panned using the modified nucleic acid, and library particles or cells displaying binding agents which bind to the modified nucleic acid are collected. In step (a), the modified nucleic acid is contacted with the display library of binding agents to allow any binding interactions to occur. In step (b) binding agents which is able to bind to the modified nucleic acid are selected.

Modified nucleic acids which may be used in panning are described further herein. Any suitable modified nucleic acid may be used as the target antigen.

Typically the modified nucleic acid target (or bait) is tagged or labelled in such a way as to allow isolation of library particles or cells which bind to it. For example, the modified nucleic acid may be immobilised, e.g. coupled to a solid support such as a microtitre plate or solid particles. When the support, charged with the nucleic acid, is contacted with the library, nucleic acid-binding particles or cells will be absorbed onto the solid support. After incubation with the library, bound particles/cells can be eluted from the support. Methods of elution are known in the art. Typically such methods involve disrupting the binding interaction between the nucleic acid and the binding agent. For example, this can be done by altering the pH, e.g. to outside the physiological range, using suitable buffers. Typically, the support is washed before elution, to remove non-specific binders.

Suitable immobilisation methods are known in the art. For example, the modified nucleic acid may comprise a biotin molecule, at for example, the 5' or 3 'end. In one aspect, the method comprises screening the library with both 5' -biotinylated and 3'biotinylated modified nucleic acid. A biotinylated nucleic acid may be coupled to a solid support via an avidin or streptavidin linker. For example, a biotinylated modified nucleic acid may be immobilised on streptavidin coated magnetic beads, or a streptavidin-coated microtitre plate. A modified nucleic acid may be tagged with a specific hapten for antibody capture, or bear a chemical coupling agent for coupling to a carrier protein for immobilisation purposes. A modified nucleic acid may be directly bound to a solid support using chemical linkages.

Before panning with modified nucleic acid, the library may be treated with a blocking solution, such as Marvel in PBS or another blocker, to remove any generally sticky binding agents which may arise during library construction.

Prior to incubation with library, a support bearing the modified nucleic acid may be washed. The support may then be treated with a blocking solution such as Marvel in PBS, and optionally, washed again

Steps (a) and (b) of the library panning method allow collection of binding agents

(displayed on library particles or cells) which are able to bind to the modified nucleic acid bait. In steps (c) and (d), a subtractive round of panning is carried out using these collected library particles/cells and the unmodified nucleic acid. In step (c) binding agents (displayed on library particles or cells) collected in (b) are contacted with unmodified nucleic acid. In step (d), library particles/cells displaying binding agents which bind to the unmodified nucleic acid are removed, and the remaining library display particles/cells, which display binding agents having binding specificity for the particular nucleic acid modification, can then be collected.

In general the unmodified nucleic acid for use in subtractive screening step (c) comprises the same base sequence as the modified nucleic acid bait used in step (a) but lacks (one or more of) the modification(s) in the modified nucleic acid. Preferably the unmodified nucleic acid lacks all of the modifications in the target modified nucleic acid, in order to screen out binding agents which bind to naturally occurring nucleic acid. Thus in one aspect the unmodified nucleic acid comprises a structure found in naturally occurring nucleic acid.

The subtractive panning steps using unmodified nucleic acid in can be carried out in the same way as described above for the panning steps with the modified nucleic acid.

However, in the subtractive steps (c) and (d), it is non-binding displayed binding agents which are to be separated and collected. Unmodified nucleic acid may thus be tagged and/or immobilised as described for the modified nucleic acid. Binding agent bound to unmodified nucleic acid can then be removed, e.g. by precipitation.

A number of subtractive steps may be carried out in order to isolate binding agents of the required specificity. For example, where a target modified nucleic acid has more than one modification, a number of subtractive screening steps may be carried out using in the subtractive steps, nucleic acids lacking one or more than one of the modifications in the target. However, in general it is preferred that at least one subtractive step is carried out using unmodified nucleic acid which lacks any modification, so that the subtractive screening step eliminates binding agents which bind to naturally occurring nucleic acids.

The library panning method may comprise one or more enrichment steps. For example, for a phage or display library, phage collected in step (d) may be amplified and steps (a) to (d) repeated using these amplified phage as the starting library in step (a). This process may be repeated 1, 2, 3 or more times.

Methods for phage amplification are known in the art. In general the method comprises infecting bacteria, typically log phase bacteria, with the phage under conditions which allow phage amplification, and collecting amplified phage. Suitable bacteria are known in the art and are described herein. These include E. coli.

Other types of library display structures may also be subjected to enrichment.

The panning method may comprise panning of multiple display libraries. Thus, in one aspect the method comprises screening more than one, such as 2, 3, 4, 5 or more libraries.

Following library panning, and selection of binding agents able to bind the modified but not the unmodified nucleic acid under the panning conditions, nucleic acid encoding the selected binding agent(s) can be extracted from the library display particles or cells. The encoding nucleic acid can be sequenced and/or used to prepare binding agents as described herein.

Methods for preparing the nucleic acid encoding binding agent from library display particles are known in the art, and described in the references given above. For example, for a phage library, phage selected in the library screening may be used to infect bacteria, such as log phase bacteria, and the infected cells plated to single colonies. Nucleic acid may be prepared from a colony using conventional methods. For example, primers based on known sequence around the insertion site of DNA encoding a binding agent may be used to prepare a sample of the inserted DNA.

Encoding nucleic acid may be used to prepare binding agents as described herein, for example, antibody fragments, or whole antibodies including chimeric, humanised or human antibodies as described herein.

Methods for preparing the binding agents from the encoding nucleic acid are known in the art and described herein. For example where the nucleic acid in the library encodes a binding agent which is an antibody fragment, such as a scFv or Fab fragment, the fragment may be reformatted into whole (chimeric) antibody and the whole antibody preparation used as a binding agent in an assay. Antibody reformatting techniques are known in the art. For example, preparation of chimeric antibodies is described in US 5,807,715. Use of whole antibodies (including the Fc region) as binding agents may have advantages where the binding agent is intended for use in clinical assays. Alternatively, where the nucleic acid in the library encodes a binding agent which is a whole antibody, a fragment of the antibody may be prepared and used as a binding agent. For example, the VH and VL domains may be used to prepare a scFv fragment.

Alternatively or additionally to extraction of nucleic acid encoding binding agent from library display particles/cells, binding agent may also be expressed from the nucleic acid in the selected display particles or cells. Such preparations of binding agent may be further tested for properties, e.g. binding properties. Methods for expressing and preparing proteins from display libraries, such as a phage or phagemid display library, are known in the art. For example, phage may be used to infect bacteria, and expression of phage- inserted DNA induced, before isolation of the expressed protein from the bacteria. In one example, phage infected bacteria are plated to single colonies, and individual colonies cultured before inducing expression of the phage-inserted DNA encoding the protein of interest, into the periplasm of the bacteria. The contents of the periplasm (peripreps), including the expressed protein, can then be isolated by conventional means.

Additional screening steps

In addition to the library panning steps (a) to (d), the present methods may comprise one or more further screening steps. The further screens may allow selection of binding agents on the basis of binding properties such as binding specificity, or sensitivity of binding to the modified nucleic acid antigen.

Further screening steps may be carried out using binding agent prepared recombinantly after recovery of nucleic acid encoding binding agent from the display library, or using binding agent expressed and prepared from display library.

Primary screen

Binding agent may be assayed for ability to bind to modified nucleic acid in a binding assay, e.g. an immunoassay such as an enzyme linked immunosorbant assay (ELISA). Typically the modified nucleic acid is the same as in the library panning steps. Any form of direct binding assay is suitable. In one such assay, the binding agent, or alternatively the nucleic acid, is labelled. Suitable labels include radioisotopes such as I¹²⁵, enzymes such as peroxidise, fluorescent labels such as fluorescein, and chemiluminescent labels. Typically the other binding partner is insolubilised, for example immobilised on a microtitre plate. After combining the labelled component with the insolubilised component, the solid phase is washed and the amount of bound label determined. Binding to the modified nucleic acid may be compared to a control, e.g. a negative control such as PBS. Typically a binding agent of the invention shows binding to a modified nucleic acid which is at least 2x, 3x, 4x or greater than the binding to the negative control.

Preferably a binding screen of this kind is carried out on a preparation of binding agent expressed from library particles or cells, before isolation of nucleic acid encoding binding agent from the library, and so form a primary screen following library panning steps (a) to (d).

Secondary screen

Where modified nucleic acid used in library panning step (a) bears a label, e.g. a biotin label, binding agents may be screened for binding to the corresponding unlabelled modified nucleic acid. For example, a competition assay may be carried out. Methods for carrying out competition assays are known in the art (see for example, Assay Guidance Manual Version 5.0, 2008, Eli Lilly and Company and NIH Chemical Genomics

Center;available online at: http://www.ncgc.nih.gov/guidance/manual_toc.html).

Competition between nucleic acids for binding to an agent can be detected using suitable labels, e.g. ELISA, or a reporter agent attached to one nucleic acid which can be detected in the presence of other untagged nucleic acid(s). Binding agent, or nucleic acid may be immobilised. Competition may be assessed in terms of the percentage inhibition of the signal obtained in a negative control, e.g. when a neutral substance such as PBS replaces competing nucleic acid. Preferably, the level of inhibition by the unlabelled modified nucleic acid is at least 40%, at least 50%, at least 60%>, at least 70%>, at least 80%>, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% under the conditions of the assay.

Preferably such a screen is carried out on a preparation of binding agent expressed from library particles or cells, before isolation of nucleic acid encoding binding agent from the library. The screen may be carried out after a primary screen, and may form a secondary screen.

Screening for clon lity and uniqueness

Library members selected in the method may be tested for clonality and uniqueness in the nucleic acid encoding binding agent which they carry. For example, nucleic acid encoding binding agent in library particles or cells may be subjected to nucleic acid restriction analysis, and only unique clones taken forward. For example, in the case of a phage display library, infected bacterial isolates may be plated to single colonies, and at least 2 colonies selected from each isolate and cultured. PCR primers specific for either side of the nucleic acid encoding the binding agent (i.e. for the insertion site in the phage DNA) may be used to amplify the inserted nucleic acid encoding the binding agent, which is then subjected to restriction digestion analysis. Clones which are shown to be unique may then be selected.

Further screening for ability to bind modified nucleic acid and not unmodified nucleic acid

Binding agent may be assayed for the ability to specifically bind modified nucleic acid and not non-modified nucleic acid, for example using a binding assay or a competition assay. Typically the modified nucleic acid is the same as in the library panning steps.

Typically in a binding assay a binding agent of the invention shows binding to a modified nucleic acid which is at least 2x, 3x, 4x or greater than the binding to the non-modified nucleic acid. Preferably the binding agent does not bind to the non-modified nucleic acid within the limits of detection of the assay.

Non-modified nucleic acid preferably shows less than 25%, 20%, 15%>, 10%>, 5%, 2% ,1%> inhibition of binding of binding agent to the target modified nucleic acid in a competition assay. Preferably, non-modified nucleic acid does not compete with target modified nucleic acid for binding to a binding agent within the limits of detection of the assay, i.e. 0% or undetectable inhibition or competition. Screening assays to select for binding agents having particular binding specificities

The library panning steps of the current method select binding agents on the basis of a specific requirement for a nucleic acid modification in order to bind a nucleic acid molecule. As already noted, these steps will pull out of the library two broad classes of binding agent: (i) binding agents for which binding is specific for the nucleic acid modification alone (irrespective of base sequence or secondary structure in the modified nucleic acid); and (ii) binding agents for which binding is specific for the nucleic acid modification and additionally for a particular base sequence and/or a particular secondary structure in the modified nucleic acid. Secondary structure dependence is particular likely where the modified nucleic acid using in panning comprises internal self-complementarity and has potential for a stem loop structure. Thus, for example, where panning step (a) is performed using a backbone modified nucleic acid (e.g. nucleic acid having a

phosphorothioate backbone), and panning step (c) is performed using the equivalent nucleic acid having an unmodified (phosophodiester) backbone, the panning steps will select for binding agents having binding specificity for the modified backbone. Some of the selected binding agents will have a binding specificity that depends only on the presence of the modified backbone, and is independent of base sequence and/or secondary structure. Other binding agents however, will depend upon the presence of the nucleic acid modification and a particular base sequence and/or secondary structure for binding to a modified nucleic acid.

Binding specificity of a binding agent can be more precisely defined, and the contributory factors determined, using further screening, for example a competition assay, or binding assay.

Typically the assay is carried out using a panel of test nucleic acid antigens as comparator nucleic acids (in a binding assay) or as competitor nucleic acids (in a competition assay). Binding of binding agent to the test nucleic acids is compared to binding of the binding agent to the modified nucleic acid to which the binding agent is known to bind (typically the modified nucleic acid used in library panning), usually with reference to a negative control, e.g. PBS in place of nucleic acid. The sequence and structure of the test nucleic acid antigens are selected to reflect the aspects of binding specificity that are being investigated.

For example, to assay for binding which is dependent only on nucleic acid modification and is independent of sequence, base sequence might be varied while keeping modification and optionally secondary structure constant. A panel may comprise 2, 3, or more modified nucleic acids having different base sequences to the original modified nucleic acid used in panning (irrelevant sequence), but the same modification. Preferably the assay includes at least one non-modified nucleic acid.

As another example, to assay for binding which is dependent on nucleic acid modification and on a particular base sequence, base sequence might be varied while keeping modification and optionally secondary structure constant. In this case though, the variant sequences are generally related in some way to the modified nucleic acid that the binding agent is known to bind to (such as the modified nucleic acid used in panning). A panel might comprise 2, 3, or more modified nucleic acids comprising (overlapping) fragments of this modified nucleic acid.

To assay for binding which is dependent on nucleic acid modification and on a secondary structure, a panel of test nucleic acids might be used in which modification and secondary structure are kept constant, and sequence varied. For example, if the modified nucleic acid used in library panning includes a stem loop structure, a panel might comprise 2, 3, or more modified polynucleotides having different base sequences but all having a stem loop.

A binding agent may of course have a binding specificity determined by all three of modification, base sequence and secondary structure, particularly where the modified nucleic acid used in library panning has secondary structure, e.g. a stem loop. To test for that, competitor or comparator nucleic acids in an assay might include the self- complementary region of the modified nucleic acid used in panning, single stranded sections of that sequence, such as overlapping fragments of the sequence, and a different (irrelevant) self-complementary sequence, preferably of the same length.

It will be readily understood that by varying the sequences and structures of the comparator or competitor nucleic acids, one can determine the contributions made by nucleic acid modification, base sequence and secondary structure, to the binding preferences of a binding agent.

By way of illustration, reference is made for example to the competition assay carried out in the present Examples using the nucleic acids of Table 1 (SEQ ID NOs: 66-74), the results of which are shown in Table 4 (Figure 7). This assay was carried out to further establish the binding specificity of binding agents selected from a display library by panning with a nucleic acid (C274 oligonucleotide) having a phosphorothioate backbone (C274PS), and with the umodified C274 oligo (C274PO). All of the binding agents have binding specificity for phosphorothioate backbone, but the competition assay was used to determine which binding agents exhibited binding which is also dependent on base sequence and/or secondary structure in the modified C274 oligo.

The comparative level of binding to the nucleic acids in a binding assay, or the % inhibition of binding by competitor nucleic acids in a competition assay, indicates the binding preferences of the binding agent. Some illustrative guidelines for interpreting the results of the assays are given below. However, the results of a binding assay or competition assay may generally be interpreted empirically to determine the specificity of a binding agent.

In a binding assay, a binding agent having sequence-independent binding to modified nucleic acid may show comparable binding to more than one, e.g. 2, 3, 4, 5, or more modified nucleic acids having the same modification but irrelevant base sequences. In general the binding agent does not bind to the corresponding non-modified nucleic acid(s) within the limits of detection of the assay. A binding agent having sequence-dependent binding to modified nucleic acid may show binding to that specific sequence which is at least 2x, 3x, 4x, 5x, or more greater than binding to other base sequences having the same modification. A binding agent having secondary structure-dependent binding to modified nucleic acid may show binding to modified nucleic acid having that specific structure which is at least 2x, 3x, 4x, 5x, or more greater than binding to modified nucleic acids not having that secondary structure. In a competition assay, a binding agent having sequence-independent binding to modified nucleic acid, typically has at least 50%, such as at least 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 92, 94, 95, 96, 99 or 100% inhibition of binding by at least 1, 2, 3, 4, 5, or more modified nucleic acids having the same modification but irrelevant base sequences, where inhibition is expressed compared to binding to a target modified nucleic acid in the absence of competitor nucleic acid. For example, the % inhibition may be 70-100%), 80-100%), or 90-100%). A binding agent having sequence- dependent binding to modified nucleic acid typically has at least 50%>, such as at least 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 92, 94, 95, 96, 99 or 100% inhibition of binding by that specific sequence, where inhibition is expressed compared to binding of a target modified nucleic acid in the absence of competitor nucleic acid. For example, the % inhibition may be 70-100%, 80-100%, or 90-100%. Preferably the % inhibition produced by that sequence is at least 2x, 3x, 4x, 5x or more greater than any % inhibition shown by another competitor sequence, e.g. by another sequence fragment of the target modified nucleic acid to which the binding agent binds. A binding agent having secondary structure-dependent typically has at least 50%>, such as at least 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 92, 94, 95, 96, 99 or 100% inhibition of binding by modified nucleic acids having that specific structure, where inhibition is expressed compared to binding of a target modified nucleic acid in the absence of competitor nucleic acid. For example, the % inhibition may be 70-100%, 80-100%, or 90- 100%.

Determining binding specificity in this way can be useful in that it enables screening for a binding agent having a particular base sequence specificity in a modified nucleic acid. For example, one can screen to identify binding agents specifically recognising different truncated versions of modified nucleic acid. These can be useful for detecting degraded forms of a nucleic acid pharmacaphore in a clinical context, and so to monitor metabolism of the pharmacaphore. Alternatively, the assays can be used to identify a, which will have broad applicability in detecting multiple nucleic acid pharmacaphores. Titration assays and screening to determine IC50 values

A binding agent may be further assayed to determine IC50 value for binding to modified nucleic acid. The IC50 value may be determined in a competition assay in which unlabelled modified nucleic acid competes with labelled modified nucleic acid for binding to the binding agent. The IC50 value is the concentration of unlabelled modified nucleic acid which is required to produce 50% inhibition of binding of the labelled modified nucleic acid, under the conditions of the assay.

Methods for carrying out competition assays are known in the art and are described herein. Typically, in a competition assay to determine IC50 value, the concentrations of binding agent and labelled modified nucleic acid are selected to be the minimal concentration that gives a reasonable signal in a detection assay, for example, A370nm > 1.0 in a

fluorescence detection assay. Minimal concentrations may be determined in a titration assay, such as a chessboard titration assay as described herein. A negative control may be PBS. A positive control may be, for example, unlabelled modified nucleic acid at excess concentration.

A competition assay to determine IC50 value is preferably carried out using recombinantly expressed binding agent after extraction of nucleic acid encoding binding agent from the display library. In one example, an IC50 assay is carried out using binding agent comprising reformatted antibody.

In general, the IC50 value for the binding agent and the target nucleic acid antigen is <5ng/ml, 4, 3, 2, 1 , 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.15, 0.14ng/ml under the conditions of the assay. For example, IC50 value may be <1.3ng/ml or <2.7ng/ml.

Minimal concentrations for use in an IC50 competition assay may be determined in a titration assay, such as a chessboard titration assay. A series of dilutions of binding agent are each incubated with a series of dilutions of (labelled) modified nucleic acid, under the same reaction conditions that will be used in the IC50 assay, e.g. temperature, length of incubation, washing conditions and detection means. The assay allows an empirical determination of the minimal concentrations of binding agent and modified nucleic acid antigen which produce a reasonable signal in the assay -for example, A370nm = 1.0 in a fluorescence detection assay.

A titration assay can also be used to determine potency of binding of a binding agent to a given modified nucleic acid. The lower the concentration of binding agent and modified nucleic acid required to produce a reasonable signal, the greater the potency of the binding agent for binding to that modified nucleic acid.

In a titration assay such as that described herein, preferably the concentrations of binding agent and (labelled) modified nucleic acid required to give A370nm =1 at room

temperature are <30ng/well binding agent and <0.3ng/well modified nucleic acid. Minimal concentrations of binding agent may be for example <30, 25, 20, 15, 10, 9, 8 or 7.5ng/well and minimal concentrations of modified nucleic acid may be for example <0.3, 0.2, 0.1, 0.05, 0.04, 0.039, 0.038ng/well of modified nucleic acid, or any combination thereof, such as <8 or <7.5ng/well binding agent and <0.038ng/well modified nucleic acid, for example, any of the combinations presented for binding agents in Table 5 (Fig 8). A titration assay described herein may also be used to determine the effect of nucleic acid secondary structure on binding between the binding agent and the modified nucleic acid antigen. Titration assays may be carried out at a range of different temperatures, and the results compared to determine any binding preferences. If temperature has no effect on results, it is likely that the binding agent binding specificity is not dependent on secondary structure. If the signal increases with increasing temperature, the binding agent likely binds preferentially to single stranded nucleic acid species. Similarly if the signal decreases with decreasing temperature, the binding agent typically binds preferentially to double stranded nucleic acid species.

Reaction conditions during the method (library panning and further screening steps)

All binding properties and binding specificities referred to are as determined under the conditions at that stage in the method, e,g during library panning, during additional screening or assaying, and within the limits of detection of the method.

In particular, binding specificities and properties are likely to be influenced by

temperature. At higher temperatures, nucleic acids are more likely to adopt single stranded conformation. Thus binding agents identified by panning at higher temperatures are more likely to have specificity for single stranded modified nucleic acids.

Preferably, where a binding agent is intended for a particular use, e.g. in diagnostics in vitro or in vivo, it is preferable to perform the present methods (e.g. panning, binding assays, competition assays) under conditions which mimic as closely as possible the conditions under which the binding agent will be used.

Thus, if a binding agent is intended for use at room temperature, for example, in clinical in vitro assaying, preferably the library screen (and any additional screening steps) are carried out at room temperature, for example, at about 20°C. Similarly, if a binding agent is intended for use in vivo, e.g. in whole body diagnostics, or in therapy, preferably the library screen (and any additional screening steps) are carried out at body temperature, for example, at about 37°C.

Similar considerations apply to other reaction conditions. For example, if a binding agent is intended for use in a clinical assay of blood plasma samples, preferably an assay to determine IC50 value is carried out in a matrix that is representative of plasma.

Further development of binding agents

Binding agents identified in the present methods may be further developed. For example, where a binding agent comprises an antibody, development may be by antibody maturation. Binding agents may be conjugated to further functional moieties, such as detectable labels or catalytic molecules. Binding agents may be further formulated as described herein, for example, into compositions or pharmaceutical compositions.

Binding agents

In addition to the present methods, the invention also relates to binding agents having binding specificity for a particular nucleic acid modification. In one aspect the binding agents may be identified or identifiable using the present methods. Functional properties of binding agents

A binding agent of the invention has binding specificity for a particular nucleic acid modification. By this is meant that the binding agent will bind specifically to a modified nucleic acid but will not bind (or will not substantially bind to) a nucleic acid having the same sequence but without the modification, e.g. naturally occurring nucleic acid. Ability to bind differentially in this way can be determined using one or more of the assays described herein. Generally this is within the limits of detection of the given assay.

Thus an agent has the ability to differentially bind to a modified nucleic acid compared to a non-modified nucleic acid, and to distinguish between them. Put in another way, a binding agent has a greater relative affinity for the modified nucleic acid compared to the non- modified nucleic, such that the agent can discriminate between the two in a clinical assay.

As already noted, in one embodiment, binding agents of the invention may exhibit binding to a nucleic acid which is solely modification specific. Alternatively, binding agents of the invention may exhibit binding to modified nucleic acid which is modification and sequence and/or secondary structure specific. A binding agent which binds specifically to a modified nucleic acid may demonstrate any of these binding specificities.

For example, binding agents of the invention may have binding specificity dependent solely on a modified backbone, e.g. a phosphorothioate backbone. These agents will recognise and bind to a nucleic acid having the modified backbone irrespective of the sequence and secondary structure, but will not bind to the equivalent nucleic acid without modified backbone. Alternatively, binding agents may have binding specificity dependent on both the modified backbone, and a particular base sequence, and/or a particular secondary structure such as a stem loop.

Binding agents which are solely modification specific (regardless of the base sequence or structure) may be particularly useful in detecting and/or measuring nucleic acid

pharmacaphores in clinical samples, since they have broad applicability. An example of such a binding agent is binding agent 3749.03 in the present Examples. Binding agents which are modification and sequence specific, may be useful in monitoring particular modified nucleic acid pharmacaphores. A panel of binding agents may be prepared, each recognising a different sequence epitope in the pharmacaphore, e.g.

corresponding to truncations of the pharmacaphore. Such a panel may be useful in monitoring degradation and clearance of a pharmacaphore. Binding of binding agent 3749.93 is influenced by both modification and sequence.

Binding specificity may depend upon secondary structure of the nucleic acid, e.g. requiring a stem loop or double stranded structure, or of single stranded structure. An example is binding agent 3745.58 in the present Examples which favours single stranded structures. Alternatively, binding may be independent of secondary structure. An example is binding agent 3742.30 in the present Examples which shows structure-independent binding.

Independence of secondary structure can be useful since it may allow an assay using the binding agent to be performed at various temperatures. Specificity for secondary structure in a modified nucleic acid may be in addition to sequence dependence, or may be sequence independent.

Nucleic acid modifications for which a binding agent may be specific are described further herein. A binding agent may have binding specificity for any of these, and may bind specifically to any of the modified nucleic acids described herein. Preferably a binding agent exhibits binding which is specific for a modified backbone, such as

phosphorothioate, or for a modified sugar component. Preferably a binding agent shows backbone modification specific binding. A binding agent may bind specifically to a modified base, other than a methylated base.

In addition to modification specific binding, a binding agent may additionally display binding specificity for a particular base sequence in modified nucleic acid, including a base sequence of any of the nucleic acid sequences or fragments thereof described herein, e.g. a sequence of any one or more of SEQ ID NOs:65, 66, 67 or 69-74. This is not limited to the base sequence in the context of a backbone modification listed for the given SEQ ID NO: in the Sequence Listing or in Table 1. For example, reference to the base sequence in SEQ ID NO: 67 refers to that base sequence having a modified phosphorothioate backbone as listed for SEQ ID NO: 67, and also to that base sequence in other contexts, e.g. having a backbone modified in another way. A binding agent may recognise an epitope comprising any of the nucleic acid sequences or fragments thereof described herein. Binding specificity may be dependent upon secondary structure in a modified nucleic acid, for example, internal self complementarity.

Binding specificity of a binding agent may be determined in one or more assays, such as those described herein. Throughout, binding and properties are as measure under the conditions of and within the limits of detection of a given assay.

A binding agent may be tested for binding to modified nucleic acid but not to unmodified nucleic acid in one or more screen(s) of one or more display library(s) such as those described in the methods herein, and within the conditions and limits of detection of the assay.

Binding may be assessed in a binding assay, such as an immunoassay. Any form of direct binding assay is suitable. In one such assay, the binding agent, or alternatively the nucleic acid, is labelled. Suitable labels include radioisotopes such as I¹²⁵, enzymes such as peroxidise, fluorescent labels such as fluorescein, and chemiluminescent labels. Typically the other binding partner is insolubilised, for example immobilised on a microtitre plate. After combining the labelled component with the insolubilised component, the solid phase is washed and the amount of bound label determined. Binding to a test nucleic acid may be compared with binding to a control, e.g. a negative control such as PBS. Typically a binding agent of the invention shows binding to a modified nucleic acid which is at least 2x, 3x, 4x or greater than the binding to the non-modified nucleic acid. Preferably the binding agent does not bind to the non-modified nucleic acid within the limits of detection of the assay. A binding agent may show binding to modified nucleic acid which is at least 2x, 3x, 4x, 5x greater than that of the control.

Binding may be determined using a competition assay. Methods for carrying out competition assays are known in the art. Competition between nucleic acid for binding to an agent can be detected using suitable labels, e.g. ELISA, or a reporter agent attached to one nucleic acid which can be detected in the presence of other untagged

polynucleotide(s). Binding agent, or nucleic acid may be immobilised. Competition may be assessed in terms of the percentage inhibition of the signal obtained in a negative control, e.g. when a neutral substance such as PBS replaces competing nucleic acid.

Non-modified nucleic acid preferably shows less than 25%, 20%, 15%, 10%, 5%, 2% ,1% inhibition of binding of binding agent to the modified nucleic acid. Preferably, non- modified nucleic acid does not compete with modified nucleic acid for binding to a binding agent within the limits of detection of the assay, i.e. 0% or undetectable inhibition or competition.

The particular type of specificity shown by a binding agent (solely modification specific, or modification and sequence and/or structure specific) can be determined using a binding assay or competition assay as described herein in relation to the methods of the invention, and with reference to the Examples. The effect of secondary structure on binding can also be determined by determining the effect of temperature on binding to the agent, as described herein.

In one aspect, it is preferred that a binding agent has an IC50 value for binding a modified nucleic acid antigen of <5ng/ml, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.15, 0.14ng/ml in a competition assay. For example, IC50 value may be <1.3ng/ml or

<2.7ng/ml.

The IC50 value may be determined in a competition assay in which unlabelled modified nucleic acid competes with labelled modified nucleic acid for binding to the binding agent. The IC50 value is the concentration of unlabelled modified nucleic acid which is required to produce 50%> inhibition of binding of the labelled nucleic acid, under the conditions of the assay.

Methods for carrying out competition assays and determining IC50 values are known in the art and are described herein in relation to the methods of the invention.

In one aspect, a binding agent has a preferred potency. Potency can be determined in a titration assay. Methods for performing a titration assay, e.g. a chessboard titration, are described herein in relation to the methods of the invention. In a titration assay such as that described herein, preferably the concentrations of binding agent and (labelled) modified nucleic acid required to give A370nm =1 at room temperature are <30ng/well binding molecule and <0.3ng/well modified polynucleotide. Minimal concentrations of binding agent may be for example <30, 25, 20, 15, 10, 9, 8, 7.5ng/well and minimal concentrations of modified nucleic acid may be for example <0.3, 0.2, 0.1, 0.05, 0.04, 0.039, 0.038ng/well of modified nucleic acid, or any combination thereof, such as <8ng or <7.5ng/well binding agent and <0.038ng/well modified nucleic acid, for example, any of the combinations presented for binding agents in Table 5 (Fig 8).

Binding agents may be characterised using the methods described in the present Examples. Structural properties of binding agents

A binding agent as described herein is able to bind to a nucleic acid molecule. Typically there is some degree of complementarity between the binding agent and the nucleic acid which facilitates binding, e.g. spatial complementarity between a surface of the binding agent and an area of the nucleic acid. In general a binding agent is encoded by DNA, and suitable for display in a display library as described herein.

A binding agent of the invention comprises an antigen binding site, for example, an antigen binding site of an antibody. An agent may comprise an antibody or a fragment or derivative thereof comprising an antibody antigen-binding site. An agent may comprise a non-antibody molecule that comprises an antigen binding site. A binding agent may be natural or wholly or partially synthetic, as described herein.

The basic structural unit of an antibody is generally a tetramer that consists of two identical pairs of immunoglobulin (Ig) chains, each pair having one light and one heavy chain.

Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains mediate effector functions. Thus these domains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors.

The variable domains or regions are the regions of both the light chain and the heavy chain of an Ig that contain antigen-binding sites. A variable region is composed of polypeptide chains containing four relatively invariant "framework regions" (FRs) and three highly variant "hypervariable regions" (HVs). Because the HVs constitute the binding site for antigen(s) and determine specificity by forming a surface complementary to the structure of the bound antigen, they are more commonly termed the "complementarity-determining regions," or CDRs, and, proceeding from the N-terminus of a heavy or light chain, are denoted CDR1, CDR2, and CDR3 (see, Sequences of Proteins of Immunological Interest, E. Kabat et al., U.S. Department of Health and Human Services, 1983). A VH domain comprises a set of HCDRs, and a VL domain comprises a set of LCDRs. Thus, VH comprises HCDR1, HCDR2 and HCDR3, and VL comprises LCDR1, LCDR2 and LCDR3.

Within the heavy and light chain, the framework regions surround the CDRs. Proceeding from the N-terminus of a heavy or light chain, the order of regions is: FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4. As used herein, the term variable domain or variable region is intended to encompass a complete set of four framework regions and three complementarity-determining regions. Thus, a sequence encoding a "variable region" or a "variable domain" would provide the sequence of a complete set of four framework regions and three complementarity-determining regions.

The CDRs are primarily responsible for binding to an epitope of an antigen and the CDR3 comprises a unique region specific for antigen-antibody binding. An antigen-binding site, therefore, may include six CDRs, comprising the CDR regions from each of a heavy and a light chain V region. Alteration of a single amino acid within a CDR region can alter the affinity of an antibody for a specific antigen (see Abbas et al., Cellular and Molecular Immunology, 4th ed. 143-5, 2000). The locations of the CDRs have been precisely defined, e.g., by Kabat et al, Sequences of Proteins of Immunologic Interest, U.S. Department of Health and Human Services, 1983. The light and heavy chains of an Ig each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. By the Kabat definition, the CDRs of the light chain are bounded by the residues at positions 24 and 34 (L-CDR1), 50 and 56 (L-CDR2), 89 and 97 (L- CDR3); the CDRs of the heavy chain are bounded by the residues at positions 31 and 35b (H-CDR1), 50 and 65 (H-CDR2), 95 and 102 (H-CDR3), using the numbering convention delineated by Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5^th Edition, Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda (NIH Publication No. 91-3242).

Reference is made to the numbering scheme from Kabat, E. A., et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987) and (1991). In these compendiums, Kabat lists many amino acid sequences for antibodies for each subclass, and lists the most commonly occurring amino acid for each residue position in that subclass. Kabat uses a method for assigning a residue number to each amino acid in a listed sequence, and this method for assigning residue numbers has become standard in the field. To assign residue numbers to a candidate antibody amino acid sequence which is not included in the Kabat compendium, one follows the following steps. Generally, the candidate sequence is aligned with any immunoglobulin sequence or any consensus sequence in Kabat. Alignment may be done by hand, or by computer using commonly accepted computer programs; an example of such a program is the Align 2 program. Alignment may be facilitated by using some amino acid residues which are common to most Fab sequences. For example, the light and heavy chains each typically have two cysteines which have the same residue numbers; in VL domain the two cysteines are typically at residue numbers 23 and 88, and in the VH domain the two cysteine residues are typically numbered 22 and 92. Framework residues generally, but not always, have approximately the same number of residues, however the CDRs will vary in size. For example, in the case of a CDR from a candidate sequence which is longer than the CDR in the sequence in Kabat to which it is aligned, typically suffixes are added to the residue number to indicate the insertion of additional residues. For candidate sequences which, for example, align with a Kabat sequence for residues 34 and 36 but have no residue between them to align with residue 35, the number 35 is simply not assigned to a residue.

CDR and framework (FR) residues may also be determined according to a structural definition (as in Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987). Where these two methods result in slightly different identifications of a CDR, the structural definition is generally preferred, but the residues identified by the sequence definition method are considered important FR residues for determination of which framework residues to import into a consensus sequence.

The framework region (FR) comprises relatively conserved sequences flanking the three highly divergent complementarity-determining regions (CDRs) within the variable regions of the heavy and light chains of an antibody. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the variable region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Without being bound by theory, the framework regions serve to hold the CDRs in an appropriate orientation for antigen binding. The numbering of the residues in the light chain and heavy chain framework regions follows the numbering convention delineated by Kabat et al., (1991, supra). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. A "human" framework region is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework region of a naturally occurring human immunoglobulin.

By way of illustration, antibody VH and VL domains, FRs and CDRs occurring in binding agents identified in the present Examples are listed in the attached sequence listing.

The constant region comprises the portion of an antibody molecule which confers effector functions. A binding agent may include constant regions derived from human immunoglobulins. The heavy chain constant region can be selected from any of five isotypes: alpha, delta, epsilon, gamma or mu. Heavy chains of various subclasses (such as the IgG subclass of heavy chains) are responsible for different effector functions. Thus, by choosing the desired heavy chain constant region, humanized antibodies with the desired effector function can be produced. The light chain constant region can be of the kappa or lambda type.

A binding agent may comprise a monoclonal antibody or a fragment thereof. Typically a monoclonal antibody refers to an antibody produced by a single clone of cells, e.g. B- lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art.

Immunoglobulins also exist in a variety of other forms including, for example, Fv, Fab, and (Fab')₂, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al, Eur. J. Immunol. 17: 105, 1987; Huston et al, Proc. Natl. Acad. Sci. U.S.A., 85:5879- 5883, 1988; Bird et al, Science IM'AT -Md, 1988; Hood et al, Immunology, Benjamin, N.Y., 2nd ed., 1984; Hunkapiller and Hood, Nature 323: 15-16, 1986).

It has been shown that such fragments of a whole antibody can perform the function of binding antigens. A binding agent of the invention may comprise an antibody fragment or derivative thereof, which comprises an antibody antigen binding site.

Examples of antigen binding fragments may include:

(i) the fragment antibody (Fab) fragment consisting of VL, VH, CL and CHI domains;

(ii) the Fd fragment consisting of the VH and CHI domains;

(iii) the Fv fragment consisting of the VL and VH domains of a single antibody;

(iv) the dAb fragment, a small monomeric antigen-binding fragment of an antibody, consisting of the VH or VL domain (Ward, E. S. Et al, Nature 341,544-546 1989; Holt et al, Trends in Biotechnology 21, 484-490, 2003);

(v) isolated CDR regions;

(vi) F(ab')₂ fragments, a bivalent fragment comprising two linked Fab fragments;

(vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426,1988 ; Huston et al, PNAS USA, 85, 5879- 5883,1988) ;

(viii) dsFV, wherein the VL and VH are chain linked by disulfide bonds; (ix) bispecific single chain Fv dimers (PCT/US92/09965); and

(x) "diabodies", multivalent or multispecific fragments constructed by gene fusion (W094/13804; P. Holliger et al, Proc. Natl. Acad. Sci. USA 90 6444-6448,1993).

Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains (Y. Reiter et al, Nature Biotech, 14, 1239-1245,1996). Minibodies comprising an scFv joined to a CH3 domain may also be made (S. Hu et al, Cancer Res., 56, 3055-3061,1996).

Other examples of binding fragments are Fab', which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region, and Fab'-SH, which is a Fab' fragment in which the cysteine residue(s) of the constant domains bear a free thiol group. (Fab')₂ fragments are the fragments of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction, or the corresponding structure obtained by genetic engineering;

Methods of making these fragments are routine in the art. For example, fragments may be obtained starting from a whole antibody molecule, by digestion with enzymes, e.g. pepsin or papain, and/or by cleavage of disulphide bridges by chemical reduction. Fragments may also be obtained by protein synthesis, by genetic recombinant methods or by nucleic acid synthesis and expression.

In the present case, where sequence is presented herein for a VH of VL domain, an scFv fragment may comprise a peptide linker between the domains, and/or a 6-His c-myc tag at the C-terminal end of the VL domain.

A binding agent may comprise a bispecific (or bifunctional) antibody, in which two different variable regions are combined in the same molecule.

Bispecific antibodies may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4,446-449 (1993)), e.g. prepared chemically or from hybrid hybridomas. Bispecific antibodies may also be any of the bispecific antibody fragments mentioned above. Thus, bispecific antibodies can be constructed as entire IgG, as bispecific Fab'2, as Fab 'PEG, as diabodies or as bispecific scFv. Further, two bispecific antibodies can be linked using routine methods known in the art to form tetravalent antibodies.

Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli.

Antibodies may be modified using recombinant methods to produce other antibodies or chimeric molecules that still bind the target antigen. A binding agent may comprise such a modified antibody or derivative.

For example, an antigen binding site of an antibody (e.g. the VH and/or VL domain, or the CDRs, of an antibody) may be fused to another polypeptide (e.g. the constant regions or constant regions plus framework regions, of a different antibody). This can be done by fusing the encoding DNAs. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.

Chimeric antibodies may be antibodies whose light and heavy chain genes comprise variable and constant regions encoded by variable and constant region genes of different species. Typically such chimeric antibodies are produced by genetic engineering. For example, the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3. In one example, a therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species can be used, or the variable region can be produced by molecular techniques. Methods of making chimeric antibodies are well known in the art, e.g., see U.S. Patent No. 5,807,715, which is herein incorporated by reference.

Humanizing is a technique in which one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody is fused to a human framework region. The non-human antibody providing the CDRs is termed a "donor" and the human antibody providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor antibody in the humanized molecule. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized molecule, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences.

A "humanized antibody" may be an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized molecule may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering, e.g., see U.S. Patent No. 5,225,539 and U.S. Patent No. 5,585,089, which are herein incorporated by reference.

A human antibody is an antibody wherein the light and heavy chain genes are of human origin.

An antigen binding site refers to the part of a molecule that binds to and is complementary to all or a part of a target antigen. An antibody may only bind to a particular part of an antigen, which is termed an epitope. An antibody antigen binding site may be provided by one or more antibody-variable domains. An antibody antigen binding site may comprise a VH and/or a VL domain as described. An antigen binding site may comprise one or more loop structures which may be analogous to one or more CDRs. An antigen binding site may comprise one or more CDRs, such as at least 1, 2, 3, 4, 5, or 6 CDRs, such as any of the CDRs described herein. An antigen binding site may comprise for example, HCDR3 and/or LCDR3. An antigen binding site may comprise a set of CDRs corresponding to the CDRs in VL or VH e.g. the set of CDRs which is (HCDR1 + HCDR2 + HCDR3) and/or the set of CDRs which is (LCDR1+ LCDR2 + LCDR3).

An epitope refers to a particular site on an antigen which is recognized by an antigen binding site. An epitope may be defined with reference to a particular sequence: an amino acid sequence in a peptide antigen; a base sequence in a nucleic acid antigen. Two binding agents, e.g. antibodies are said to bind to the same epitope if each competitively inhibits (blocks) binding of the other to the antigen as measured in a competitive binding assay (see, e.g., Junghans et al, Cancer Res. 50: 1495-1502, 1990). Alternatively, two binding agents, e.g. antibodies have the same epitope if most mutations in the antigen that reduce or eliminate binding of one binding agent reduce or eliminate binding of the other.

A binding agent may comprise an antigen binding site in a non-antibody scaffold. An antigen binding site may be formed by positioning one or more CDRs on a non-antibody scaffold, e.g. by grafting in one or more CDRs. An antigen binding site may be formed by rational or random mutation of amino acids in the molecule, often of amino acids in one or more loop structures, or of surface residues, in the scaffold molecule which is/are involved in binding, to provide a particular binding specificity. Such loop structures can be analogous to the CDRs of an antibody. Thus, novel binding sites can be engineered, and libraries of binding agents can be prepared and screened for binding capability.

Typically, molecules for use as scaffolds are those which are involved with binding proteins or other ligands under natural conditions. For example, protein display scaffolds are reviewed in Hosse, R.J. et al, 2006, Protein Science, 15: 14-27. These include:

(a) scaffolds with a-helical frameworks, such as Affibodies, Immunity proteins (e.g the E coli colicin E7 immunity protein ImmE7), Cytochrome b₅62, peptide a₂p8, repeat proteins;

(b) scaffolds with few or irregular secondary structures, such as insect defensin A, Kunitz domain inhibitors, PDZ domain proteins (e.g. Ras-binding protein AF-6), scorpion toxins (e.g. Charybdotoxin), the plant homeodomain (PHD) finger protein from the transcriptional cofactor Μί2β, TEM-1 β-lactamase; and

(c) scaffolds with β-sheet frameworks, such as 10^th fibronectin type III domain (¹⁰Fn3), human cytotoxic lymphocyte associated protein 4 (CTLA-4) (which comprises CDR-like loops similar to antibodies), T-cell receptors, Knottins, Neocarzinostatin (the neocarzinostatin protein component (NCS) has two loops, structurally equivalent to CDR1 and CDR3 of an antibody), carbohydrate binding module 4-2 (CBM4-2, derived from the Rhodothermus marinus xylanase XynlOA)), Tendamistat (an inhibitor of a-amylase from Streptomyces tendae), Lipocalins, or green fluorescent protein (GFP).

A summary of the scaffolds is provided in Table 1 of Hosse et al, supra, the contents of which are hereby incorporated by reference.

Affibodies are an engineered version (Z domain) of one of the five stable three-a-helix bundle domains from the immunoglobulin Fc-binding region of staphylococcal protein A (Hosse et al, supra). Affibodies, and affibody display libraries are reviewed in Nygren, Per- Ake, 2008, FEBS Journal 275: 2668-2676. For examepl, naive (or unbiased) libraries of candidate affibody binding proteins have been constructed through the genetic randomisation of 13 surface located positions of the Z-protein-domain scaffold (Nygren, 2008 supra).

Repeat motif proteins offer the opportunity to vary the size of the binding interface by varying the number of repeats. An example of repeat motif proteins are those with ankyrin repeat domains. Typically these consist of repetitive structural units of 33 residues comprising a β-turn followed by two anti-parallel a-helices and a loop linking to the turn of the next repeat. Library construction of designed ankyrin repeat proteins (DARPins) has been described; artificial consensus sequence motifs have been designed and nonconserved residues suitable for restricted randomizations have been determined. Leucine rich-repeat proteins have also been utilised (Hosse et al, supra).

Kunitz domain inhibitors are generally protease inhibitor proteins, possessing loop strctures that can be mutated without destabilising the structural framework. Examples include: bovine pancreatic trypsin inhibitor (BPTI); human pancreatic secretory trypsin inhibitor (PSTI); Alzheimers amyloid β-protein precursor inhibitor (APPI); the leech derived trypsin inhibitor (LTDI); the mustard trypsin inhibitor II (MTI II); the periplasmic E coli protease inhibitor ecotin; and the human lipoprotein associated coagulation inhibitor (LACI) . Use of fibronectin type III domain as a scaffold, and the production of libraries of scaffold molecules bearing randomised loops (analogous to antibody CDRs) to provide varied binding capabilities is described in WO 00/34784.

Knottins are proteins, some of which function as protease inhibitors, comprising disulphide bonds which lead to a knotted topology, and interspersed variable peptide loops. More data concerning knottins is available on the "knottin web" (http ://knottin. com) (J. Gracy et al. Nucleic Acids Res. 2008, 36:D314-9, and J.-C. Gelly et al. Nucleic Acids Res. 2004, 32:D156-9.) Examples include the trypsin inhibitor from the squirting cucumber Ecballium elaterium (EETI-II), the C-terminal cellulose binding domain (CBD) of cellobiohydrolase I from the fungus Trichoderma reesei, and Min-23, a derivative of EETI-II.

Lipocalins are proteins, typically about 160-180 residues, involved in storage or transport of hydrophobic and/or chemically sensitive organic compounds. They consist of a β-barrel of eight anti-parallel β-strands, which form a conical structure. The entrance to the ligand binding pocket is composed of four hypervariable loops connecting the β-strands in a pairwise fashion at the open end of this central folding motif. Typically for generation of libraries for binding to a low-molecular weight ligand, residues in the cavity (ligand binding pocket) are mutated/randomised; for protein targets, loop residues are mutated/randomised. The engineered versions are generally referred to as anticalins, reviewed in Skerra, A., 2008, FEBS Journal 275: 2677-2683. Examples of liopcalin scaffolds include the bilin-binding protein (BBP) from the butterfly Pieris brassicae, human apolipoprotein D (ApoD), and the bovine heart fatty acid-binding protein (FABP).

A suitable scaffold into which to graft one or more CDRs may be provided by any domain member of the immunoglobulin gene superfamily.

A scaffold may be a human or non- human protein. Preferably, where a binding agent is intended for use in a human, the scaffold is a human scaffold.

Non-antibody scaffolds are often smaller and/or structurally less complex than antibodies, which can be advantageous. Although CDRs can be carried by non-antibody scaffolds, the structure for carrying a CDR, e.g. CDR3, or a set of CDRs in a binding agent will generally be an antibody heavy or light chain sequence or a substantial portion thereof in which the CDR or set of CDRs is located at a location corresponding to the CDR or set of CDRs of naturally occurring VH and VL antibody variable domains. The structures and locations of immunoglobulin variable domains may be determined by reference to Kabat (supra).

Illustrative examples of binding agents

It is apparent that the present methods can be used to identify binding agents having specificity for any specific modification or modified nucleic acid. The methods thus allow specific detection and targeting of many pharmacaphores. By way of example, for illustrative efficacy, the inventors developed binding agents specific for modified C274 oligonucleotide having a fully modified phosphorothioate backbone (C274PS). The C274 oligonucleotide contains unmethylated CpG motifs, and mimics non-mammalian DNA- binding to Toll-like receptor 9 (TLR9), inducing proinflammatory cytokines and leading to a T helper type 1 (Thl) immune response. C274PS is a 22mer phosphorothioate oligonucleotide with a potential for dimer formation between 12 internal bases. The nucleotide sequence of C274 is presented in SEQ ID NO: 65 (unmodified backbone C274PO) and in SEQ ID NO: 66 (modified backbone C274PS).

The present Examples describe identification of a number of binding agents which bind specifically to the C274PS oligonucleotide having a phosphorothioate backbone, but not to the corresponding unmodified C274PO oligo (having a non-modified phosphodiester backbone). A selection of the binding agents were assayed to determine the nature of their binding specificity (see Table 4), and to determine IC50 value (see Table 6).

A binding agent may comprise an antigen-binding site of, or derived from, any of the binding agents in the Examples such as any of binding agents 3749.03, 3742.30, 3745.58 or 3749.93. A binding agent may exhibit the binding specificity, potency, and/or IC50 value of any of the binding agents in the Examples, such as any of binding agents 3749.03, 3742.30 3745.58 or 3749.93. For example, a binding agent may comprise an antigen binding site of or derived from that of binding agent 3749.03. In one aspect such a binding agent has binding specificity for a phosphorothioate backbone independent of sequence context, as described herein. In one aspect such a binding agent has a potency, and/or IC50 value which is at least as favourable as that of binding agent 3749.03, as determined in the present Examples.

Such a binding agent may comprise a VH domain having the sequence of SEQ ID NO: 15 or a functional variant thereof and/or a VL domain having the sequence of SEQ ID NO: 16, or a functional variant thereof.

Such a binding agent may comprise any one or more of, such as at least 1, 2, 3, 4, 5, or 6 of, the CDRs in SEQ ID NOS: 1-6. For example, a binding agent may comprise at least SEQ ID NO: 3 and SEQ ID NO: 6, and/or may comprise SEQ ID NOS: 1-3 and/or SEQ ID NOS: 4-6. Also envisaged are functional variant CDR sequences. A binding agent may comprise a HCDR3 having less than 10, 9, 8 amino acids.

In another example, a binding agent may comprise an antigen binding site of or derived from that of binding agent 3742.30. In one aspect such a binding agent has binding specificity for phosphorothioate backbone independent of secondary structure. In one aspect, binding of the binding agent to modified nucleic acid is substantially unaffected by temperature. In one aspect such a binding agent has a potency, and/or IC50 value which is at least as favourable as that of binding agent 3742.30, as determined in the present Examples.

Such a binding agent may comprise a VH domain having the sequence of SEQ ID NO: 31 or a functional variant thereof and/or a VL domain having the sequence of SEQ ID NO: 32, or a functional variant thereof.

Such a binding agent may comprise any one or more of, such as at least 1, 2, 3, 4, 5, or 6 of, the CDRs in SEQ ID NOS: 17-22. For example, a binding agent may comprise at least SEQ ID NO: 19 and SEQ ID NO: 22, and/or may comprise SEQ ID NOS: 17-19 and/or SEQ ID NOS: 20-22. Also envisaged are functional variant CDR sequences. In another example, a binding agent may comprise an antigen binding site of or derived from that of binding agent 3745.58. In one aspect such a binding agent has binding specificity dependent on a phosphorothioate backbone and secondary structure, e.g. stem loop structure. In one aspect, binding of the binding agent to modified nucleic acid antigen is substantially increased by lowering temperature, for example, from 37°C to 22°C and/or from 22°C to 4°C . In one aspect such a binding agent has a potency, and/or IC50 value which is at least as strong as that of binding agent 3745.58, as determined in the present Examples.

Such a binding agent may comprise a VH domain having the sequence of SEQ ID NO: 47 or a functional variant thereof and/or a VL domain having the sequence of SEQ ID NO: 48, or a functional variant thereof.

Such a binding agent may comprise any one or more of, such as at least 1, 2, 3, 4, 5, or 6 of, the CDRs in SEQ ID NOS: 33-38. For example, a binding agent may comprise at least SEQ ID NO: 35 and SEQ ID NO: 38, and/or may comprise SEQ ID NOS: 33-35 and/or SEQ ID NOS: 36-38. Also envisaged are functional variant CDR sequences.

In another example, a binding agent may comprise an antigen binding site of or derived from that of binding agent 3749.93. In one aspect such a binding agent has binding specificity dependent on a phosphorothioate backbone and base sequence. In one aspect such a binding agent has a potency, and/or IC50 value which is at least as strong as that of binding agent 3749.93, as determined in the present Examples.

Such a binding agent may comprise a VH domain having the sequence of SEQ ID NO: 63 or a functional variant thereof and/or a VL domain having the sequence of SEQ ID NO: 64, or a functional variant thereof.

Such a binding agent may comprise any one or more of, such as at least 1, 2, 3, 4, 5, or 6 of, the CDRs in SEQ ID NOS: 49-54. For example, a binding agent may comprise at least SEQ ID NO: 51 and SEQ ID NO:54, and/or may comprise SEQ ID NOS: 49-51 and/or SEQ ID NOS: 52-54. Also envisaged are functional variant CDR sequences.

Nucleic acids encoding binding agents The present invention further relates to an isolated nucleic acid encoding a binding agent such as an antibody or antibody fragment, e.g. a VH and/or a VL domain, or a HCDR or LCDR, as described herein. The invention further relates to a vector, such as an expression vector comprising such a nucleic acid sequence, and to a host cell transformed with such a nucleic acid or vector.

Binding agents and nucleic acids encoding binding agents according to the invention are generally provided in isolated or purified form. Typically the agents or nucleic acids encoding the agents have been isolated or purified from their natural environment, in substantially pure or homogeneous form, or, in the case of encoding nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding the binding agent. Isolated binding agents and isolated nucleic acid encoding binding agent will be free or substantially free of material with which they are naturally associated, such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo.

Binding agents or nucleic acid encoding binding agents may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example the binding agents will normally be mixed with gelatin or other carriers if used to coat microtitre plates, e.g. for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.

The invention does not relate to antibodies or other binding agents in their natural environment. Antibodies or fragments thereof, or other binding agents have been isolated or obtained by purification from natural sources, or else obtained by genetic recombination or by chemical synthesis. The invention does not relate to antibodies in polyclonal sera.

Compositions and articles comprising binding agents

It will be apparent that, for end use, e.g. in diagnostics or therapy, it may be desirable to add one or more functional activities (other than nucleic acid binding) to a binding agent. Accordingly a binding agent may be coupled to or conjugated to another functional or bioactive moiety. Alternatively or additionally, a binding agent may comprise amino acid sequence encoding such a moiety. Examples of such active molecules include detectable labels, enzymes or catalytic sites, toxins or targeting moieties.

Therefore, for example, in addition to antibody sequences and/or an antigen-binding site, a binding agent according to the present invention may comprise other amino acids, encoding a peptide or polypeptide that provides another function in addition to antigen binding, e.g. a catalytic site. A binding agent may comprise an effector region, such as an Fc region, which include a particular additional function, for example, catalytic activity.

Binding agents of the invention may carry a detectable label, or may be conjugated to a toxin or a targeting moiety or enzyme (e.g. via a peptidyl bond or linker). A binding agent of the invention may comprise a detectable label.

Binding agents may be formulated into a composition, such as a pharmaceutical composition, comprising one or more binding agents according to the invention, of the same or different binding specificities. Each binding agent in the composition may bind the same target modified nucleic acid, or the composition may comprise at least one binding agent for each of at least 2, 3, 4, 5, 6 or more modified nucleic acid targets.

In one aspect, a composition comprises binding agents having different binding specificities. For example, the composition may comprise at least one binding agent having specificity for a modification, e.g. a modified backbone, irrespective of sequence, and at least one binding agent having specificity for a particular sequence having that modification. A composition may comprise a panel of binding agents, each specific for different sequence position in a nucleic acid pharmacaphore. For example, there may be a panel of binding agents each with specificity for a different truncated version of a given pharamacaphore.

The invention further relates to binding agents immobilised on a solid surface. For example, binding agent may be adsorbed onto or covalently linked to a microtitre plate, dipstick, or other surface, such as colloidal gold particles. Thus in one aspect the invention related to an article of manufacture comprising a binding agent of the invention as an active component. Typically the article is for use in diagnostics or screening or therapy. Binding agent in or on the article may comprise a detectable label.

In one aspect the invention provides a diagnostic kit comprising a binding agent

(optionally having a detectable label), composition or article of manufacture described herein. Typically the kit is for use in detecting, or measuring the concentration of a nucleic acid pharmacaphore. A kit may additionally comprise one or more components for detecting binding agent, and/or one or more standards for calibration.

Modified and unmodified nucleic acids

A nucleic acid as referred to herein may comprises DNA and/or R A. A nucleic acid may be single stranded or double stranded. A nucleic acid may in some cases be referred to as a polynucleotide or an oligonucleotide.

A nucleic acid (modified or unmodified) may comprise any suitable length. For example, a nucleic acid may be from 6 to 100 bases, e.g. from 10 to 50 bases, 10 to 40, 10 to 30, 10 to 20 bases in length. A nucleic acid may be for example, 12, 14, 15, 16, 17, 18, 19 or 20 bases in length, or 21, 22, 23, 24, 25, bases in length.

A modified nucleic acid comprises one or more nucleic acid modifications. A

modification as referred to herein describes an alteration, typically a chemical alteration, which is present in the modified nucleic acid molecule compared to the equivalent commonly or naturally occurring nucleic acid molecule. Thus a modified nucleic acid or nucleic acid analogue typically comprises a nucleic acid modification which does not occur naturally, for example, in human or animals.

In one aspect the modified nucleic acid, or the given modification, is not detectable in naturally occurring nucleic acid in a clinical sample taken from a human or animal.

Suitable samples include those of a type which would be taken for the purposes of detecting a nucleic acid pharmacaphore, but without prior administration of the

pharmacaphore. Nucleic acid pharmacaphores often comprise modified nucleic acid. The modification may alter the biophysical properties of the nucleic acid, such as resistance to degradation or immunogenicity. A modified nucleic acid may comprise such a modification and may comprise, for example, an antisense, antigene, ribozyme, aptamer, decoy, siRNA, transition state analogue molecule or immunostimulatory sequence oligonucleotide (ISS). In one aspect, a modified nucleic acid is not an aptamer.

A modified nucleic acid generally comprises a synthetic molecule.

Modifications may be, for example, of the 3ΌΗ or 5ΌΗ group, of the backbone

(generally of the phosphate group), of the sugar component, or of the nucleotide base(s). Modifications may include addition of non-naturally occurring linker molecules and/or of interstrand or intrastrand cross links.

In one aspect, the modified nucleic acid comprises modification of one or more of the 3ΌΗ or 5ΌΗ group, the backbone (generally of the phosphate group), the sugar component, or the nucleotide base(s), and /or addition of non-naturally occurring linker molecules. Preferably the modification is of the backbone or the sugar component. It is particularly preferred that the modification is of the backbone.

In one aspect a modified backbone comprises a backbone other than a phosphodiester backbone. In one aspect a modified sugar comprises a sugar other than deoxyribose (in modified DNA) or other than ribose (modified RNA). In one aspect a modified base comprises a base other than adenine, guanine, cytosine or thymine (in modified DNA) or a base other than adenine, guanine, cytosine or uracil (in modified RNA).

Typically a modification comprises a chemical modification.

In one aspect, the modification is not of the nucleotide base, in particular is not a methylation of a base. In one aspect, a modification as referred to herein is not, and/or the modified nucleic acid does not comprise, one or more of: Z-form DNA, A- form DNA, an RNA/DNA hybrid; triple helical DNA; UV-irradiated DNA or a modification caused thereby; photooxidised DNA or a modification caused thereby; a DNA adduct formed by alkylating agent or platinum derivative; or carcinogen modified DNA or a modification caused thereby.

Backbone modifications

Backbone phosphate modifications to nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging),

phosphotriester and phosphorodithioate and may be used in any combination. Other non- phosphate linkages may also be used.

Backbone phosphate group modifications (e.g., methylphosphonate, phosphorothioate, phosphoroamidate and phosphorodithioate internucleotide linkages) can in some cases confer immunomodulatory activity on the modified nucleic acid and/or enhance their stability in vivo, making them particularly useful in therapeutic applications. A particularly useful phosphate group modification is the conversion to the phosphorothioate or phosphorodithioate forms of the oligonucleotides. In addition to their potentially immunomodulatory properties, phosphorothioates and phosphorodithioates are often more resistant to degradation in vivo than their unmodified oligonucleotide counterparts.

Synthesis of polynucleotides containing modified phosphate linkages or non-phosphate linkages is known in the art. For a review, see Matteucci (1997) "Oligonucleotide Analogs: an Overview" in Oligonucleotides as Therapeutic Agents, (DJ. Chadwick and G. Cardew, ed.) John Wiley and Sons, New York, NY. The phosphorous derivative (or modified phosphate group) which can be attached to the sugar or sugar analog moiety in the polynucleotides can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate or the like. The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per se, is also known. Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al. (1996) Nucleic Acids Res. 24:2318-2323; and Schultz et al. (1996) Nucleic Acids Res. 24:2966-2973. For example, synthesis of phosphorothioate oligonucleotides is similar to that for naturally occurring

oligonucleotides except that the oxidation step is replaced by a sulfurization step (Zon (1993) "Oligonucleoside Phosphorothioates" in Protocols for Oligonucleotides and Analogs, Synthesis and Properties (Agrawal, ed.) Humana Press, pp. 165-190). Similarly the synthesis of other phosphate analogs, such as phosphotriester (Miller et al. (1971) JACS 93:6657-6665), non- bridging phosphoramidates (Jager et al. (1988) Biochem. 27:7247-7246), N3^* to P5^* phosphoramidiates (Nelson et al. (1997) JOC 62:7278-7287) and phosphorodithioates (U.S. Patent No. 5,453,496) has also been described.

Backbone modifications are further described in Micklefield, J. 2001, Current Medicinal Chemistry 8: 1157-1179, the contents of which are hereby incorporated by reference. Backbone modifications may be those which most closely resemble native DNA:

phosphorothioate, phosphodithioate and boranophosphate.

Backbone modification may comprise replacing the phosphodiester linkage with an alternative moiety such as an anionic, neutral or cationic group. Such modification may result in increased stability to nuclease digestion. Examples of such modification include: anionic internucleoside linkage; N3' to P5' phosphoramidate modification;

boranophosphate DNA; prooligonucleotides, in which phosphodiester or phosphorothioate linkages are masked with a bioreversible protecting group resulting in neutral

phosphotriester or phosphorothioate trimester pro-drugs; neutral internucleoside linkages such as methylphosphonates; amide linked DNA; methylene(methylimino) linkages;

formacetal and thioformacetal linkages; backbones containing sulfonyl groups; and positively charged deoxyribonucleic guanidine (DNG) oligos, all as described at pages 1161 to 1171 of Mickelfield supra. A modified nucleic acid may comprise a chimeric or mixed backbone comprising one or more modifications, e.g. a combination of phosphate linkages such as a combination of phosphodiester and phosphorothioate linkages.

Some modification may comprise replacement of the whole sugar-phosphodiester backbone with an alternative moiety. Examples include morpholino oligos and peptide nucleic acids (PNA), as described at pages 1171 to 1174 of Micklefield, supra.

It is preferred that the modification for which binding agents have binding specificity is a modification of the backbone. However, it will be apparent that alternatively, or additionally, other modifications can be made. Sugar modifications

A modified nucleic acid may comprise modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, and a sugar "analog" cyclopentyl group. The sugar can be in pyranosyl or in a furanosyl form. The sugar moiety may be the furanoside of ribose, deoxyribose, arabinose or 2'-0- alkylribose, and the sugar can be attached to the respective heterocyclic bases either in [alpha] or [beta] anomeric

configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA/DNA chimeras. For example, a sugar modification may include, 2'-0-methyl-uridine and 2'-0-methyl-cytidine. Sugar modifications include 2'-0-alkyl-substituted deoxyribonucleosides and 2'-0- ethyleneglycol like ribonucleosides. The preparation of these sugars or sugar analogs and the respective "nucleosides" wherein such sugars or analogs are attached to a heterocyclic base (nucleic acid base) is known. Sugar modifications may also be made and combined with any phosphate modification.

Base modifications

The nucleic acid may comprise at least one modified base. Preferably modification is to a modified form of adenine, guanine cytosine or thymine (in modified DNA) or a modified form of adenine, guanine cytosine or uracil (modified RNA).

Examples of base modifications include, but are not limited to, addition of an electron- withdrawing moiety to C- 5 and/or C-6 of a cytosine of the nucleic acid. Preferably, the electron- withdrawing moiety is a halogen. Such modified cytosines can include, but are not limited to, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5- fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, uracil, and any other pyrimidine analog or modified pyrimidine. Other examples of base modifications include, but are not limited to, addition of an electron- withdrawing moiety to C- 5 and/or C-6 of a uracil. Preferably, the electron- withdrawing moiety is a halogen. Such modified uracils can include, but are not limited to, 5- bromouracil, 5-chlorouracil, 5- fluorouracil, and 5-iodouracil. See, for example, WO 99/62923.

Other examples of base modifications include the addition of one or more thiol groups to the base including, but not limited to, 2-amino-adenine, 6-thio-guanine, 2-thio- thymine, 4- thio-thymine, 5-propynyl-uracil, and 4-thio-uracil. Other examples of base modifications include, but are not limited to, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8- azaguanine and 5-hydroxycytosine. See, for example, Kandimalla et al. (2001) Bioorg. Med. Chem. 9:807-813. A nucleic acid may include 2'-deoxyuridine and/or 2- amino-2'- deoxyadenosine.

A large number of "synthetic" non-natural nucleosides comprising various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and the nucleic acid can include one or several heterocyclic bases other than the principal five base components of naturally- occurring nucleic acids. For example, the heterocyclic base may include uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin- 7-yl, guanin-8-yl, 4- aminopyrrolo [2.3-d] pyrimidin-5-yl, 2-amino-4-oxopyrolo [2, 3-d] pyrimidin-5-yl, 2- amino-4-oxopyrrolo [2.3-d] pyrimidin-3-yl groups, where the purines are attached to the sugar moiety of the nucleic acid via the 9-position, the pyrimidines via the 1 -position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the 1 -position.

The preparation of base-modified nucleosides, and the synthesis of modified

oligonucleotides using said base-modified nucleosides as precursors, has been described, for example, in U.S. Patents 4,910,300, 4,948,882, and 5,093,232. These base-modified nucleosides have been designed so that they can be incorporated by chemical synthesis into either terminal or internal positions of an oligonucleotide. Such base-modified nucleosides, present at either terminal or internal positions of an oligonucleotide, can serve as sites for attachment of a peptide or other antigen. Nucleosides modified in their sugar moiety have also been described (including, but not limited to, e.g., U.S. Patent Nos. 4,849,513;

5,015,733; 5,118,800; and 5,118,802) and can be used similarly. Linkers

Examples of linker molecules or non-nucleoside spacers which may be included in the modified nucleic acid include hexaethylene glycol (HEG), glycerol, triethylene glycol (TEG), propanediol and trebler.

Crosslinking

A further type of modification comprises chemical crosslinking of a nucleic acid molecule to lock the nucleic acid into either a duplex or hairpin form. Duplex (i.e., double stranded) and hairpin forms of most nucleic acids are in dynamic equilibrium, with the hairpin form generally favoured at low polynucleotide concentration and higher temperatures. Covalent interstrand or intrastrand cross-links increases duplex or hairpin stability, respectively, towards thermal-, ionic-, pH-, and concentration-induced conformational changes.

Chemical cross-links can be used to lock the polynucleotide into either the duplex or the hairpin form for physicochemical and biological characterization. Cross-linked nucleic acids that are conformationally homogeneous and are "locked" in their most active form (either duplex or hairpin form) could potentially be more active than their uncross -linked counterparts. Accordingly, some modified nucleic acids contain covalent interstrand and/or intrastrand crosslinks. A variety of ways to chemically cross-link duplex DNA are known in the art. Any cross-linking method may be used as long as the cross-linked

polynucleotide product possesses the desired activity of the nucleic acid. Synthesis

In general and as described above, modified nucleic acid can be synthesized using techniques and nucleic acid synthesis equipment which are known in the art including, but not limited to, enzymatic methods, chemical methods, and the degradation of larger oligonucleotide sequences. See, for example, Ausubel et al. (1987) and Sambrook et al. (1989). When assembled enzymatically, the individual units can be ligated, for example, with a ligase such as T4 DNA or RNA ligase. See, for example, U.S. Patent No. 5,124,246. Oligonucleotide degradation can be accomplished through the exposure of an

oligonucleotide to a nuclease, as exemplified in U.S. Patent No. 4,650,675. Modified nucleic acids in nucleic acid pharmacaphores

Nucleic acid pharmacaphores often comprise modified nucleic acid. The modification may alter the biophysical properties of the nucleic acid compared to the native DNA or RNA, For example, increased stability to nuclease enzymes, increased uptake into cells, increased affinity, kinetics and base pairing specificity upon binding to nucleic acid targets, increased immunogenicity, enhanced in vivo tissue distribution, metabolism or clearance.

Preferably a nucleic acid modification referred to herein is of a type that is usually found in nucleic acid pharmacaphores. A modified nucleic acid may comprise, for example, an antisense, antigene, ribozyme, aptamer, decoy, siRNA, transition state analogue molecule or immunostimulatory sequence oligonucleotide (ISS). In one aspect, a modified nucleic acid is not an aptamer.

Modifications of RNA molecules for use in therapeutics are discussed in Tremblay & Oldfield 2009 (Bioanalysis 1(3): 595-609) the contents of which are incorporated by reference. For example, at pages 596-598 (Fig 1A-1G) modifications include

phosphorothioate backbone modification, 2'-fluoro modification, 2'-0-methyl

modification, 2'-o-methoxy ethyl modification, a "locked nucleic acid" modification in which the ribose moiety is kept in a C3'-endo conformation, and morpholino modification, in which the ribose sugar is replaced with morpholino rings and the anionic phosphodiester linkage is replaced with non-ionic phosphorodiamidate groups.

A modified nucleic acid may comprise an antisense molecule. An antisense molecule may be for example, 10-30 bases in length, such as 15-25, or 10-20 bases in length. An antisense molecule may comprise for example, at least 12, 13, 14, 15, 16, 17 or 18 nucleotides.

A modified nucleic acid may comprise an immunostimulatory sequence oligonucleotide (ISS-ODN) as described herein. An ISS-ODN typically has an immunostimulatory effect in humans and often has a modified backbone, e.g. phosphorothioate.

An ISS may comprise a CpG (unmethlyated cytosine-phosphate-guanosine) motif. It is believed that CpG ISSs bind to Toll-like receptor 9 (TLR9) in the endosomal compartment. Thus a CpG ISS-ODN is typically a TLR9 agonist. Examples of ISSs have been described in the art. (Dorn & Kippenberger, 2008, Current Opinion in Molecular Therapeutics 10(1): 10-20; Fearon et al, 2003, Eur J Immunol 33: 2114-2122; Marshall et al, 2003, Journal of Leukocyte Biology, 73: 781-792; Marshall et al, 2003 Nucleic Acids Research 31 : 5122- 5133; Krieg A M, 2006, Nature Reviews, 5: 471-484).

A CpG ISS ODN may comprise a consensus hexamer sequence: for example, GTCGTT for use in humans; or GACGTT for mice (Krieg, 2006, supra). A CpG ISS ODN may comprise a number of repeats of the hexamer, for example, 2-10, 2-8, 2-6 or 2-4 repeats of the CpG motif. The CpG motifs are typically spaced by, for example, 6, 5, 4, 3, or 2 intervening bases, such as thymine residues. In one aspect, an ISS-ODN may have 2 to 4 repeats of the motif, spaced by 2 thymine residues. An ISS-ODN may comprise a 5 ' TpC dinucleotide and/or may comprise at least partially modified, e.g. phosphorothiate backbone (Kreig 2006, supra).

CpG-DNA ISSs have been grouped by some authors into 3 classes: CpG-A, CpG-B and CpG-C. A modified nucleic acid may comprise any of these oligonucleotides as characterised herein.

CpG-A ODNs are described as generally potent inducers of IFNa. Examples include ODN-2216 (Coley Pharmaceutical Group Inc ).

CpG-B ODNs are described as tending to stimulate B cells and promote secretion of TNFa. Examples include agatolimod sodium (PF-3512676/CpG-7909; Pfizer Inc) and CpG 1018 (Marshall et al 2003, Journal of Leukocyte Biology supra).

CpG-C ODNs are described as combining the immunological effects of the A and B classes as they are said to induce intermediate levels of IFNa, and strong activation of B cells. Examples include CpG-2395 (Dorn & Kippenberger, 2008, page 12 Table 1 supra). Further examples of CpG ODNs which are undergoing clinical development are presented in Table 2 of Dorn & Kippenberger, 2008, supra.

A first member of this class C, C274 was characterised in Marshall et al 2003, Journal of Leukocyte Biology supra. The authors identified key features of Class C ISS-ODNs as optimally including: 1 to 2 TCG trinucleotides at or close to the 5 'end of the ISS-ODN; a palindromic region of at least 10 to 12 bases, which contains at least 2 additional CG dinucleotides preferably spaced 0 to 3 bases apart. This class of ISS-ODN may comprise a phosphorothioate backbone and may have a 3 '-palindromic sequence that enables formation of duplex.

A CpG ISS ODN may comprise or be included in a chimeric immunomodulatory compound (CIC), which contains multiple heptameric ISSs connected by non-nucleoside spacers, in branched or linear configuration (Marshall et al, 2003 Nucleic Acids Research 31 : 5122-5133).

Such a CIC may, for example, contain one or more copies of the sequence 5'-TCGXCGC and/or 5'-TCGXTCG, where X is any nucleotide. Such a CIC may additionally comprise free 5' ends and/or long hydrophilic spacers, such as HEG (hexaethylene glycol). Such a CIC may, for example, be useful for inducing IFN-a (Marshall et al, 2003 Nucleic Acids Research 31 : 5122-5133)..

Alternatively a CIC may contain one or more copies of the sequence 5'-TCGTXXX and/or 5'-AACGTTC. Such a CIC may be useful for stimulating B cell activity (Marshall et al, 2003 Nucleic Acids Research 31 : 5122-5133).

In one aspect a modified nucleic acid may comprise sequence of any one or more of HEX 1 (SEQ ID NO: 69), HEX 2 (SEQ ID NO: 70), HEX 3 (SEQ ID NO: 71), HEX 4 (SEQ ID NO: 72), HEX 5 (SEQ ID NO: 73), and HEX 6 (SEQ ID NO: 74). Such a modified nucleic acid may be a CpG ISS ODN. Such a nucleic acid may comprise a modified backbone, such as a phosphorothioate backbone.

The modified nucleic acid may comprise self-complementary sequence with potential to form a stem loop structure, for example, at the 5' or 3' end of the molecule. For example, the nucleic acid may comprise the sequence of SEQ ID NO: 67 (consisting of HEX 2, 3, and 4).

A modified nucleic acid may comprise sequence of any one or more of HEX 1 (SEQ ID NO: 69), HEX 2 (SEQ ID NO: 70), HEX 3 (SEQ ID NO: 71), HEX 4 (SEQ ID NO: 72), HEX 5 (SEQ ID NO: 73), and HEX 6 (SEQ ID NO: 74), in one or more copies. The ODN may comprise hexamer containing sequence separated by non-nucleoside linkers, such as HEG, glycol or TEG, as described herein.

This is not limited to the base sequence in the context of a backbone modification listed for the given SEQ ID NO: in the Sequence Listing or in Table 1. For example, reference to the base sequence in SEQ ID NO: 67 refers to that base sequence having a modified phosphorothioate backbone as listed for SEQ ID NO: 67, and also to that base sequence in other contexts, e.g. having a backbone modified in another way.

In one example, the modified nucleic acid comprises HEX 3.

A modified nucleic acid such as a CpG ISS ODN may comprise or consist of, for example, 6-30, nucleotides in length, such as 10-30, 12-30, 15-27, 17-27, 18-25, nucleotide in length, optionally with non-nucleoside linkers.

In one aspect, a modified nucleic acid comprises at least 40% GC nucleotides.

In one aspect a modified nucleic acid, such as a CpG ISS-ODN, comprises a modified backbone, such as a phosphorothioate backbone.

Non-CpG ISSs are also known. A family of motifs that convey immune stimulation in these ODNs has been identified and consist of a family of motifs of 6 nucleotides length with the sequence 5'-G3xG₂-3' (where x is any base). Non-CpG ODNs which exert immunosuppressive effects have also been identified, including those comprising the hexanucleotide repeats with the TTAGGG consensus sequence. (Dorn & Kippenberger, 2008, supra)

In one aspect the modified nucleic acid referred to herein is not bound to a protein or in a nucleic acid-protein complex.

Any one or more properties of the modified nucleic acid described herein may apply in any combination thereof. Unmodified nucleic acid

Un-modified or non-modified nucleic acid as referred to herein lacks one or all of the modification(s) present in the modified nucleic acid. The nucleic acid may comprise unmodified backbone, base and/or sugar components.

Preferably the unmodified nucleic acid lacks all modification present in the modified nucleic acid. Preferably the un-modified nucleic acid comprises naturally occurring nucleic acid. This does not necessarily imply that the base sequence is naturally occurring, but that the nucleic acid structure is naturally occurring. For example, the backbone, sugar and bases are naturally occurring.

In one aspect an unmodified nucleic acid comprises a phosphodiester backbone. In one aspect unmodified nucleic acid comprises deoxyribose sugar (in DNA) or ribose sugar (in R A). In one aspect unmodified nucleic acid comprises the bases adenine, guanine, cytosine and thymine (in DNA) or the bases adenine, guanine, cytosine or uracil (in RNA). Preferably the unmodified nucleic acid comprises all three stated preferences.

Nucleic acids occurring naturally may include those in clinical samples taken from humans or animals, for example, samples of a type that would be taken in order to assay for the presence of a pharmacaphore, but without prior administration of the

pharmacaphore to the subject. Such samples are described herein.

Unmodified nucleic acid may have the same base sequence as the modified nucleic acid, and may have any of the base sequences defined herein for the modified nucleic acid, but without the modification. In the identification and screening methods herein, non- modified nucleic acid preferably comprises the same base sequence as the modified nucleic acid antigen.

Applications

Binding agents of the present invention can be used to detect and target nucleic acid pharamacaphores in diagnostics and therapy. Nucleic acid pharmacaphores are nucleic acids having biological activity and useful as drugs. Examples of such therapeutic agents include for example, antisense molecules, antigene molecules, ribozymes, aptamers, decoys, siRNA molecules, transition state analogue molecule or immunostimulatory sequences. As already noted these often include modified nucleic acids.

Oligonucleotide therapy such as these requires a means of accurately detecting, tracking, quantifying and neutralising pharmacaphore, be that in preclinical discovery and research, or in clinical trials, or in clinical use. For example, it may be necessary to study biophysical properties of the pharmacaphore of its formulations in vitro, or to monitor localisation in cell culture. It may be necessary to monitor localisation, metabolism and toxicity in animal models, or in clinical trial patients. It may be necessary to monitor pharmacaphore levels in samples taken from patients, or tissue localisation in patients, or indeed to target administered pharmacaphore.

The present binding agents can be used in methods for determining the presence and/or amount of a nucleic acid biopharmaceutical.

Since the binding agents are able to discriminate between modified nucleic acids (nucleic acid analogues) and naturally occurring nucleic acids the binding agents will not bind to the naturally occurring background nucleic acids, avoiding interference in detection methods by background naturally circulating DNA or RNA.

Binding agents can be prepared which bind to a modified nucleic acid pharmacaphore with sufficient sensitivity to be used in a clinical assay. For example, a binding agent for use in a clinical assay for humans preferably displays a binding sensitivity of 1 to ΙΟΟρΜ, for example, at least 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 2, or 1.5pM, or any combination thereof, such as 1 to 50pM or 5-70pM, as described herein. A binding agent for use in an assay in preclinical animal studies to establish the toxicity of the pharmacophore (tox species) preferably displays a binding sensitivity of 0.1 to InM, for example, at least 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2nM, or any combination thereof, such as 0.1 to 0.5nM. Any suitable binding assay may be used to determine sensitivity. Furthermore binding agents can be identified which specifically recognise particular forms of a pharmacaphore which may not be detected using conventional hybridisation methods. For example, degraded or truncated forms can be specifically detected, making it possible to discriminate between a biopharmaceutical and its metabolites, and so to monitor breakdown of the biopharmaceutical. Similarly nucleic acids comprising short runs of sequence separated by organic linkers, or nucleic acids comprising secondary structure, can be detected using the binding agents herein.

Accordingly in one aspect the invention provides the use of a binding agent of the invention in a method for detecting, tracking, quantifying and/or targeting one or more nucleic acids biopharmaceuticals in vitro or in vivo. The invention also provides a binding agent as described herein, or a composition, e.g. a pharmaceutical composition, comprising such a binding agent, for use in medicine. Such use may be in a method of treatment of the human or animal body by surgery, therapy or in a diagnostic method practised on the human or animal body.

In the uses described herein, it is understood that the pharmacaphore or biopharmaceutical comprises modified nucleic acid and that the binding agent is able to specifically bind to the modified nucleic acid as described herein.

Use of binding agents to detect or quantify modified nucleic acids in samples

Binding agent may be used in an assay method to determine the presence and/or concentration of nucleic acid analogue. Typically such an assay is carried out in vitro, or ex vivo on a suitable sample.

A sample may be, for example, a biological sample taken from a human or animal subject. Alternatively a sample may comprise a laboratory test sample produced in vitro, for example comprising a test formulation of a modified nucleic acid pharmacaphore, or a cell culture comprising the pharmacaphore. A sample may be used for example, in an assay to test release of a modified nucleic acid from a delivery vehicle, by comparing results for formulated and unformulated samples. A biological sample may comprise any bodily sample in which the nucleic acid pharmacaphore may be found, for example a plasma, urine, bile, faeces, solid tissue (typically kidney, liver, brain or spleen), skin or vitreous humor sample

For a blood sample, measurement may be made in whole blood. However, the blood may be further processed before an assay is performed. For instance, an anticoagulant, such as heparin, citrate, EDTA, and others may be added. Alternatively, the blood sample may be centrifuged or filtered to prepare a plasma or serum fraction for further analysis.

Generally the method of bioanalysis will take into account the delivery vehicle and formulation type of the biopharmaceutical. For example, use of mild non-ionic detergent may be needed to release nucleic acid from lipids or polymers.

For a tissue sample, an extraction step may be needed to provide a nucleic acid sample for testing.

An assay generally comprises contacting the sample with binding agent and determining (the amount of) modified nucleic acid-bound binding agent.

Any suitable assay format may be used. For example, immunoassay techniques are known in the art and described for example in Self CH and Cook DB, 1996, Current Opinion in Biotechnology 7: 60-65, the contents of which are incorporated herein by reference.

In one aspect, a competitive assay is employed. Typically this comprises use of a competitor nucleic acid, which competes with the target pharmacaphore for binding to the binding agent. The greater the concentration of target in a sample, the less competitor bound to the binding agent. Generally the competitor comprises a detectable label to allow detection and measurement of bound competitor. Binding agent may also bear a label, for example, such that a signal is produced only when competitor is bound to binding agent. Binding agent may be immobilised as described herein.

In another aspect, a non-competitive assay is employed. Typically, a test sample is contacted with binding agent and complexes of bound modified nucleic acid-binding agent are detected and/or quantified. Examples of such an assay include a sandwich assay, an anti-immune complex assay and an idiometric assay.

A sandwich assay comprises use of two different binding agents recognising different epitopes in the target pharmacaphore. One binding agent acts as a capture molecule, and is typically immobilised on a solid support. The other binding agent acts as a detector molecule, and is typically labelled with a detectable label. In one example, both capture and detector binding agents comprise labels which interact to produce a signal only when brought into proximity by binding of the detector binding agent to the already bound target nucleic acid.

An anti-immune complex assay (Self & Cook supra) comprises use of a first binding agent specific for the target pharmacaphore, and a binding agent specific for the complex of "pharmacaphore + first binding agent". The first binding agent can be used as a capture molecule, and may be immobilised. The second binding agent can be used as a detector molecule, and is typically labelled with a detectable label. In one example, both capture and detector binding agents comprise labels which interact to produce a signal only when brought into proximity by binding of the detector binding agent to the complex.

In an idiometric assay (Self & Cook supra) a first binding agent is used to capture modified nucleic acid antigen. Antigen-bound capture binding agent is then detected by addition of a reagent that binds to non-bound sites (e.g. an analogue of the target nucleic acid) and prevents binding of a second binding agent. An immunoassay may be used. Examples of immunoassays include immunofluorescence techniques known to the skilled technician, immunohistochemistry, ELISA, radioimmunoassay analyses. Labels include reporter enzymes such as alkaline phosphatase, horse radish peroxidise (HRP) and colorimetric or fluorometric substrates, as well as electrochemical detection methods (Self & Cook supra).

An assay may use a binding agent which comprises a bispecific antibody.

The assays may need one or more "reference samples", such as a negative and/or positive control, or one or more standard samples to calibrate the assay. Examples of negative controls include a corresponding sample taken from an animal not exposed to the pharmacaphore (optionally also suffering from the condition being treated); a corresponding sample taken from test patient before the pharmacaphore was administered; an unrelated sample known not to contain the pharmacaphore, e.g. PBS or water. A positive control typically comprises a preparation of the pharmacaphore.

Sensitivity of an assay may be improved by optimising parameters such as the

concentration of binding agent used. For example a calibration may be carried out using a series of quality control (QC) samples. Preferably the calibration standard curve ranges between a sample representing the lower limits of quantification (LLOQ) and a sample representing the upper limits of quantification (ULOQ), with at least 3 QC samples in the intervening range (QC1, QC2, QC3). The QC samples are generally prepared in the same biological matrix as the anticipated study samples. Preferably samples are assayed in replicates of at least 3.

An assay may be calibrated using a dilution series of pharmacaphore, preferably in the same biological matrix as the anticipated study samples, and a dilution series of binding agent. This enables selection of an optimal concentration of binding agent for use in the assay. For example, a chessboard titration such as that described herein may be employed.

The signal obtained in the test assay can be compared with the signals in the calibration assays (or standard curve) to determine the concentration of pharmacaphore in the test sample.

Typically a test assay is repeated at least 2, 3, or 4 times, on repeat samples. Assay results may then be averaged.

An assay may be homogeneous or heterogenerous. A heterogeneous assay typically comprises a step of phase separation of bound and unbound analyte before signal detection, and may comprise a washing step. An assay may be designed to be useful at "point of need", for example utilising a dipstick or detector strip.

Binding agents as standards

It has been shown that complexes of oligonucleotide and DNA binding proteins sometimes used in therapy can be immunogenic (Ramussen et ah, 1996 ibid; Cerutti et al., 2008, ibid). Binding agents may also find use as anti-DNA antibody standards for use in calibrating immunogenicity assays carried out to measure patient's immune response to administered pharmacaphores.

Use of binding agents to track or localise modified nucleic acids

Binding agent may alternatively be used as a marker to track movement and/or localisation of a pharmacaphore, typically in a cell or tissue culture, or in vivo, for example to track tissue localisation of a pharmacaphore in a human or animal subject.

Thus in one aspect the invention provides a method for detecting a pharmacaphore in a human or animal subject to whom has been administered a pharmacaphore and detectably labelled binding agent according to the invention, the method comprising detecting the position of the binding agent in the patient body by means of the detectable label.

The invention also provides a binding agent of the invention, or a pharmaceutical composition comprising a binding agent of the invention, for use in a method of detecting a pharmacaphore in a human or animal subject.

The invention additionally provides use of a binding agent of the invention for the preparation of a medicament for detecting a pharmacaphore in a human or animal subject.

Generally detecting may involve tracking and/or locating the pharmacaphore.

Typically the binding agent comprises a detectable label suitable for administration to a human or animal. Suitable labels are known in the art, e.g. radioisotopes. Preferably the detection means in the method is non-invasive, e.g. a scanning procedure. Examples are known in the art, e.g. whole body radiography or nuclear imaging emission tomography (Tremblay & Oldfield 2009, Bioanalysis 1(3): 595-609). In one aspect the method includes administering the binding agent to the patient. Typically this is done by means of a suitable pharmaceutical composition as described herein.

Binding agents of the invention may also be used to monitor pharmacaphore behaviour and properties in vitro, for example, in the discovery and development phase of nucleotide pharmacaphore research. Thus in one aspect the invention provides use of the present binding agent in a method for analysing a pharmacaphore in vitro, for example, in tracking localisation of the pharmacaphore in cell culture, or monitoring release of the

pharmacaphore from a particular delivery vehicle or formulation.

Use of binding agents in therapy

Binding agents described herein may also find use in therapy.

Thus in one aspect the invention provide a binding agent of the invention, or a

pharmaceutical composition comprising a binding agent of the invention, for use in a method of oligonucleotide or pharmacaphore therapy in a human or animal subject.

The invention further provides use of a binding agent of the invention for the preparation of a medicament for use in oligonucleotide or pharmacaphore therapy in a human or animal subject.

Generally the binding agents are used to selectively target the pharmacaphore. The agents may be used, for example, as neutralising agents to rectify overdosing in oligonucleotide therapy. This may be needed for example, to limit immunogenicity issues associated with the therapy. Agents may also be used to target other functions or molecules to the pharmacaphore. In such cases, binding agents may comprise an additional function in addition to antigen binding.

Subjects, compositions and administration

A human or animal subject referred to herein is generally one to whom a nucleic acid pharmacaphore has been administered. Examples include: a test animal used as an animal model during testing of a pharmacaphore, e.g. in toxicity studies; a participant in a clinical trial; a human patient or animal undergoing clinical treatment.

Compositions for use in the methods may comprise more than one type of binding agent as described herein. Thus a composition may include multiple binding agents having different binding specificities, allowing the detection, monitoring and/or targeting of a pharmacaphore in multiple ways, or of more than one pharmacaphore, or of different forms of a pharmacaphore. Typically in such a composition the binding agents are labelled so as to be detectably distinct.

For administration to a human or animal subject, binding agent is typically formulated in a pharmaceutical composition. Typically such a composition includes one or more binding agent(s) of the invention as active ingredient(s),and a pharmaceutically acceptable excipient carrier or diluent.

The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carriers useful in the methods disclosed herein are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co, Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the agents herein disclosed.

Such formulations may further routinely contain pharmaceutically acceptable

concentrations of salt, buffering agents, preservatives, antioxidants, compatible carriers, and optionally other therapeutic agents.

A pharmaceutical composition may have a number of different forms depending on, for example, how the composition is to be administered.

Any suitable administration route and/or delivery means may be used to deliver binding agent or a binding agent composition to a subject. In one example, the agents may be formulated for parenteral administration. Parenteral preparations can be administered by one or more routes, such as intravenous, subcutaneous, intradermal and infusion; a particular example is intravenous. A formulation disclosed herein may be administered using a syringe, injector, plunger for solid formulations, pump, or any other device recognized in the art for parenteral administration. Actual dosage levels of binding agent in pharmaceutical compositions may be varied so as to obtain an amount of agent(s) that is effective to achieve the desired therapeutic response or detection level, for a particular subject, composition, and mode of administration (referred to herein as a "therapeutically or diagnostically effective amount").

The selected dosage level may, for example, depend upon the binding sensitivity of the agent, the dose of pharmacaphore administered to the subject and the condition and prior medical history of the subject being treated. However, it is within the skill of the art to start doses of the agent at levels lower than required for to achieve the desired effect and to gradually increase the dosage until the desired effect is achieved.

Typically binding agents which are intended for administration to a human comprise humanised or fully human molecules. This is to help to avoid issues with immunogenicity in the body. Thus, for example, binding agents comprising antibodies or fragments thereof which are intended for administration to a human generally comprise humanised or fully human antibodies. Similar considerations apply to use in other species. Thus, for example,, where a binding agent comprises an antibody or fragment thereof, the

framework is preferably derived from the species in which the binding agent is to be used. Thus, if a constant region is present, it is preferably substantially identical to a constant region in that species, for example, 85-90% or at least 95% identical.

Sequence variants

A variant amino acid or nucleic acid sequence referred to herein generally has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% or more identity with the reference sequence. Variants also include insertions, deletions, and substitutions, either conservative or non-conservative. Preferably, a variant CDR sequence has alteration of no more than 10, 8, 6, 5, 4, 3, 2 or 1 amino acid residues compared to the reference sequence.

Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. In terms of amino acids, small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore by "conservative

substitutions" is intended to include combinations such as Gly, Ala; Val, He, Leu; Asp, GIu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.

A functional variant is one in which the changes made with respect to the reference sequence do not substantially alter protein activity, in particular ability to bind antigen, and binding specificity.

Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences may be performed as follows.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%>, even more preferably at least 60%>, and even more preferably at least 70%>, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o, 99%), or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In one embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

Examples

The subject of this study, is an oligonucleotide Toll-like receptor 9 (TLR9) agonist (Hyashi 2006 ibid). TLR9 is expressed in plasmacytoid dendritic cells and B cells (Marshall et al., J. Leukocyte Biol, 73:781-792, 2003). Oligonucleotides containing unmethylated CpG motifs mimic non-mammalian DNA-binding to TLR9, inducing proinflammatory cytokines and leading to a T helper type 1 (Thl) immune response (Kreig Nature Rev. Drug Disc. 5:471-484, 2006; Dorn et al, Curr. Opin. Mol. Ther., 10: 10-20, 2008). C- 274PS is an example of such an oligonucleotide (Fearon et al., 2003, ibid). It is a 22mer fully modified phosphorothioate oligonucleotide with a potential for duplex formation between 12 internal bases.

The experiments below describe isolation and characterisation of a panel of binding agents that bind to C274PS oligonucleotide having a phosphorothioate backbone (SEQ ID NO: 66) . Phage and phagemid display libraries comprising scFvs and Fabs fragments were panned, using as a modified nucleic acid a C274 olionucleotide having a phoshorothioate backbone (C274PS). Subtractive panning was carried out using a non-modified C274 olionucleotide (C274PO). Binding agents selected in library panning were subjected to primary and secondary screening, and selected clones checked for uniqueness.

Periplasmic preparations of each selected binding agent were then subjected to a competition assay to determine more precisely the binding specificity of the binding agents. One of the binding agents showed binding specificity dependent only on the phosphorothioate backbone modification. Others showed binding specificity dependent additionally on base sequence and/or stem loop structure within the C274 oligonucleotide. A panel of binding agents with different binding specificities was selected, and the corresponding scFv or Fab fragments reformatted to produce whole antibodies. These whole antibody binding agents were subjected to titration assay, and competition assay to determine potency and IC50 value for binding to the C274PS oligonucleotide.

The experiments are provided to illustrate the invention.

Abbreviations

2TY- AG 2x TY broth supplemented with Ampicillin and Glucose

3'bC274 C-274 biotinylated at the 3 'end

5'bC274 C-274 biotinylated at the 5 'end

PS Phosphorothioate

PO Phosphodiester

TEA Triethylamine

TH1 T-helper 1

TLR9 Toll-Like Receptor 9 Materials and Methods Oligonucleotides

All oligonucleotides used in this study and listed in Table 1 (Figure 4) were purchased from Eurogentec. On receipt all were suspended at lmg/ml in water, dispensed in aliquots and stored at -80°C.

Isolation of binding agents by library panning

Single chain fragment variable (scFv) of Fab moieties that bind to C274PS were identified following two or three rounds of selection using three phagemid libraries from the

Medimmune Cambridge (formerly Cambridge Antibody Technology): the Bone Marrow Vaughan (BMV) library (Vaughan et al. Nature Biotechnol. 14:309-314, 1996); the combined spleen (CS) library (Lloyd et al, Protein Eng. Des. Sel. 22: 159-168, 2009); the DP47 library (Groves et al, J. Immunol. Meth. 313, 129-139, 2006) and the Fab-phage library, pCES-1 from Dyax Corp (de Haard et al, J. Biol. Chem., 274: 18218-3, 1999). All libraries are phagemid-based and require rescue with helper phage M13K07 for the production of scFv (or Fab) bearing phage.

For each round of phage-based selection, lOOul of streptavidin coated magnetic beads (Dynabeads Streptavidin M-280, Invitrogen) were charged with lug of either 5'bC274PS or 3'bC274PS. After washing the charged beads were blocked by the addition of 3% Marvel in PBS, for 1 hour. Each aliquot of phage library was blocked with 4% Marvel in PBS for 1 hour. The charged beads were washed and re-suspended in an aliquot of blocked library and then mixed for one hour before washing twice with 3% Marvel in PBS, ten times in PBS-Tween (0.1%) and twice in PBS. Phage antibodies were eluted from the beads with 500ul lOOmM TEA for 10 minutes before neutralising the supernate by the addition of 250ul 1M Tris-HCl, pH 7.5. The eluted phage-antibodies were absorbed with streptavidin magnetic beads charged with 5'bC274PO in order to remove phage which would bind to natural DNA. The resulting absorbed phage were used to infect log phase E.coli TGI for amplification and preparation of fresh phagemids (de Haard et al., 1999 ibid). After two or three rounds of selection the absorbed phage preparation were used to infect log phase E.coli HB2151. Infected cells were diluted and a range of concentrations were spread onto 2TY- AG agar and grown overnight at 30°C. Individual colonies were picked into lOOul of 2TY-AG medium per well in 96-well microtitre plates and grown at 37°C until A600nm = 0.8 before inducing expression of ScFv or Fab into the periplasm by the addition of IPTG to ImM. Plates were incubated at 30°C and 250rpm overnight before centrifuging and removing the supernates. The contents of the bacterial periplasm

(peripreps) were released by osmotic shock. Briefly, the pellets were re-suspended in 30ul per well of ice cold TES (Tris-EDTA Sucrose pH8.0) before shaking for 10 minutes, followed by the addition of 70ul of ice cold lOmM MgSC^ to each well. The plates were shaken for a further 10 minutes before centifugation and harvesting the supernates containing the selected binding agents (scFvs or Fabs.)

Primary screening of selected binding agents

Immunoassay plates (Nunc, Maxi-sorb) were coated with lOOul per well of biotinylated BSA at 2ug/ml in 50mM carbonate buffer, pH8.5 for 2 hours at 37°C. After washing three times with PBS-Tween (0.1%), lOOul of streptavidin at lOug/ml in PBS was added to each well for 1 hour at room temperature. The plates were washed and biotinylated C274PS antigen was added at 0.5ug/ml in PBS for 30 minutes before blocking by the addition of 200ml per well of 2% Marvel in PBS.

Coated, blocked plates were washed three times with PBS-Tween (0.1%>).and peripreps (prepared as described above), diluted in PBS with 2% Marvel, were applied to the assay plates. After an hour at room temperature, plates were washed three times with PBS- Tween (0.1%) and a 1 : 10000 dilution of rabbit anti-cmyc HRP conjugate (AM 9312 , Abeam) secondary antibody was added to detect bound agent (scFv or Fab). Plates were incubated for a further hour at room temperature and washed three times with PBS-Tween (O. P/o) prior to developing by the addition of TMB substrate (Sigma). Absorbances were read at 370nm on a Molecular Devices VERSAmax plate reader. Secondary Screening of selected agents

A pair of immunoassay plates (Nunc, Maxi-sorb) were coated with 5 'bC274PS as described above. To the first plate 50ul per well of C274PS (not biotinylated) at lOug/ml in PBS was added whereas the second plate contained 50ul per well PBS alone. Peripreps from colonies which were positive in primary screening were diluted 1 in 10 in PBS and 50μ1 was added to each of the two plates before incubating at room temperature for 1 hour. Plates were washed and secondary antibody was added before processing as described above.

Clone Check

All isolates selected from the primary and secondary screens were plated for single colonies on 2TY-AG agar before incubating overnight at 32°C. Eight colonies were picked from each isolate into lOOul of 2TY-AG medium per well in 96-well microtitre plates and grown overnight at 30°C. The content of each well was amplified by PCR using primers either side of the antibody fragment gene(s) (de Haard et al., 1999 ibid), before digesting with BstNI. The products were separated on 3% agarose before assessing the resulting fingerprints for both the clonality and uniqueness of each isolate.

Determination of binding specificity of binding agents by competition assay

Immunoassay plates (Nunc, Maxi-sorb) were coated with 5'bC274PS at 12.5ng per well as described above. Periplasmic preparations for each clone were titrated and a dilution which gave approximately 80% of the maximum signal was selected. A range of oligonucleotides at 125ng per well in 50ul were added to new 5'bC274PS coated plates. The selected dilution of each periprep was then added and the plates incubated at room temperature for one hour. Plates were washed and secondary antibody was added before processing as described above.

Reformatting of svFv or Fab fragments as whole antibodies

Selected antibody fragments were reformatted into whole chimeric antibodies. DNA was prepared for each of the selected clones (Qiagen miniprep kit) before sequencing both strands of the variable heavy and variable light regions. The variable heavy chain region was cloned into a mammalian expression vector containing the rabbit constant heavy region to produce a single polypeptide chain containing the variable region derived from the selected antibody fragment coupled to the constant heavy chain region of the rabbit. The variable light chain region was cloned into a mammalian expression vector containing the human light chain constant region, either Kappa or Lambda (In-Fusion™ Advantage PCR Cloning Kit, Clontech). The two vectors were co-transfected into HEK EBNA cells using PEI (Polysciences), after 7 days supernates were harvested and the antibodies purified by protein A affinity chromatography.

Competition assays using the reformatted whole antibody binding agents

For each selected reformatted antibody binding agent, a chessboard titration was performed in which lOOul of dilutions of the antibody (400, 200, 100, Ong/ml) were used to coat the rows of a protein G coated plate (Pierce). After washing with PBS-Tween (0.1%), dilutions of 5'bC274PS oligonucleotide (12, 6, 3, 1.5, 0.75, Ong/ml) were added to the columns of the plates. After incubating for 1 hour the plates were washed with PBS- Tween (0.1%) and lOOul of 1 :2000 Streptavidin-HRP (Calbiochem) was added to each well before incubating for a further hour. Plates were washed three times with PBS-Tween (O. P/o) prior to developing by the addition of TMB substrate.

From the assay described above the lowest concentrations of binding agent and 5'bC274PS producing a signal of A370nm =1, were selected. Binding agent antibodies, at the chosen dilution (lOOul), were added in duplicate to the rows of a protein G coated plate. After blocking by the addition of 200ul 2% Marvel in PBS and washing three times with PBS- Tween (0.1%o), 50ul of C274PS dilutions were added to the columns of the plate. Plates were incubated for 1 hour before the addition of 50ul of 5'b C274PS to each well at the dilution determined in the chessboard assay. After incubating for 1 hour the plates were washed three times with PBS-Tween (0.1%) and lOOul of 1 :2000 Streptavidin-HRP added to each well before incubating for a further hour. Plates were washed three times with PBS-Tween (0.1 %>) prior to developing by the addition of TMB substrate. Results

1. Selection and screening of antibody fragment phage display libraries

Each of three scFv-phage libraries (BMV, CS, DP47) and one Fab-phage library (PCESl) were panned against modified nucleic acid (both 5'bC274PS, and 3'bC274PS DNA sequences). For both of these antigens the outputs from each round of panning were absorbed with the equivalent nonmodified nucleic acid (5 ' biotinylated phosphodiester sequence) in an attempt to isolate antibody fragments specific for the pharmacophore. A total of 8, 96-well micro-titre plates were prepared (corresponding to single colonies picked into one plate per library per antigen).

In a primary screen, each plate was screened by ELISA in order to define which of the binding agent clones bound to 5'bC274PS, 3'bC274PS and a control plate with no oligonucleotide present. Setting a cut-off of twice the signal obtained on the control plate, those clones giving a positive signal for each combination of library and modified C274 antigen were selected (Table 2 ; Figure 5). All panning combinations produced isolates that bound to both 5'bC274PS and 3'bC274PS. A total of 252 positive clones were isolated. All positive clones were transferred to new plates before conducting secondary screening.

2. Secondary screening of antigen positive clones

When using labelled molecules (biotinylated in this case) for panning, it is entirely possible to isolate binding agents that bind the labelled version but not the unlabelled version of the antigen. Because recognition of the unlabelled antigen was required, a competition assay was performed as the secondary screen. The ability of unlabelled C274PS to block the binding of all antibody fragments to either 5'bC274PS, or 3'bC274PS was tested by pre- incubating diluted peripreps from all positive clones with either lOug/ml C274PS or PBS before transferring to plates coated with 5'bC274PS. Thirty one clones showing greater than 40% inhibition of binding to 5'bC274PS in the presence of C274PS (Table 3; Figure 6) were selected for further study. It is worth noting that if a particular periprep contained excessive amounts of antibody fragment, the assay format would not reveal desirable antibody fragments. However, in only one instance did the dilution of supernate used produce a signal (A370nm) greater than 1.5 in the absence of C274PS (with a signal of 0.25 in the presence of C274PS), thus it is unlikely that any useful binding agents were missed.

3. Further determination of binding agent specificity

Having identified clones capable of binding to unlabelled C274PS pharmacophore in solution, and verified that they were both unique and clonal (data not shown), an attempt was made to determine their epitope specificity. To achieve this a more refined competition assay was undertaken, using the range of oligonucleotides shown in Table 1 (Figure 4).

Initially, the lowest quantity of 5'bC274PS that gave a strong signal in ELISA, was determined; this was found to be 0.125ug/ml or 12.5ng/well. Periplasmic preparations of each binding agent clone were then titrated, from which a dilution that gave approximately 80% of the maximum signal was subsequently selected. Pre-incubation of this dilution with 125ng of competitive oligonucleotide would produce a 90% reduction in signal if competition was complete. The range of phosphorothioate oligonucleotides chosen to test comprised C274PS (SEQ ID NO: 66), the double-stranded core of C274 (C274 Core - SEQ ID NO: 67), a double stranded oligonucleotide of the same length but with a different sequence (Irrelevant Core - SEQ ID NO:68) and six overlapping single stranded hexamers which cover the sequence of C274 (Table 1 - SEQ ID NOs 69-74). The results of these competition assays are presented in Table 4 (Figure 7). All are expressed as percentage inhibition compared with the signal produced when PBS alone replaced pre-incubation with a particular oligonucleotide. Figure 1 shows two examples, the first of which, clone 3749.03, shows almost complete inhibition by all phosphorothioate oligonucleotides tested indicating specificity for the phosphorothioate backbone, whereas, the second, clone 3749.22 was predominantly inhibited by C274PS and C274 Core indicating an additional double stranded sequence specificity. From the results, those clones with similar specificities were grouped and a diverse panel of seven selected for further study . 4. Competition assays using selected binding agents

The purpose of this study was to evaluate the potential of binding agents to form the basis of an assay to determine the concentration of novel oligonucleotide pharmacophores in pre-clinical and clinical samples. To this end, selected antibody fragments were reformatted and expressed as whole chimeric antibodies and used to coat a protein G plate.

The first step was to determine suitable concentrations of both anti-C274 antibody binding agent and biotinylated antigen (5'bC274PS) to use in such an assay. As the proportion of potential secondary structures adopted by C274PS is uncertain, the assay was performed at three different temperatures, reasoning that the proportion of single stranded species would increase with temperature: 4°C; room temperature (~ 22°C); and 37°C. It is clear that in some cases changing the temperature at which the assay was performed made negligible or no difference (Figure 2). For example, the titration for 3742.30 did not significantly vary across the temperature range tested, but 3745.58 produced a greater signal the lower the temperature at which the assay was performed and 3739.93 was best performed at room temperature or 4°C. The results from these assays allowed us to determine the minimal concentrations of each binding agent and labelled antigen which produced a signal of A370nm =1, at room temperature. All of the binding agents tested required 30ng/well or less of binding agent, and 0.3ng/well or less of 5'bC274PS (Table 5; Figure 8). Clone 3749.93 seemed particularly potent requiring only 7.5ng./well binding agent and 0.0375ng of 5'bC274PS to produce a suitable signal.

Finally, the potential of each of the selected binding agent to form the basis of an assay to determine the concentration of novel oligonucleotide pharmacophores in pre-clinical and clinical samples was evaluated. Each competition assay was carried out at room temperature and the results are presented in Table 6 (Figure 9). In these assays the signal obtained when the binding agent was pre-incubated with lug/ml C274PS is taken as 100% inhibition, and when pre-incubated with PBS alone is taken as no inhibition. In Figure 3 the IC50 for 3749.93 can be estimated atl30pg/ml whereas both the other antibodies presented (3749.03 and 3749.22) showed lower sensitivity with estimated IC50 of

270pg/ml demonstrating that in these assay conditions sensitivities of sub nanogram are possible. Discussion

These experiments describe isolation of a panel of binding agents, based on scFvs and Fabs that bound to C274PS. Furthermore, several of the clones isolated were demonstrated to have different specificities for various sequences based upon C274. A subset of selected Fabs and scFvs were reformatted into whole chimeric antibody binding agents, and after determining useful concentrations of both binding agent and labelled nucleic acid antigen, their suitability as detection reagents to measure free modified C274PS in a competition assay was assessed. In the experiments described here, one of the selected binding agents (3749.93) was used to produce an assay capable of measuring C274PS in the 15-300pg/ml range (2 -40pM). This value compares with a limit of detection of 900pM reported for a traditional hybridisation assay for a 15mer phosphodiester oligonucleotide. (DeVerre et αί, Nucleic Acid Res. 25:3584-3589, 1997). It is likely that the sensitivity achievable in more complex matrices such as human plasma would be lower, although modification of various parameters of this assay, such as increasing sample size (whilst reducing volume of labelled target), changing incubation times or amplifying the signal, may improve the useful detection range further.

One of the problems facing the conventional hybridisation assay is the presence of varying amounts of genomic DNA in patients' plasma (van der Vaart. 2007 ibid). This potential limitation would also need to be considered when using antibodies that bind to particular nucleotide base sequences. However, the isolation of binding agents that are specific for the unnatural backbone frequently used in polynucleotide pharmacophores would avoid this particular problem. Indeed, to this end, all the binding agents we selected were chosen because they failed to bind to the C274 sequence when it was synthesised in a

phosphodiester backbone. Although this study was limited to a phosphothiorate modified backbone, there is no reason why this could not be extended to other modifications such as phosphoroamidate or boranophosphate (Micklefield 2001 ibid). Furthermore,

oligonucleotide pharamacaphores have been described which have short runs of nucleotides (5-7 bases) joined by a variety of organic linkers (Marshall et ah, Nucleic Acid Res. 31 :5122-5133, 2003). The inadequate opportunity for hybridisation offered by such moieties limits the utility of classical hybridisation assay and provides an opportunity to develop assays using the present binding agent approach. Determining the precise binding specificity of each of the binding agents isolated in this study was not straight-forward. C274, the pharmacophore studied here, has the potential to adopt different secondary structures such as single-stranded, double-stranded or hairpin. Furthermore, the proportion of each structure that is present during the assay could depend on many factors including temperature and matrix components. Concerns about the precise structure of the pharmacophore studied here led to the carrying out of chessboard titrations at a range of temperatures. The rationale being that at 4°C the species would essentially be double stranded with both 3' and 5' single stranded overhangs, whereas at 37°C the species would be predominantly single-stranded. In many cases, room temperature would be the most convenient condition to perform an assay. However, at this temperature it is possible that a sample of C274 would comprise a mixture of species, and thus an assay based on a particular binding agent may sub-optimal. Some of the panel of binding agents identified herein performed equally well at all three temperatures, indicating that the epitope they recognised was present in both double-stranded and single-stranded forms of the pharmacophore. Other binding agents performed poorly at the higher- temperature indicating the epitope they recognised may be double-stranded. The set of binding agent isolations described herein were all performed at room temperature. If the temperature of isolation was changed it is likely that a different range of binding agents would be isolated. For instance if the phage libraries were panned at 37°C the binding agents isolated may be expected to predominantly recognise single-stranded structures.

It has been demonstrated that the binding agents isolated in this study can be used in competition assay format. For larger pharmacophores it may be possible and desirable to isolate pairs of binding agents capable of binding the pharmacophore simultaneously but at different sites, allowing the development of a sandwich assay. In addition, it may be feasible to isolate binding agents that are able to bind to an binding agent-pharmacaphore complex but are unable to bind to either individually (Self et al., Curr. Opin. Biotech. 7:60-65, 1996). Such an approach would lead to the development of more sensitive and robust assays.

Claims

1. A method for identifying a binding agent having binding specificity for a nucleic acid modification, the method comprising:

2. A method according to claim 1 wherein the nucleic acid modification is of nucleic acid backbone, such as a phosphorothioate backbone.

3. A method according to claim 1 or 2 wherein each binding agent in the library comprises an antibody or a fragment or derivative thereof comprising an antigen binding site, such as an antibody VH domain, an antibody VH and VL domain, an antibody scFv fragment or an antibody Fab fragment.

4. A method according to any of the preceding claims which further comprises screening the binding agents selected in step (d) for sequence dependent and/or secondary structure dependent binding specificity.

5. A method according to any of the preceding claims which further comprises screening binding agents selected in step (d) to determine IC50 value for binding to the modified nucleic acid, and selecting binding agents having IC50 <5ng/ml.

6. A method according to any of the preceding claims wherein the modified nucleic acid comprises a modified backbone and comprises a sequence of any one or more of SEQ ID NOS 65, 66, 67 or HEX1 - HEX6.

7. A binding agent identified using a method according to any of the preceding claims.

8. A binding agent having binding specificity for a nucleic acid modification, wherein the binding agent comprises an antigen binding site which binds to a nucleic acid having the modification (modified nucleic acid), and wherein the nucleic acid modification comprises a modified backbone, or a modified sugar component.

9. A binding agent according to claim 8 which comprises an antibody or a fragment or derivative thereof comprising the antigen binding site.

10. A binding agent according to claim 8 or 9 wherein the nucleic acid modification comprises a phosphorothioate backbone.

11. A binding agent according to any of claims 7 to 10 wherein the binding specificity of the agent is:

(a) dependent solely on the nucleic acid modification; or

(b) dependent on the nucleic acid modification and is also dependent on the base sequence and/or secondary structure in the modified nucleic acid

12. A binding agent according to claim 11(a) which comprises: a) one or more antibody CDR sequences selected from SEQ ID NOS: 1-6 or a functional variant of any thereof; or b) an antibody VH domain having the sequence of SEQ ID NO: 15 or a functional variant thereof and/or an antibody VL domain having the sequence of SEQ ID NO: 16, or a functional variant thereof.

13. A binding agent according to any of claims 7 to 12 which binds specifically to a modified nucleic acid:

(a) comprising an immunostimulatory sequence oligonucleotide (ISS-ODN); and/or

(b) comprising any one or more of SEQ ID NOS 65, 66, 68, and HEX1 - HEX6.

14. A pharmaceutical composition comprising a binding agent according to any of claims 7 to 13 and a pharmaceutically acceptable diluent, excipient or carrier.

15. Use of a binding agent according to any of claims 7 to 13 in an in vitro assay to determine the presence and/or concentration of a pharmacaphore in a sample, wherein the pharmacaphore comprises a modified nucleic acid and wherein the binding agent binds specifically to the modified nucleic acid.

16. A method for detecting or quantifying a pharmacaphore in a human or animal subject to which the pharmacaphore has been administered, the method comprising: a) providing a sample taken from the subject; b) contacting the sample with binding agent according to any of claims 7 to 13; and c) detecting pharmacaphore -bound binding agent; wherein the pharmacaphore comprises a modified nucleic acid and wherein the binding agent binds specifically to the modified nucleic acid.

17. A binding agent according to any of claims 7 to 13, or a pharmaceutical composition according to claim 14:

(a) for use in medicine;

(b) for use in a method of detecting a pharmacaphore in a human or animal subject, wherein the pharmacaphore comprises a modified nucleic acid and wherein the binding agent binds specifically to the modified nucleic acid; or

(c) for use in a method of oligonucleotide therapy in a human or animal subject.

18. A method for detecting a pharmacaphore in a human or animal subject to which has been administered a pharmacaphore and detectably labelled binding agent according to any of claims 7 to 13, wherein the pharmacaphore comprises a modified nucleic acid and wherein the binding agent binds specifically to the modified nucleic acid, the method comprising detecting the position of the binding agent in the patient body by means of the detectable label.

19. A method of treating a human or animal subject with a therapeutic oligonucleotide comprising administration of a binding agent according to any of claims 7 to 13, or a pharmaceutical composition according to claim 14, wherein the therapeutic oligonucleotide comprises a modified nucleic acid and wherein the binding agent binds specifically to the modified nucleic acid.

20. A kit for determining the presence and/or concentration of a pharmacaphore in a sample, the kit comprising a binding agent according to any one of claims 7 to 13, wherein the pharmacaphore comprises a modified nucleic acid and wherein the binding agent binds specifically to the modified nucleic acid.