WO2013027063A1

WO2013027063A1 - Chemically programmable immunity

Info

Publication number: WO2013027063A1
Application number: PCT/GB2012/052073
Authority: WO
Inventors: Kary MULLIS; Charles Edward Selkirk Roberts
Original assignee: Altermune Technologies, Llc
Priority date: 2011-08-24
Filing date: 2012-08-23
Publication date: 2013-02-28
Also published as: GB201114662D0

Abstract

The invention relates to immunity linker molecules which target Clostridium difficile or a Clostridium difficile toxin, methods of prophylaxis and/or treatment of disease or infection from Clostridium difficile, methods for providing immediate immunity to Clostridium difficile or a Clostridium difficile toxin and pharmaceutical compositions comprising said immunity linker molecules.

Description

CHEMICALLY PROGRAMMABLE IMMUNITY

FIELD OF THE INVENTION

The invention relates to immunity linker molecules which target Clostridium difficile or a Clostridium difficile toxin, methods of prophylaxis and/or treatment of disease or infection from Clostridium difficile, methods for providing immediate immunity to Clostridium difficile or a Clostridium difficile toxin and pharmaceutical compositions comprising said immunity linker molecules. BACKGROUND OF THE INVENTION

Clostridium difficile (C. difficile) is a species of Gram-positive bacteria of the genus Clostridium that causes severe diarrhea and other intestinal disease when competing bacteria in the gut flora have been disrupted by antibiotics, such as ampicillin, amoxicillin, cephalosporins, and clindamycin (Kelly and Lamont, Annu. Rev. Med ., 49 : 375-90, 1998). C. difficile is the most common cause of infectious diarrhea in hospital patients, and is one of the most common nosocomial infections overall (Kelly et al., New Eng. J. Med., 330 : 257-62, 1994).

C. difficile disease typically occurs four to nine days after antibiotic treatment begins, but can also occur after discontinuation of antibiotic therapy. C. difficile can produce symptoms ranging from mild to severe diarrhea and colitis, including pseudomembranous colitis (PMC), a severe form of colitis characterized by abdominal pain, watery diarrhea, and systemic illness (e.g ., fever, nausea). Relapsing disease can occur in up to 20% of patients treated for a first episode of disease, and those who relapse are at a greater risk for additional relapses (Kelly and Lamont, Annu. Rev. Med ., 49 : 375-90, 1998).

C. difficile disease is believed to be caused by the actions of two exotoxins, toxin A and toxin B, on gut epithelium. Both toxins are high molecular weight proteins (280-300 kDa) that catalyze covalent modification of Rho proteins, small GTP- binding proteins involved in actin polymerization, in host cells. Modification of Rho proteins by the toxins inactivates them, leading to depolymerization of actin filaments and cell death. Both toxins are lethal to mice when injected parenterally (Kelly and Lamont 1998, supra). It is believed that toxin B is essential for virulence of C. difficile (Lyras et al. (2009) Nature Letters 458, 1176-1181).

C. difficile treatment is complicated by the fact that antibiotics trigger C. difficile associated disease. Nevertheless, antibiotics are the currently preferred treatment option. Antibiotics such as vancomycin and metronidazole are frequently used, since they treat, and are least likely to worsen, C. difficile. Vancomycin resistance evolving in other microorganisms is a cause for concern in using this antibiotic for treatment, as it is the only effective treatment for infection with certain other microorganisms (Gerding, Curr. Top. Microbiol. Immunol ., 250 : 127-39, 2000). Probiotic approaches, in which a subject is administered non-pathogenic microorganisms that presumably compete for niches with the pathogenic bacteria, are also used. For example, treatment with a combination of vancomycin and Saccharomyces boulardii has been reported (McFarland et al., JAMA., 271(24) : 1913-8, 1994. Erratum in : JAMA, 272(7) : 518, 1994).

Vaccines have been developed that protect animals from lethal challenge in infectious models of disease (Torres et al., Infect. Immun. 63(12) :4619-27, 1995). In addition, polyclonal antibodies have been shown to protect hamsters from disease when administered by injection or feeding (Giannasca et a/., Infect. Immun. 67(2) : 527-38, 1999; Kink and Williams, Infect. Immun., 66(5) : 2018- 25, 1998). Murine monoclonal antibodies have been isolated that bind to C. difficile toxins and neutralize their activities in vivo and in vitro (Corthier et al., Infect. Immun., 59(3) : 1192-5, 1991). There are some reports that human polyclonal antibodies containing toxin neutralizing antibodies can prevent C. difficile relapse (Salcedo et al., Gut., 41(3) : 366-70, 1997). Antibody response against toxin A has been correlated with disease outcome, indicating the efficacy of humoral responses in controlling infection. Individuals with robust toxin A ELISA responses had less severe disease compared to individuals with low toxin A antibody levels (Kyne et al., Lancet, 357(9251) : 189-93, 2001).

It is therefore an object of the invention, to provide alternative and effective treatments for Clostridium difficile infection. SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided an immunity linker molecule comprising at least one first binding site that binds to an immune system molecule and at least one second binding site that is an aptamer which binds to Clostridium difficile or a Clostridium difficile toxin.

According to a second aspect of the invention, there is provided a method of prophylaxis and/or treatment of disease or infection from Clostridium difficile which comprises administering to a subject in need thereof an immunity linker molecule as defined herein.

According to a third aspect of the invention, there is provided a method of immunizing a human or animal against Clostridium difficile or a Clostridium difficile toxin comprising administering to the human or animal an immunity linker molecule, the immunity linker molecule containing at least one first site that binds to an immune system molecule, and a second site that is an aptamer which binds to Clostridium difficile or the Clostridium difficile toxin. According to a fourth aspect of the invention, there is provided a method of diverting an immune response to Clostridium difficile or a Clostridium difficile toxin in an individual comprising, administering to the individual an effective amount of a composition comprising one or more immunity linkers, wherein the linker molecules comprise at least one first binding site and at least one second binding site, wherein the individual has a pre-existing immune response to the first binding site, and wherein the first binding site binds to an immune system molecule and the second binding site is an aptamer which binds to Clostridium difficile or the Clostridium difficile toxin. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 Confirmation of aptamer inserts for TcdA.

Figure 2 Effect of TcdA on Raw 264.7 cells.

Figure 3 Neutralisation assay for selected aptamers.

Figure 4 In vitro effect of aptamer alone on RAW 264.7 cell viability. Figure 5 : Confirmation of aptamer inserts for TcdB by agarose gel electrophoresis.

Figure 6: Effect of TcdB on RAW 264.7 cells.

Figure 7: Inhibition of TcdB cytotoxicity by selected TcdB aptamers. Figure 8: In vitro effect of aptamer alone on RAW 264.7 cell viability.

Figure 9: 100 % Vero cell rounding due to toxicity from C. difficile supernatant.

Figure 10: Healthy Vero cells showing no toxicity. DETAILED DESCRIPTION OF THE INVENTION

Clostridium difficile is a species of Gram-positive bacteria of the genus

Clostridium that causes severe diarrhea and other intestinal disease. Pathogenic C. difficile strains produce several known toxins. The most well-characterized are enterotoxin (toxin A) and cytotoxin (toxin B), both of which are responsible for the diarrhea and inflammation seen in infected patients, although their relative contributions have been debated. Toxins A and B are glucosyltransferases that target and inactivate the Rho family of GTPases. Toxin B (cytotoxin) induces actin depolymerization by a mechanism correlated with a decrease in the ADP- ribosylation of the low molecular mass GTP-binding Rho proteins. Another toxin, binary toxin, has also been described, but its role in disease is not yet fully understood. The therapeutic targetting of Clostridium difficile toxins has been previously described by using monoclonal antibodies (Lowy et al., (2010) N Engl J Med 362(3), 197-205). However, it is not yet clear that adjuvant antibody therapy targeting Clostridium difficile toxins will be effective in all clinical settings (Parks and Gkrania-Klotsas (2010) N Engl J Med 362(15), 1444-1446). The invention provides a solution to the limitations facing other investigational and approved therapies that target Clostridium difficile infections. For example, the invention provides immunity linkers, compositions and methods for a programmable immunity that can provide a substantially immediate immune response by an individual against a Clostridium difficile toxin that possess stability for in vivo therapeutic applications and target specificity. Since an immediate effective immune response is achieved, these compositions may be administered to an individual any time prior to the individual's contact with a Clostridium difficile toxin or even soon after the individual's contact with a Clostridium difficile toxin.

The immunity linkers, compositions and methods of the invention also provide an advantage over traditional immunization techniques because the methods do not require that the Clostridium difficile toxin or a portion thereof be administered to an individual for effective immunization against the Clostridium difficile toxin.

The invention finds greatest utility when using an existing immune response in an individual and is capable of redirecting the immune response to a different target to provide a stable and specific immediate immunity. Thus, if an individual is already immune to a particular antigen, an immunity linker molecule can be made that has a first binding site comprising or corresponding to the antigen to which the individual is immune and the second binding site can be directed to the Clostridium difficile toxin. In this embodiment, the invention carries the significant advantage of removing the need for vaccinations entirely, with the benefit of increased safety and the ability to use the immune system to treat individuals without the advance notice period required to give vaccination.

In one alternative and optional embodiment, the linker compositions of the invention may make use of a pre-existing immune response in an individual and link that pre-existing immune response to a different target (i.e. the Clostridium difficile toxin), which is unrelated to the pre-existing immune response except for the connection between the two provided by the immunity linker. The preexisting immune response is directed to an antigen containing the first binding site of the immunity linker and can be induced in the individual by administration of a universal immunogen containing the first binding site. Tying the immune response to the Clostridium difficile toxin allows for an immediate, linked immune response without the requirement for a primary immune response against the Clostridium difficile toxin. Following the initiation or generation of a pre-existing immune response, if the individual is exposed, or is suspected of being exposed, to a Clostridium difficile toxin for which immediate immunity is desired, the individual is administered an immunity linker described herein that contains a first binding site that will be recognized corresponds to the universal immunogen and a second binding site that is an aptamer which binds to a Clostridium difficile toxin. The immunity linker binds at the one first binding site to the immune response components, for instance antibodies, produced during the pre-existing immune response, and also binds to the Clostridium difficile toxin at the at least one second binding site thereby providing an immune complex of the immune response component bound to the immunity linker which is also bound to the Clostridium difficile toxin. The immune system of the individual recognizes these immunity linker complexes and removes or clears them from the body, for instance when sufficient numbers of them exist in close proximity to each other that they can 'crosslink' and be recognized by cells of the immune system.

Thus, by administering a composition comprising an immunity linker described herein, the pre-existing immune response of the individual is re-directed from the universal immunogen to the Clostridium difficile toxin. As mentioned above, another benefit of the invention is that only one initial immunizing molecule or universal immunogen is required for priming an individual's immune system for a later antigen-specific immune response. Thus, the invention may decrease the number (and possibly the complexity of formulation) of vaccinations currently recommended or required for individuals. A further benefit of the invention is the ease of preparation of the immunity linker and the universal immunogen. The immunity linkers of the invention can be easily assembled and provided to health care professionals for rapid response to such public health needs as pandemic infections, bioterroristic threats, or limited outbreaks of specific pathogens. Thus, in one embodiment the invention solves many of the problems facing the military regarding protection of their personnel from agents of bioterrorism. The invention relies upon the previously disclosed concept of chemically programmable immunity or programmable immunity. Programmable immunity differs from classical immunity in that programmable immunity allows for the re- direction of a pre-existing immune response directed toward one antigen, to the target. The immune response is re- directed using an immunity linker of the invention.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly indicates otherwise. Thus, for example, reference to a "linker" is a reference to one or more such linkers and includes equivalents thereof known to those skilled in the art, and so forth. Immunity linkers of the invention comprise at least two sites; (1) a first binding site that binds to at least one immune response component of an individual, and (2) a second binding site that binds to the Clostridium difficile toxin. The immune response component is one that exists in the individual prior to administration of the immunity linker to the individual . For example, the immune response component can be an antibody that was part of a previous immune response to the first binding site, or to a molecule, or a large assembly of molecules, or even a micro-organism containing the first binding site.

Accordingly, as used herein, the term "pre-existing immune response" refers to an immune response that is directed toward the first binding site or an epitope that is immunologically similar to the first binding site. In other words, a "pre-existing immune response" is an immune response in which immune response components are generated or exist that bind to the first binding site. The pre-existing immune response can be generated by a previous administration to the individual of a universal immunogen that corresponds to a first binding site or can exist in the individual without such administration .

Accordingly, the invention includes a method of diverting a pre-existing immune response (or increasing an effective immune response) in an individual from a first antigen to Clostridium difficile or a Clostridium difficile toxin comprising, administering to the individual an effective amount of a composition comprising one or more immunity linkers, wherein the linkers comprise at least one first binding site and at least one second binding site, wherein the second binding site is an aptamer which binds to Clostridium difficile or a Clostridium difficile toxin and wherein the first antigen comprises the first binding site or an immunological equivalent thereof.

The invention also encompasses a method of diverting an immune response to Clostridium difficile or a Clostridium difficile toxin comprising, administering to the individual an effective amount of a composition comprising one or more immunity linkers, wherein the linkers comprise at least one first binding site and at least one second binding site, wherein the second binding site is an aptamer which binds to Clostridium difficile or a Clostridium difficile toxin and wherein the individual has a pre-existing immune response to the first binding site or an immunological equivalent thereof.

Universal Immunogens

A universal immunogen that "corresponds to" a first binding site can be identical to the first binding site, can contain the entire first binding site, can contain a portion of the first binding site, or can be an immunological equivalent of the first binding site. When referring to two or more molecules, the term "immunological equivalent" refers herein to molecules that are bound by the same immune response component. The invention only requires that the immune response component raised by the universal immunogen also bind to the first binding site. In one embodiment, the universal immunogen binds to the immune response component with sufficient affinity to result in the production of a complex that is capable of initiating or participating in an immune response. In a preferred embodiment, the cross-reactivity of the immune response component to molecules other than the universal immunogen and the first binding site is minimal.

The universal immunogen can be any molecule, organism or compound to which an individual mounts an immune response and can be administered via any route. The universal immunogen can be, but is not limited to, a molecule, a microbe, or a toxin or a toxoid derived therefrom; a protein or polypeptide; a polynucleotide; a mono- to poly-saccharide; a synthetic material or a combination thereof.

In one embodiment, the universal immunogen causes an immune response in an individual that provides for long-lasting immune memory, can be re- administered to individuals in booster doses, and does not cause disease, pathology or long-term illness in individuals. An immunogen that comprises a portion of a pathogen or a modified portion of a pathogen can be a universal immunogen, but a universal immunogen is not required to bear any relationship to anything except the complementary immune response which it elicits. For example, humans are routinely immunized with immunogenic antigens from mumps virus, measles virus, tetanus toxoid, and polio virus. Animals, such as cats and dogs, are routinely immunized with immunogenic antigens from rabies virus. These and other traditional immunogens can be used as universal immunogens, however, this would be a matter of convenience, not necessity.

Alternatively, non-traditional immunogens may be used as the universal immunogen. In one embodiment, a non-traditional immunogen does not contain either a portion or a modified portion of a pathogen. In one embodiment, the universal immunogen is a protein, or a portion of a protein, to which a hapten is bound . A "hapten" is defined herein as a molecule that reacts with a specific antibody, but cannot induce the formation or generation of additional antibodies unless bound to a carrier protein or other large antigenic molecule. Most haptens are small molecules, but some macromolecules can also function as haptens. In one embodiment, the hapten is a phenylarsonate and the universal immunogen is a phenylarsonylated protein (as described in Example 1 of WO 2010/129666). In an alternative embodiment, the universal immunogen comprises a bacteriophage or an epitope of a bacteriophage. An immune response component can bind to any part of the bacteriophage and in one embodiment, binds to a peptide that is expressed on the surface of the bacteriophage. A bacteriophage universal immunogen can be administered to an individual via any route and in some embodiments, the bacteriophage can be contained within a bacteria as a convenient means of administration.

First and Second Binding Sites and Spacers of an Immunity Linker

The immunity linker can be any type of chemical or biological material including a microbe, a bacteriophage, a protein, a nucleic acid, a mono- to polysaccharide, a synthetic material or a combination thereof. In one embodiment, the at least one first binding site is physically or chemically linked or conjugated to a molecule comprising the at least one second binding site. In this embodiment, a spacer molecule may reside between the first binding site and the second binding site. In another embodiment, the immunity linker is a single molecule containing the at least one first binding site and the at least one second binding site.

The invention is able to re-direct a pre-existing immune response directed toward a universal immunogen to a different antigen, in part, because the universal immunogen corresponds to a first binding site of an immunity linker. Since the first binding site is a part of both the universal immunogen and the immunity linker molecule, the pre-existing immune response, or the pre-existing immune system components, that are directed to the universal immunogen also recognize the immunity linker.

The first binding site of the immunity linker can comprise a polypeptide, a polynucleotide, a mono- to poly-saccharide, an organic chemical, a microorganism such as a bacteriophage, a bacterium, a virus or viral particle, or a protozoa, any fragment or portion of the foregoing, any combination of the foregoing, or any other composition that is recognized by the immune system of an individual or bound by an immune response component in an individual. In one embodiment, the first binding site is an oligosaccharide such as the alpha-Gal epitope, i.e., galactosyl-alpha-l,3-galactosyl-beta-l,4-N-acetylglucos- amine as is described in Galili, U . and Avila, J. L., Alpha-Gal and Anti-Gal, Subcellular Biochemistry, Vol. 32, 1999. Xenotransplantation studies have determined that humans mount an immune response to the alpha-galactosyl epitope, which itself is not normally found in humans, but is found in other animals and many microorganisms.

However, it is believed that Enterotoxin A of C. difficile may interact with alpha- Gal epitopes, therefore, in an alternative, more advantageous, embodiment the first binding site is a mono- or oligo-saccharide such as rhamnose, i.e. L- rhamnose. Anti-rhamnose (anti-Rha) antibodies have been reported to be present in human sera (Chen et al. (2011) ACS Chem Biol 6(2), 185-191) and rhamnose glycoconjugates are naturally found in plants and in some bacteria (e.g . Mycobacterium tuberculosis). Data is shown herein in Example 3 that rhamnose conjugated aptamers surprisingly demonstrated effective neutralizing activity against C. difficile Toxin A and Toxin B.

In another embodiment, the first binding site comprises a portion of a bacteriophage, and more preferably, a polypeptide that is expressed on the surface of a bacteriophage.

In one embodiment, the aptamer of the second binding site comprises one or more chemical modifications. Such modifications can be in individual nucleotides prior to amplification or synthesis of the nucleic acids, or can be added to nucleotides after incorporation into multimers. Such modifications include, but are not limited to, modifications at cytosine, exocyclic amines, substitution of 5- bromo-uracil, backbone modifications, methylations, unusual base- pairing combinations and others known to those skilled in the art.

In one embodiment, the aptamer comprises a modified phosphate backbone. Such modifications have been shown in WO 2010/129666 to provide increased stability for in vivo administration. Such modifications include phosphate backbone modifications including substantially all phosphorothioates or phosphorodithioates. Phosphorothioates or phosphorodithioates can be included in substantially all of the polynucleotide phosphate backbone or a part of the phosphate backbone. In some embodiments at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of the phosphate background is thiolated. These modified aptamer target-binding sites are found to have significant stability and circulating half-life for therapeutic administration with target binding specificity. These modified aptamer, more specifically thioaptamer, immunity linkers offer an improvement over previously described immunity linkers that includes significant stability with concomitant binding specificity where it was previously believed to be a trade-off.

As described above, the immunity linker includes any type of molecule or organism that contains a first binding site capable of binding to an immune response component, and contains a second binding site capable of binding a Clostridium difficile toxin. In some embodiments, the immunity linkers can contain more than one first binding site and/or more than one second binding site. The multiple first binding sites can be identical or can be different. The multiple second binding sites can also be identical or different. Binding sites may differ in their specificity for different molecules or their affinity for the same molecule. The immunity linker can also be modified to reduce its own immunogenicity.

Binding by the first and second binding sites to the immune response component and Clostridium difficile toxin, respectively, can be accomplished through any interaction including through binding provided by other molecules, such as mono- to poly-saccharides or nucleic acids. In one embodiment, a first binding site is specific for an immune response molecule and a second binding site is specific for a Clostridium difficile toxin. As described above, a molecule is "specific for" another molecule when the two molecules bind with sufficient affinity to result in the production of a functional complex for purposes of the immune system. In a further embodiment, the cross-reactivity of one second binding site with molecules other than a Clostridium difficile toxin is minimal. In an alternative embodiment, the cross-reactivity of one first binding site with molecules other than an immune response component is minimal .

Following administration of the immunity linker to the individual, an immunity linker complex comprising the immune response component, the immunity linker, and the Clostridium difficile toxin is formed . The immunity linker can bind the Clostridium difficile toxin prior or subsequent to the binding of the immunity linker to an immune system component. Following formation of the immunity linker complex, the Clostridium difficile toxin is cleared via immune system pathways. A "clearing" of a Clostridium difficile toxin refers herein to the removal, inactivation or modification of the Clostridium difficile toxin such that it is no longer harmful to the body.

It will be appreciated by the skilled person that the therapeutic approach of the invention toward the Clostridium difficile toxins is a viable approach in view of the enabling disclosure in WO 2010/129666 where corresponding toxin-specific aptamers (containing an alpha-gal linker) to a similarly toxinogenic bacteria, Bacillus anthracis, were clearly shown to be effective at increasing the survival rate of mice exposed to Bacillus anthracis (as can be seen in Example 6 and Figure 3 of WO 2010/129666; the contents and data of which are herein incorporated by reference).

In another embodiment, the immunity linker comprises a first binding site that corresponds to an alpha-galactosyl, or alpha-Gal epitope. In one particular embodiment of the invention, the alpha-galactosyl epitope is conjugated to a second binding site that comprises an aptamer which binds to a Clostridium difficile toxin.

In another embodiment, the immunity linker comprises a first binding site that corresponds to a rhamnose, or L-rhamnose, epitope. In one particular embodiment of the invention, the L-rhamnose epitope is conjugated to a second binding site that comprises an aptamer which binds to a Clostridium difficile toxin.

In other embodiments, the first and second binding sites comprise an aptamer nucleic acid, and more preferably an aptamer that has been produced by the SELEX process. SELEX stands for Systemic Evolution of Ligands by Exponential enrichment. SELEX methods are known in the art and are described in at least the following issued U .S. Patents: U .S. Patent Nos. 5,475,096; 6,261,774; 6,395,888; 6,387,635; 6,387,620; 6,376,474; 6,346,611; 6,344,321; 6,344,318; 6,331,398; 6,331,394; 6,329,145; 6,300,074; 6,280,943; 6,280,943; 6,280,932; 6,261,783; and 6,232,071.

In general, the SELEX method relates to identifying nucleic acids that specifically bind to three dimensional targets. Nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers such that some sequences can be found that bind specifically with virtually any chemical compound. For purposes of stability in biological fluids, a preferred aptamer contains one or more modified nucleotides such as 2'-fluoro- or 2'-amino-2'-deoxypyrimidines. Nucleic acids using these bases are much more stable in vivo than naturally occurring nucleic acids. See, M . Famulok and G. Mayer, Cur. Top. Micro. Immunobiol. 243 : 123-146, 1999. Spiegelmers (see

Vater, A. and Klussmann, S. Current Opin. Drug Discov Devel . 2003 Mar; 6(2) : 253- 61) derived by similar methods may also be employed for their inherent stability in serum.

In one embodiment, oligonucleotide libraries for use in the SELEX method are made using commercially available kits from Roche (Mannheim, Germany) such as the GS FLX Titanium series protocols and reagents such as the Amplicon Library Preparation protocol . GS FLX Titanium fusion primers (Roche, Mannheim, Germany) may be used to sequence aptamers that are identified although other sequencing methods are known in the art and may be used as well . In addition to the SELEX method there are a number of other methods which may suitable be used for aptamer selection, such as those using microbeads, microfluidics, and sequencing technologies. It will be appreciated that the invention applies equally, regardless of the source or method of selection of the aptamer component of the molecule and is not intended to be limited by the method of selection, whether one of those methods stated herein or subsequent selection methods later developed.

The aptamers of the invention may suitably comprise ribonucleotides only (RNA aptamers), deoxyribonucleotides only (DNA aptamers), or a combination of ribonucleotides and deoxyribonucleotides. The nucleotides may be naturally occurring nucleotides (e.g ., ATP, TTP, GTP, CTP, UTP) or modified nucleotides. Modified nucleotides refers to nucleotides comprising bases such as, for example, adenine, guanine, cytosine, thymine, and uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups. Some examples of types of modifications that can comprise nucleotides that are modified with respect to the base moieties, include but are not limited to, alkylated, halogenated, thiolated, dithiolated, aminated, amidated, or acetylated bases, in various combinations. More specific examples include 5- propynyluridine, 5-propynylcytidine, 6-methyladenine, 6- methylguanine, N5N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2- aminoadenine, 1 -methylinosine, 3- methyluridine, 5-methylcytidine, 5- methyluridine and other nucleotides having a modification at the 5 position, 5- (2-amino)propyl uridine, 5-halocytidine, 5-halouridine, 4- acetylcytidine, 1 - methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2- methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5- methylaminoethyluridine, 5- methyloxyuridine, deazanucleotides such as 7- deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2- thiouridine, other thio bases such as 2-thiouridine and 4- thiouridine and 2- thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety (e.g ., 2'-fluoro or 2'-0-methyl nucleotides), as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide is also meant to include what are known in the art as universal bases. By way of example, universal bases include but are not limited to 3-nitropyrrole, 5 -nitro indole, or nebularine. Modified nucleotides include labeled nucleotides such as radioactively, enzymatically, or chromogenically labeled nucleotides). In one particular embodiment, the DNA or RNA oligonucleotides are thiolated or dithiolated. Such thiolation provides the advantage of ensuring that the oligonucleotide is resistant to endonuclease activity.

In a further particular embodiment, the aptamers comprise spiegelmers. Spiegelmers are oligonucleotide mirror images (L-form nucleotides), which due to their optical re-configuration confer resistance to endonuclease activity whilst maintaining specificity of the 'regular' (D-form) type of oligonucelotides. Spiegelmers are well characterized and have been shown to demonstrate high affinity for specific targets, such as gonadotrophin (Wlotzka et a/, PNAS June 25, 2002 vol. 99 no. 13 8898-8902).

In one embodiment, the Clostridium difficile toxin is toxin A.

In one embodiment, when the Clostridium difficile toxin is toxin A, the aptamer which binds to Clostridium difficile toxin A comprises any one of the following aptamer sequences:

CDA-4 - 5'-CCA TAC TTT CCC AAG ACT TAT ACT AAG AAC ATA CCC GTT TAC-3' (SEQ ID NO: 14);

CDA-5 - 5'- ATA ATA TAA CCG TAC AAT ACC CGA CTC TAA CAT AAA GTT CAC-3' (SEQ ID NO: 15);

CDA-7 - 5'-TAA ATC CAC ACA AAT ATC CTT TTA CCA ACC TTA TTG CCA CAT-3' (SEQ ID NO: 16);

CDA-8 - 5'-CCT ATT TAC TAA GAC CAT TCC TCC TCT TTA TGA CCC TCA ATA- 3' (SEQ ID NO : 17);

CDA-10 - 5'-CAA AGC ACT TTA TAA CAC AAC CTA CAT GAC AAA ACC GCC CTC- 3' (SEQ ID NO: 18);

CDA-11 - 5'-AAG ATA AAC CGA CTC TTC TAA CTT TCC ACC GAA TAA CGT ATT- 3' (SEQ ID NO: 19);

CDA-12 & CDA-18 - 5'-GAA GCA TGG CTC TAC ATC CCT TTA CCT CCC TCT TTT ACT ACA-3' (SEQ ID NO: 20); CDA-13 - 5'-TCC TGA TTT TCA TAC AAC TTA ATA CTA TCT CCG CTC CGT ACC- 3' (SEQ ID NO: 21);

CDA-14 - 5'- ACT TTT CTC CCT CTT TTT TTG TAA ATT CTC AGA CAC ACA CCC CAG-3' (SEQ ID NO: 22);

CDA-15 - 5'-CAC TCA CTC ACA AGA TCA TCT CGA CTC AAA CTT CAT CAA AAT-3' (SEQ ID NO: 23);

CDA-16 - 5'-ACT GCT CAA CCA TCA ACA CCT TCT TTC TTC ACT TA-3' (SEQ ID NO: 24);

CDA-17 - 5'- AAC TTA ACT TAC CAT CTA TAG TCT GCC CAC TAT AAC TAT TGA- 3' (SEQ ID NO: 25);

CDA-19 - 5'-ATT CTA TGA CTA CCT ACA CCT CAC TGT CTC TCC ACC TTT TTG-3' (SEQ ID NO: 1);

CDA-21 - 5'-CAC CCG ACC GAA CTT CTC TTT TTC CTA CAT ATA GCA TCG TAA-3' (SEQ ID NO: 2);

CDA-23 - 5'-ACC CAC GAA TTT CCA CAC ACT ATC CTA TCT CCA CTA ATC TA-3' (SEQ ID NO: 3);

CDA-24 - 5'-CAC CCT TTC CAC TTC CCC ATA GAC CAT TCC CTC TAC ATA CCC-3' (SEQ ID NO: 26);

CDA-25 - 5'-CCT CCT CAA TTA TAC CTC TTC ATA AAC CCT TGA CAT CTG ACA-3' (SEQ ID NO: 27);

CDA-26 - 5'-CCA CTA TTA GTT CTT TTA TAC CCA TTG TAC GGA AGA CTC GCC- 3' (SEQ ID NO: 28);

CDA-27 - 5'-CCT ATC CCT CCT ACC ATA TTC CCT ACA ATT ACG CGT CCA TCA-3' (SEQ ID NO: 29);

CDA-28 - 5'- ATA GTA CAC ATT TAT AAC ATT TGC AAA GAT TAC TAA CCG TTT- 3' (SEQ ID NO: 30);

CDA-29 - 5'-CCC TAA CAA ACA TGT CAA TTC AGA GAT TTT TAC CTA ACA TGC-3' (SEQ ID NO: 4);

CDA-30 - 5'-ACC CAA TGA CAA ATT AAT TAA ATC CTT TTA ACC AAT ACC TTT- 3' (SEQ ID NO: 31);

CDA-33 - 5'-GAA CAT TAA CAC TCG CCG GAA TAT TCC AAC TAC CTT TTA CCT-3' (SEQ ID NO: 5);

CDA-34 - 5'-GTT TGG ATT GTA CCA CCA TTC AAT TAA CTT ACT ATA CTA TTA- 3' (SEQ ID NO: 6); CDA-36 - 5'-ACC TCC TCC CAT ACT AAG CTC AAC CCA ATC ACT TAT TAC CCT-3' (SEQ ID NO: 32);

CDA-37 - 5'-TCC CAT ACC ACA TTC CCT TCT CAA ACT ATC AAA AGC TCA GGG- 3' (SEQ ID NO: 7);

CDA-38 - 5'-TTT ACA CTA AAT TTT ACC CTG CGA GCA ACC TTC AAC TAA TTA- 3' (SEQ ID NO: 33);

CDA-39 - 5'-TAC CAC TCC TTT TAT CCT CAA CTT CGT GAA CTT TTT CTA TAA- 3' (SEQ ID NO: 34);

CDA-40 - 5'-CTT ATA TGA CTC ACT CCA ACT TTT CCA TAC TTC TTA AAA CTA-3' (SEQ ID NO: 35);

CDA-41 - 5'-CAG TCC ATA CTA ACT TCA ATC TTC CTA TGA CTT AAT TAT TAA- 3' (SEQ ID NO: 36);

CDA-42 - 5'-AGA AAT GTT AGG TTG AAA TAG AAA TCC CTT CGA AGA ATT GTG- 3' (SEQ ID NO: 37);

CDA-43 - 5'-CCC TCT GTA CCC TTC ACC CTA TGT TAA CTT ATT TTC ACT ACA-3' (SEQ ID NO: 38);

CDA-44 - 5'-CGC ACT CTA ACT ACC ATA ACA CTT ATT CTC ATT ACT CAC CAT-3' (SEQ ID NO: 39);

CDA-46 - 5'-ACA TTA CTC ATT AAC TAC CAC TCC TCA TTA CAA CTC TAT GAG-3' (SEQ ID NO: 40);

CDA-47 - 5'-CCC TTA AAT TCA ATT ACG ACC TCA TTC CCT TAC ACA AAT GCG-3' (SEQ ID NO: 41);

CDA-49 - 5'-ATC CCC ACA TCC CTT TAA TAA ATT ACT GAA TGA ACA TAC CTC-3' (SEQ ID NO: 42);

CDA-51 - 5'-CAC TAG GGC CAT CTT CTT TCA TTA CAC CTT TCA TCC ACT ACA-3' (SEQ ID NO: 43);

CDA-53 - 5'-CAC CAC ACT TTT CCT CAT TCA ACT AAC GTT CGT CAC CGC AAT- 3' (SEQ ID NO: 8); or

CDA-57 - 5'-CGA AAC ATT CAA CCC AAA TCA TTT TCT ATC ACA TTT GCC CAT-3' (SEQ ID NO: 44).

In a further embodiment, when the Clostridium difficile toxin is toxin A, the aptamer which binds to Clostridium difficile toxin A comprises any one of the following aptamer sequences: CDA-19 - 5'-ATT CTA TGA CTA CCT ACA CCT CAC TGT CTC TCC ACC TTT TTG-3' (SEQ ID NO: 1);

CDA-21 - 5'-CAC CCG ACC GAA CTT CTC TTT TTC CTA CAT ATA GCA TCG TAA- 3'(SEQ ID NO: 2);

CDA-23 - 5'-ACC CAC GAA TTT CCA CAC ACT ATC CTA TCT CCA CTA ATC TA- 3'(SEQ ID NO: 3);

CDA-29 - 5'-CCC TAA CAA ACA TGT CAA TTC AGA GAT TTT TAC CTA ACA TGC- 3'(SEQ ID NO: 4);

CDA-33 - 5'-GAA CAT TAA CAC TCG CCG GAA TAT TCC AAC TAC CTT TTA CCT- 3'(SEQ ID NO: 5);

CDA-34 - 5'-GTT TGG ATT GTA CCA CCA TTC AAT TAA CTT ACT ATA CTA TTA- 3'(SEQ ID NO: 6);

CDA-37 - 5'-TCC CAT ACC ACA TTC CCT TCT CAA ACT ATC AAA AGC TCA GGG- 3'(SEQ ID NO: 7); or

CDA-53 - 5'-CAC CAC ACT TTT CCT CAT TCA ACT AAC GTT CGT CAC CGC AAT- 3'(SEQ ID NO: 8).

In a yet further embodiment, when the Clostridium difficile toxin is toxin A, the aptamer which binds to Clostridium difficile toxin A comprises any one of the following aptamer sequences:

CDA-34 - 5'-GTT TGG ATT GTA CCA CCA TTC AAT TAA CTT ACT ATA CTA TTA- 3'(SEQ ID NO: 6); or

In a still yet further embodiment, when the Clostridium difficile toxin is toxin A, the aptamer which binds to Clostridium difficile toxin A comprises CDA-34 - 5'- GTT TGG ATT GTA CCA CCA TTC AAT TAA CTT ACT ATA CTA TTA-3'(SEQ ID NO : 6).

In one embodiment, the aptamer which binds to Clostridium difficile toxin A has at least 50% sequence identity with any one of SEQ ID NOS 1 to 8. In a further embodiment, the aptamer which binds to Clostridium difficile toxin A has at least 75% sequence identity with any one of SEQ ID NOS 1 to 8. In a yet further embodiment, the aptamer which binds to Clostridium difficile toxin A has at least 80, 85, 90, 95 or 99% sequence identity with any one of SEQ ID NOS 1 to 8.

In one embodiment, the Clostridium difficile toxin is toxin B.

In a further embodiment, when the Clostridium difficile toxin is toxin B, the aptamer which binds to Clostridium difficile toxin B comprises any one of the following aptamer sequences:

CDB-1 - 5'-CTT ACC TTT TAC ACA TAA CAA CTT GGC ATT CAA CCT TTC ACA-3' (SEQ ID NO: 45);

CDB-2 - 5'-TTA TTT TGG TCT TCT TTA CTT TTT TTT CTT TTC CTT TTT CTA-3' (SEQ ID NO: 46);

CDB-3 - 5'-TCA CAC GTT TAT TAC ACA TTT CCA CAT AAC CTC CAT TAA GAA-3' (SEQ ID NO: 47);

CDB-4 - 5'-TAA CAA TTT CTT TTA CTT CCA TTT CCT TAT GCA CTA AAT CTC-3' (SEQ ID NO: 9);

CDB-5 - 5'-AAC ACT CTT TTC TTT ATT TAT TGT CTC TTT TAC TTT TTT TT-3' (SEQ ID NO: 10);

CDB-6 - 5'-CCA CAC CTC TAA CAC ACA TCA CTG TAA ACA TTT TAA CCA ACT-3' (SEQ ID NO: 48);

CDB-7 - 5'-TTC TCT TTC TTC TGT TTT TCT TTT CTT TTC GTC TTA TAC TT-3' (SEQ ID NO: 49);

CDB-9 - 5'-TTC TCC TGT GTT TTA TTT ATA TTT ACT TCT TTG TTT TTC TTT-3' (SEQ ID NO: 50);

CDB-10 - 5'-CTT ACC GAT CTC TCC ATT TTT TCT TTC TTC TTT TTT CTA TT-3' (SEQ ID NO: 51);

CDB-11 - 5'-AAC TAT TTA TAT GTC TAC ACG TAA TTA TTT CTT CCC TAA CCA-3' (SEQ ID NO: 52);

CDB-12 & CDB-30 - 5'-TAC ACC TTT TTT TAA TCC CTT ACA TTT ACC ATT CTT TAT CA-3' (SEQ ID NO: 11);

CDB-13 - 5'-TCT CTA CAA GAC TAA TTT CCT ATT TTT TAT TTC TTT AAC TAT- 3' (SEQ ID NO: 53);

CDB-14 - 5'-CGA TTT ATA TTA TTC TTT TTC TTT CTT CTA CTT TTT TTT TT-3' (SEQ ID NO: 54); CDB-15 - 5'-CTC ATT CCC TTC ATC CGT TTT TTT TAG TTA TCT ATT TCC TAC-3' (SEQ ID NO: 55);

CDB-16 - 5'-ATT TAC CAC CTT TCT CTT TTT CCC GCC TTT CCA TAG CAA CAG-3' (SEQ ID NO: 56);

CDB-17 & CDB-28 - 5'-AGA AAC AAA ATA AAC ACA ACC ACA CTT ACT CAT TTG GAT ACA-3' (SEQ ID NO: 57);

CDB-18 - 5'-TAC CTT TTA TTC TCC CCC TTA ACC AAT ATT TCG TTT CAT TAA-3' (SEQ ID NO: 58);

CDB-20 - 5'-CTC ATT CTC TCT TTC CGT CAT TTT TAT TTC TTC TTA TTT TTT- 3' (SEQ ID NO: 59);

CDB-21 - 5'-TTG TTC ATT TTA TTT TCT CTT TTT TAT CTC TTT TTT TTA TTT- 3' (SEQ ID NO: 60);

CDB-22 - 5'-TCT ATC TTT TTA TTT TTA CTT CCT CTT TCT ATC TTT TTC TAT- 3' (SEQ ID NO: 61);

CDB-23 - 5'-ATT TTT TTT CTT TCT TTT CTC TAT TCA TTC TTT CTT TTT- 3' (SEQ ID NO: 62);

CDB-24 - 5'-CTT ATT ACG TTT CCT CTT TTG TCT ACA ATT CTA CTC CAC CAA-3' (SEQ ID NO: 63);

CDB-25 - 5'-TGA TTT TTT TCC TTC TTT ATT TCT TTT TTT TTT ATT TTT TT-3' (SEQ ID NO: 64);

CDB-26 - 5'-CTT CCT TGA TTA CAC AAA TTT CAC TTA AAC CAT TCC CTA TT-3' (SEQ ID NO: 65);

CDB-27 - 5'-TCC TTT TTC TAT TAA TCC TTT CTT TTT TTT TTT TTG ATT TTT T-3' (SEQ ID NO: 66);

CDB-29 - 5'-TAG ACT CAT ATT TCC TTC TAC TCG TCC TCA TTC ATT TTC TGA-3' (SEQ ID NO: 67);

CDB-32 - 5'-GAT TCC CCT AAA TTC CTT CTT CTT ATT AAA CTC TCC CCC CC-3' (SEQ ID NO: 68);

CDB-33 - 5'-ATC ATT TTT TTT TGT TCG TTG TCT TTT CTT TTT CTC TTA TTT-3' (SEQ ID NO: 69);

CDB-34 - 5'-ACT TTC CTT ATT ATC TTT CCC TAT TTT TTG TTT CTA TTC TAT- 3' (SEQ ID NO: 12);

CDB-35 - 5'-CTT ATA CCC TCT TTG TTG CTT TTC TTT TAT CTT CTT TCT TTC-3' (SEQ ID NO: 70); CDB-36 - 5'-TTT ACT TTC TGT ATT TTC AAT GAT ACT AAC CAA AAT CCC CA-3' (SEQ ID NO: 71);

CDB-38 - 5'-ACC TCT ATC TTA CTC TTA CTA GGC TTA TTC TTC TCC CAA GCA-3' (SEQ ID NO: 72);

CDB-39 - 5'-TCT TTA TTA TCT TTG TGT TCT TAT AGG TTT ATA CTC TTT TTT T-3' (SEQ ID NO: 73);

CDB-40 - 5'-TTT TTA CTT TTT TTT TCT TTT TTT TTT TAT TTT TTT TGT TC- 3' (SEQ ID NO: 74);

CDB-41 - 5'-ATT TTT TTT CTT TTT TTT CTT TTT TGT TTT TAG TTC TTC TT-3' (SEQ ID NO: 75);

CDB-42 - 5'-TAA CTA CAC CCA TTC TTT ACA TTA ATA TTC TGC CAT TTT AGC-3' (SEQ ID NO: 76);

CDB-43 - 5'-TCT TTT CTA TTT TCT TTC TTT CTT TTT TTT TTT TTT TT-3' (SEQ ID NO: 77);

CDB-44 - 5'-TCA TTG GTC GTT TTC TTT TTT TCT TTT TTT TTA TAT TTT TAT- 3' (SEQ ID NO: 78);

CDB-45 - 5'-TAG TCT TTT GGT CTT TCT GTT TCT CTT TTT GTT TCT TTT TTT-3' (SEQ ID NO: 79);

CDB-46 - 5'-TTT TAC TCA TCC CAA TTA CCT CTT TTC TAA TAA CTC GCC CTT-3' (SEQ ID NO: 13);

CDB-47 - 5'-TAA AAG CTA TTT ATA TTA CCC ACT ATC TTC CTC ATG TAA CCA-3' (SEQ ID NO: 80);

CDB-48 - 5'-CCA ACA TGT TCT ACT CAC TTT TCC TAT TAT AAC TAC AAA CTC-3' (SEQ ID NO: 81); or

CDB-49 - 5'-TAC TCA TGG TTA ACA ATC ATA TTC TAT TTA TTC ACC CTA CCT-3' (SEQ ID NO: 82).

CDB-4 - 5'-TAA CAA TTT CTT TTA CTT CCA TTT CCT TAT GCA CTA AAT CTC- 3'(SEQ ID NO: 9);

CDB-5 - 5'-AAC ACT CTT TTC TTT ATT TAT TGT CTC TTT TAC TTT TTT TT-3'(SEQ ID NO: 10); CDB-12 - 5'-TAC ACC TTT TTT TAA TCC CTT ACA TTT ACC ATT CTT TAT CA- 3'(SEQ ID NO: 11);

CDB-34 - 5'-ACT TTC CTT ATT ATC TTT CCC TAT TTT TTG TTT CTA TTC TAT- 3'(SEQ ID NO: 12); or

CDB-46 - 5'-TTT TAC TCA TCC CAA TTA CCT CTT TTC TAA TAA CTC GCC CTT- 3'(SEQ ID NO: 13).

In a yet further embodiment, when the Clostridium difficile toxin is toxin B, the aptamer which binds to Clostridium difficile toxin B comprises CDB-5 - 5'-AAC ACT CTT TTC TTT ATT TAT TGT CTC TTT TAC TTT TTT TT-3'(SEQ ID NO: 10).

In one embodiment, the aptamer which binds to Clostridium difficile toxin A has at least 50% sequence identity with any one of SEQ ID NOS 9 to 13. In a further embodiment, the aptamer which binds to Clostridium difficile toxin A has at least 75% sequence identity with any one of SEQ ID NOS 9 to 13. In a yet further embodiment, the aptamer which binds to Clostridium difficile toxin A has at least 80, 85, 90, 95 or 99% sequence identity with any one of SEQ ID NOS 9 to 13. It will be appreciated that the aptamer sequences described herein contain the target binding site sequences for Toxin A or Toxin B of Clostridium difficile and the presence of additional nucleotides or modified nucleotides at either end would not substantially impact upon the efficacy of the invention. Percent sequence identity is determined by conventional methods. Aptamers having at least 50% sequence identity to the aptamers described herein are characterized as having one or more substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative substitutions and other substitutions that do not significantly affect the target binding activity of the aptamer.

Toxin B of Clostridium difficile has been demonstrated to be essential for the virulence of Clostridium difficile (Lyras et al (2009) Nature 458, 1176-1181; the contents of which are herein incorporated by reference) therefore Toxin B represents a particularly attractive target of the invention .

In one embodiment, the Clostridium difficile toxin is binary toxin.

It will be appreciated that the invention finds particular utility with respect to aptamers which specifically target a Clostridium difficile toxin, however, also within the scope of the invention are aptamers which specifically target Clostridium difficile and are designed to result in whole cell destruction of Clostridium difficile.

The first and second binding sites of the immunity linker described herein may be linked, or conjugated, by any means known to one of skill in the art. The terms "conjugated" and "conjugation" are defined herein to refer to a covalent or other form of linking two or more molecules. Conjugation can be accomplished by any means including, but not limited to, chemical means, genetic engineering means, or in vivo by biologic means. The first and second binding sites may be linked by a double stranded nucleic acid, a polypeptide, a chemical structure, or any other appropriate structure, or may be linked by a simple chemical bond . In one particular embodiment the first and second binding sites of a linker are evolved in vitro in such as way that the first binding site will only interact with the immune response component after the second binding site has bound to the Clostridium difficile toxin. Allosteric interactions leading to such behavior are well-known in proteins and other macromolecules, and could be a component of the selection process in the in vitro evolution of the linker.

Immune Response Components

As stated above, the one or more first binding sites of the immunity linker bind to an immune response component. The term "immune response component" is used herein to refer to any molecule or cell involved in an immune response of an individual. The term "individual" encompasses both animals and humans. Non-limiting examples of immune response components are antibodies; lymphocytes including, but not limited to, T cells, B cells and natural killer cells; macrophages; granulocytes including, but not limited to, neutrophils, basophils and eosinophils; and receptors on any of the foregoing cells including, but not limited to, T cell receptors and B cell receptors. The term antibody includes all of the classes and subclasses of antibodies, IgG, IgM, IgA, IgD, IgE, etc., secretory and excreted forms of the antibodies, fragments of antibodies, including variable, hypervariable and constant regions, heavy and light chains, combinations of fragments and mixtures of fragments and whole antibodies.

Such antibodies can be humanized, polyclonal or monoclonal, naturally derived or synthetic antibodies.

In one embodiment, at least one first binding site binds to the active binding site of the immune response component. For example, if the immune response component is an antibody such as an IgG molecule, the first binding site of the immunity linker is the antigenic epitope to which the active binding site of the variable region of the IgG molecule normally binds.

Immunity Linker Populations

As indicated above, the immunity linkers of the invention can have more than one first binding site and/or more than one second binding site. The invention also encompasses the use of one or more populations of immunity linkers wherein each population has a different first binding site and/or second binding site. The multiple binding sites may differ either in their specificity for different molecules or epitopes or their affinity for the same molecule or epitope. In one embodiment, the immunity linker comprises two or more second binding sites, each specific for a different Clostridium difficile toxin. In an alternative embodiment, the immunity linker comprises two or more second binding sites, each specific for different epitopes on the same Clostridium difficile toxin. In yet another embodiment, the immunity linker comprises two or more second binding sites, each specific for the same epitope on a Clostridium difficile toxin but having different affinities for the Clostridium difficile toxin. In still other or further embodiments, the immunity linker comprises two or more first binding sites, each capable of binding to a different immune response component. In yet another embodiment, the immunity linker comprises two or more first binding sites, each capable of binding to different sub-structures of the same immune response component. In another embodiment, the immunity linker comprises two or more first binding sites, each capable of binding to the same sub-structure of an immune response component but having different affinities for the immune response component.

The immunity linkers of the invention can have any combination of the aforementioned multiple first binding sites and second binding sites. The invention also encompasses the administration of different populations of immunity linkers, each population having any combination of the aforementioned multiple first binding sites and second binding sites.

In one embodiment, a population of immunity linkers is administered to an individual, wherein each linker has an identical first binding site and the second binding sites are all aptamers, that bind to the same Clostridium difficile toxin, but with different affinities for the Clostridium difficile toxin.

The invention contemplates populations of immunity linkers that comprise at least one first binding site described herein. Such populations can have immunity linkers all having first binding sites having the same binding specificity or combinations of binding specificities. Further, the binding may be accomplished by first binding sites of the same type, such as all being nucleic acid molecules or all proteins, which may have the same or different binding specificities. The binding may be accomplished by first binding sites of different types on one immunity linker or a population of different immunity linkers with differing first binding sites. The first binding sites of different types can have the same or different binding specificities for one or more immune response components.

Additionally, the invention contemplates populations of immunity linkers that comprise at least one second binding site described herein. Such compositions comprise immunity linkers all having second binding sites having the same binding specificity or combinations of binding specificities. Further, the binding may be accomplished by second binding sites of the same type, such as all being nucleic acid molecules, i.e. aptamers, which may have the same or different binding specificities. The binding may be accomplished by second binding sites of different types on one immunity linker or a population of different immunity linkers with differing second binding sites. The second binding sites of different types can have the same or different binding specificities for one or more Clostridium difficile toxins.

Thus, the compositions comprise immunity linkers in which the binding specificity of the at least one first binding site and the binding specificity of the at least one second binding sites are all uniform, that is, each first binding site has the same binding specificity for its binding partner and each second binding site has the same binding specificity for its binding partner. Alternatively, the compositions may comprise multiple immunity linker populations each population having first binding sites with differing binding specificities and also having second binding sites with differing binding specificities.

In one embodiment, a pharmaceutical composition is provided which comprises at least one immunity linker molecule wherein the aptamer binds to Clostridium difficile toxin A in combination with at least one immunity linker molecule wherein the aptamer binds to Clostridium difficile toxin B. Such an embodiment is believed to provide a synergistic treatment of Clostridium difficile infection by virtue of combining a targeted therapeutic approach upon both toxin A and toxin B. It will also be appreciated that the pharmaceutical compositions of this embodiment may also be administered in combination with antibiotics as defined herein.

Methods of Use

The invention comprises methods and compositions for diverting a pre-existing immune response in an individual from a first antigen to a second target, i.e. a Clostridium difficile toxin. Since the first antigen, or an immunological equivalent of the first antigen, is present in the linker molecule, the "diverting" of an immune response does not require a cessation of the immune response to the first antigen. The invention further provides methods and compositions for diverting an immune response to a Clostridium difficile toxin in an individual. A previous immune response to the Clostridium difficile toxin may or may not already exist in the individual. The invention also provides chemically programmable immunity for individuals that provide for the immediate and specific immunization of the individual against a Clostridium difficile toxin. According to the invention, the individual is first immunized with a universal immunogen. The individual can then be immediately immunized against a chosen Clostridium difficile toxin simply by administering to the individual a composition comprising an immunity linker with at least one first binding site that binds to an immune response component and a second binding site that binds to a Clostridium difficile toxin. Any combination of universal immunogen and immunity linker described herein can be used with the only requirement that the first binding site of the immunity linker will be bound by some of the immune response components produced as a result of inoculation by the universal immunogen. Immunity to the universal immunogen may occur as a result of an intentional inoculation or, as in the case of the alpha-Gal or L-rhamnose epitope and its attendant anti-Gal and anti-Rha immunity, by natural processes.

The invention may also be particularly useful in elderly populations having been treated with an array of broad spectrum antibiotics during prolonged hospital stays, and developing Clostridium difficile infection and suffering the effects of the toxin. One particular advantage of the invention in this regard is that it is a targeted treatment specific to the pathogen of choice, and while neutralizing the effect of Clostridium difficile, it has no collaterally damaging effect on other commensal bacteria, unlike the broad spectrum antibiotics which often give rise to the Clostridium difficile infection in the first place.

The invention can be used to prevent and/or treat disease or infection from Clostridium difficile. The immunity lasts as long as the individual continues to maintain adequate in vivo concentrations of immunity linkers. In one embodiment, immunity linkers are administered to the individuals on a continuing basis in order to maintain adequate in vivo concentrations of immunity linkers. Immunity linkers can be administered at any interval including, but not limited to, hourly, daily, weekly, or monthly intervals. In the case of immunity linkers that must necessarily be administered for a long period of time, linkers are sought wherein the second binding site is not itself immunogenic. Once the threat is passed, administration of immunity linkers is stopped . Possible side effects of the invention are therefore temporary, unlike traditional immunizations which often generate long- lasting side effects or complications in immunized humans or animals. With regard to the more general population, pharmacies can have a library of different immunity linkers available for a variety of different Clostridium difficile toxins. Once an individual is pre-immunized with a universal immunogen, administration of one or more of these different immunity linkers results in the generation of a protective immune response against the variety of different Clostridium difficile toxins.

In order to maximize the treatment effect, and minimize the need for regular repeated administration, all embodiments of the invention contemplate methods for protecting nucleic acids to confer advantageous properties, such as increased serum stability. Such methods will typically comprise modifications to the aptamers of the invention. Suitable examples of such modifications are exactly as hereinbefore described and in particular include, but are not limited to, alkylated, halogenated, thiolated, dithiolated, aminated, amidated, or acetylated bases, in various combinations. The method of protection, and the amount of serum half-life of intact molecule thus conferred, may be tailored specifically to the treatment environment. The invention further comprises adjunct or combination therapy with existing anti-bacterial treatment. Thus, according to one aspect of the invention there is provided a method of treating a Clostridium difficile infection which comprises administering an immunity linker according to the invention in combination with an anti-bacterial agent. It will be appreciated that this aspect of the invention may not only be used to treat the Clostridium difficile infection per se but also finds particular utility in the treatment indications which are secondary to Clostridium difficile infection, such as infectious diarrhea or Clostridium difficile induced pseudomembranous colitis. It is believed that the combination of the immunity linkers of the invention (particularly those which are able to target a specific Clostridium difficile toxin) will have a synergistic effect upon existing therapies for treatment of Clostridium difficile infection. Such a synergy has already been observed by administering monoclonal antibodies against Clostridium difficile toxins A and B in combination with antibiotics such as vancomycin or metronidazole (Lowy et al. (2010) N Engl J Med 362(3), 197-205; Parks and Gkrania-Klotsas (2010) N Engl J Med 362(15), 1444).

In one embodiment, the anti-bacterial agent is an antibiotic which specifically targets Clostridium difficile. In a further embodiment, the antibiotic is selected from vancomycin or metronidazole.

The invention further comprises methods for removing Clostridium difficile toxins from the body of a human or animal by administering a composition comprising an immunity linker.

In the event of an adverse reaction to the aptamers of the invention, it will be appreciated that an oligonucleotide "antidote" may be administered to an individual suffering an adverse reaction to minimize such adverse reaction or provide the necessary clearance of the aptamer. Such an antidote may typically comprise an unprotected reverse complement to the aptamer sequence to ensure that the antidote hybridizes to the therapeutic aptamer of the invention to result in inactivation or ensure that it is rapidly cleared from the system.

The terms "treatment," "treating," "treat," and the like are used herein to refer generally to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially transferring immunity from one antigen to a Clostridium difficile toxin and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers using the immune response directed to one antigen for the control of Clostridium difficile toxin or its effects such as any treatment of a disease in a subject, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom, but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.

The terms "treatment," "treating," "treat," and the like also include the reduction, control or containment of a Clostridium difficile toxin, in an individual . Reduction of a substance may be determined by any method .

The expression "therapeutically effective amount" refers to an amount of, for example, a composition disclosed herein, that is effective for preventing, ameliorating, treating or delaying the onset of a disease or condition. A "prophylactically effective amount" refers to an amount of, for example, a composition disclosed herein that is effective for preventing a disease or condition. Methods of Administration

According to the invention, a universal immunogen is administered to an individual prior to administration of a corresponding immunity linker. A universal immunogen can be naturally encountered, or deliberately administered at a suitable time prior to administration of a corresponding immunity linker (ideally immediately prior to administration of the immunity linker) and may be administered multiple times prior to administration of a corresponding immunity linker. These multiple administrations may be referred to as "booster" administrations. One method contemplated by the invention comprises multiple administrations of different universal immunogens. With administrations of different universal immunogens, the repertoire of possible immune linkers is increased.

Multiple administrations of immunity linkers are also included in the invention. Methods include immunization of an individual using one universal immunogen followed by one or more administrations of the same or different immunity linkers. Methods also include immunization of an individual using several different universal immunogens followed by one or more administrations of the same or different immunity linkers. It is preferred that immunity linkers are administered to an individual for as long as is needed and at appropriate intervals to maintain adequate in vivo concentrations of the immunity linkers to treat an infection or disease or to remove sufficient amounts of an unwanted material from the individual. Immunity linkers can be administered at any interval including, but not limited to, hourly, daily, weekly, or monthly intervals, or any division thereof.

Appropriate administration intervals can be determined by those of ordinary skill in the art and are based on the identity of the target or pathogen, the amount of target or pathogen detected in the individual, duration of exposure, immune linker pharmacokinetics, characteristics of the individual such as age, weight, gender, etc., and any other relevant factors. The time of administration of immunity linker will need to be empirically determined and could vary with particular Clostridium difficile toxin etc, duration of exposure, linker pharmacokinetics, etc.

The universal immunogens and immunity linkers of the invention are administered to individuals using any appropriate route. Appropriate routes of administration include, but are not limited to, oral, inhalation, parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraoccular, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, subcutaneous, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, transmucosal, intranasal, iontophoretic means, and transdermal means. Differing types of immune response are sometimes triggered by different routes of administration of an antigen, and the preferred route for the particular immune response is known to those skilled in the art. The invention is not limited by the route of administration of the universal immunogen or immunity linker. With regard to the bacteriophage linker molecules and bacteriophage universal immunogens, both can be administered as the purified phage or as a bacterial clone containing it. In a preferred embodiment, a lytic bacteriophage is administered to an individual as a portion of, or contained within, a bacterium. The bacteriophage can be delivered by known administration methods that would allow for an optimum response to the target.

The compositions described herein are also contemplated to include pharmaceutical compositions comprising immunity linkers or universal immunogens and at least one pharmaceutically acceptable excipient such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Pharmaceutically acceptable excipients are preferred. Examples and methods of preparing such sterile solutions are well known in the art and can be found in well known texts such as, but not limited to, REMINGTON'S PHARMACEUTICAL SCIENCES (Gennaro, Ed ., 18th Edition, Mack Publishing Co. (1990)). Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the compound. Pharmaceutical excipients and additives useful in the invention include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates. The pharmaceutical compositions comprising the compounds of the invention can also include a buffer or a pH adjusting agent. Additionally, pharmaceutical compositions of the invention can include polymeric excipients/additives.

The term "adjuvant" as used herein is any substance whose admixture with the universal immunogen increases or otherwise modifies the immune response generated thereby. Any adjuvant system known in the art can be used in the composition of the invention. Such adjuvants include, but are not limited to, Freund's incomplete adjuvant, Freund's complete adjuvant, polydispersed B-(l, 4) linked acetylated mannan ("Acemannan"), Titermax® (polyoxyethylene- polyoxypropylene copolymer adjuvants from CytRx Corporation), modified lipid adjuvants from Chiron Corporation, saponin derivative adjuvants from Cambridge Biotech, killed Bordatella pertussis, the lipopoly saccharide (LPS) of gram-negative bacteria, large polymeric anions such as dextran sulfate, and inorganic gels such as alum, aluminum hydroxide, or aluminum phosphate.

For oral administration, pharmaceutical compositions can be in the form of a tablet or capsule, such as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the immunity linkers; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion and as a bolus, etc. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. The tablets may be optionally coated or scored and may be formulated so as to provide a slow or controlled release of the active ingredient therein. In one embodiment, the immunity linker or universal immunogen is provided by orally administering E. coli infected with a bacteriophage immunity linker or bacteriophage universal immunogen.

In addition, the compositions of the invention may be incorporated into biodegradable polymers allowing for sustained release of the immunity linkers, for example, the polymers being implanted for slow release of the immunity linkers. Biodegradable polymers and their uses are described, for example, in detail in Brem et al., 74 J. NEUROSURG. 441-46 (1991).

Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Formulations suitable for topical administration in the mouth include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the immunity linkers or universal immunogens to be administered in a suitable liquid carrier. The liquid forms may include suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Formulations suitable for vaginal administration may be presented as pessaries, tamports, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

The compositions of the invention may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions (REMINGTON'S PHARMACEUTICAL SCIENCES (A. Osol ed., 16th ed . (1980)). The invention provides stable formulations as well as preserved solutions and formulations containing a preservative as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use, comprising the immunity linker compositions disclosed herein in a pharmaceutically acceptable formulation.

In general, the compositions disclosed herein may be used alone or in concert with therapeutic agents at appropriate dosages defined by routine testing in order to obtain optimal efficacy while minimizing any potential toxicity. The dosage regimen utilizing a composition of the invention may be selected in accordance with a variety of factors including type, species, age, weight, sex, medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular composition or therapeutic agent employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the immunity linker and/or universal immunogen required to prevent, counter, or arrest the progress of the condition.

The dosages of a composition disclosed herein may be adjusted when combined to achieve desired effects. Methods are known in the art for determining effective doses for therapeutic and prophylactic purposes for the disclosed pharmaceutical compositions. More specifically, the pharmaceutical compositions may be administered in a single dose, or a single daily dose or the total daily dosage may be administered in divided doses of two, three, or four times daily. The dosage of the compositions may be varied over a wide range from about 0.0001 to about 1,000 mg per individual or until an effective response is achieved. The range may more particularly be from about 0.001 mg/kg to 10 mg/kg of body weight, about 0.1- 100 mg, about 1.0-50 mg or about 1.0-20 mg, for adults (at about 60 kg). The compositions may be administered on a regimen of about 1 to about 10 times per day, for one or multiple days, or once a week or once a month, or until an effective response is achieved. The pharmaceutical compositions of the invention may be administered at least once a week over the course of several weeks or months. Data obtained from cell culture assays and animal studies may be used in formulating a range of dosages for use in humans. In addition, co-administration or sequential administration of the compositions of the invention and other therapeutic agents may be desirable. A composition described herein can be administered during, before or after administration of any other therapeutic agent. Methods of Production

Immunity linkers can be made in many ways, several of which are described herein and are not to be seen as limiting the methods of making immunity linkers. The universal immunogen, or first binding site, can be physically linked or conjugated, such as with known chemical conjugation methods or molecules, to the at least one second binding site that binds the Clostridium difficile toxin.

In another embodiment, the immunity linker can be produced or manufactured as a single molecule containing the first and second binding sites. The immunity linker may also comprise an organism. In yet another embodiment, the immunity linker consists of two active binding sites connected together by a rigid or flexible spacer such as a double helical region of RNA or DNA. A function of the spacer is to hold the two ends of the linker together, while preventing them from interacting.

The first and second binding sites of the invention may be identified and isolated by any method. Methods for making the nucleic acid aptamers, which form one embodiment of a first and/or second binding site are known in the art and are taught at least in several patents referenced above. In general, the methods comprise making a nucleic acid ligand for the Clostridium difficile toxin. The methods involve selection from a mixture of nucleic acid candidates and step-wise iteration of structural improvement using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. For example, the SELEX method allows for isolation of a single sequence variant in a mixture containing from at least 10¹⁰ to 10¹⁴ sequence variants. Aptamers generated using the SELEX methods or improvements or other methods are then used as the second binding sites for immunity linkers. The aptamers to any target can be generated quickly, linked to the linker portion and the first binding site of the immunity linker, and provided for protection of a population.

EXAMPLES

Example 1: Selection and Characterisation of DNA aptamers against Clostridium difficile Toxin A (Ted A)

The purpose of this study was to identify aptamers that bind to C. difficile TcdA in vitro. Iterative rounds of selection and amplification of ss-DNA aptamers identified 40 potential TcdA aptamers. The binding of the aptamers to TcdA was determined by testing their ability to inhibit TcdA-induced cellular cytotoxicity of RAW 264.7 cells. (A) MATERIALS AND METHODS

(i) Cell lines and reagents

All of the cell lines were obtained from the American Type Culture Collection, Manassas, VA. Cell culture plates and petri dishes were purchased from Fisher Scientific, Pittsburgh, PA. All of the chemicals were purchased from Sigma- Aldrich, St. Louis, MO. Fetal bovine serum (FBS) was obtained from Atlanta Biologicals, Lawrenceville, GA. Clostridium difficile (C. difficile) toxin A (TcdA) was obtained from List Biological Laboratories, Campbell, CA. Random library and primers were synthesized by Sigma-Genosys, The Woodlands, TX or

Integrated DNA Technologies, Coralville, IA. TOPO TA cloning kits were purchased from Invitrogen, Carlesbad, CA. QIAprep Spin Miniprep Kit was obtained from Qiagen, Valencia, CA. XTT Cell Proliferation Kit II was obtained from Roche Applied Science Indianapolis, IN. Aptamers, with each base modified with phosphorothioate, were purchased from SIGMA-Genosys (The Woodlands, TX) and Integrated DNA Technologies (Coralville, IA). HAWP filters (0.45 prn pore density) were purchased from Millipore, Bedford, MA. Tri Reagent was purchased from Fischer Scientific.

(ii) Random library and primers

A 102 base single-strand DNA (ss-DNA) template containing 42 bases of random sequence flanked by defined primer-binding sites 5' ACC CCT GCA GGA TCC TTT GCT GGT ACC NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN AGT ATC GCT AAT CAG TCT AGA GGG CCC CAG AAT 3' (SEQ ID NO: 83) were synthesized . The pool was then amplified via polymerase chain reaction (PCR) using forward primers and 5' biotinylated reverse primers. The pool was immobilized on SA beads and the unbiotinylated strand was stripped from the biotin immobilized strand by mild base (0.3M NaOH) and captured resulting in a primarily ssDNA pool used in the in vitro selection process. (iii) In vitro selection procedure

Iterative rounds of selection and amplification of ss-DNA aptamers were performed as described previously by Vivekananda and Kiel, Laboratory

Investigation, Vol . 86, pp. 610-618, (2006). In brief, to exclude filter binding ss- DNA sequences from the pool, the DNA was passed through a 0.45 prn HAWP filter and washed with an equal volume of binding buffer containing 20 mM Tris- hydrochloric acid (HCL), pH 7.5, 45 mM sodium chloride (NaCI), 3 mM

magnesium chloride, 1 mM EDTA, 1 mM dithiothreitol (DTT). In the present study, ss-DNA pools of 500 pmol for initial rounds and 200 pmol for later rounds were used in the selection process. Single-stranded DNA pools were denatured at 94 ^°C for 3 minutes, and then cooled immediately to 4 ^°C in binding buffer. Selection was performed by incubating ss-DNA pools with equimolar

concentration of TcdA at room temperature for one hour in binding buffer with gentle rotation. After 1 hour, the aptamer-toxin complex was vacuum-filtered over a HAWP filter at 5 psi and washed three times with binding buffer. Single- stranded DNA retained on the filter was eluted with 7 M urea, 100 mM 4- morpholine-ethansulfonic acid (MES), pH 5.5 and 3 mM EDTA by boiling. Eluted ss-DNA was then precipitated with an equal volume of isopropyl alcohol.

Selected ss-DNAs were amplified by PCR and used for the next round of selection. After round 10, the pool was amplified by PCR and the product was cloned using a TOPO TA Cloning Kit. The cloned sequences were transformed into One Shot® TOP10 chemically competent E. coli. Approximately fifty colonies from the TcdA pool were picked randomly, PCR was performed to verify aptamer insert (Figure 1), and the plasmid DNA was purified by DNA mini-prep kit and sequenced (Beckman Coulter, Danvers, MA). A total of 59 TcdA unique sequences were obtained and 40 TcdA aptamers were selected for the in vitro neutralization study.

(iv) In vitro cytotoxicity assay

To test the cytotoxicity of TcdA, RAW 264.7 (mouse macrophage) cells were seeded into 24 well plates at 1.0x10^s cells per well and grown to 80%

confluence. These cell lines were cultured in Dulbecco's modified Eagles's Medium (DMEM), supplemented with 100 units/ml penicillin and streptomycin and 10 % FBS for the duration of the experiment. The cells were maintained at 37 °C in a humidified incubator with 5% C0₂ and 95 % air. To determine the lethal dose of 50 % (LD₅₀), 200 μΙ media alone or increasing concentrations of TcdA from 0 through 1000 ng/ml in media was added to the cells. TcdA is 280 kDa and thus lOOOng/ml is a molar concentration of 3.57nM . To assess cell viability, the XTT Cell Proliferation Kit II protocol was performed 24 hours post challenge by combining 56 μΙ_ of electron coupling reagent with 2.8 ml_ of XTT labeling reagent for each 24 well plate. A volume of 100 μΙ_ was then added to each well . The amount of soluble formazan salt produced by the cleavage of the tetrazolium salt XTT by viable cells was measured using the Synergy HT microplate reader from BIO-TEK (Winooski, VT) at 450 nm. The percent viability for each group was determined by dividing the absorbance of the treated cells by the average of the untreated media control absorbance. Multiple experiments were combined and the average ± standard error of mean was graphed. The standard error of the mean was calculated by dividing the standard deviation of the samples by the square root of the number of samples. Significant differences in cell viability were determined using the Student's t test function of Microsoft Excel.

(v) In vitro TcdA neutralization assay by aptamer

For neutralization assays, the aptamers listed above were synthesized and the backbone was fully mono-phosphorothioated to enhance nuclease resistance. Only the ~42mer oligonucleotides were synthesized corresponding to the random region and did not include the original primer sequences from the selected pool. To analyze the ability of aptamers to inhibit the cytotoxic effects of TcdA, phosphorothioate modified aptamers at molar excess of 500X, 1000X, 2500X and 5000X and TcdA at 500ng/pl (1.78nM) in 200μΙ media was added directly to RAW 264.7 cells. The XTT Cell Proliferation assay was utilized as described above to determine percent viability compared to untreated media control.

(vi) Effect of Aptamer on RAW 264.7 Cell Viability

To analyze the effect of aptamer alone on the viability of RAW 264.7 cells, phosphorothioate modified aptamers were added to the media and 200 μΙ_ of the solution was added directly to RAW 264.7 cells as previously described in section (v). Two aptamers, CDA-35 and CDA-53, were tested at 500 and 1000 fold (X) the concentrations as previously described in section (v). The XTT Cell

Proliferation assay was utilized as described above to determine percent viability compared to untreated media control.

(B) RESULTS AND DISCUSSION

(i) TcdA In Vitro Cytotoxicity Assay

Values from six independent RAW 264.7 cytotoxicity experiments were combined and the average ± SEM were graphed (Figure 2). Significant decreases in cell viability were detected with exposure of RAW 264.7 cells to as little as 1 ng/ml TcdA. An average 50% (range 32%-71%) reduction in cell viability was obtained following exposure of the RAW 264.7 cells to 500 ng/ml TcdA.

(ii) In vitro Aptamer Characterization

In vitro Aptamer Neutralization of TcdA

Forty TcdA aptamers were initially tested for the ability to neutralize the effect of TcdA on Raw 264.7 cells. Of the 40 aptamers tested, 24 provided at least a 15% increase in viability (when compared to TcdA-treated cells alone). Additional aptamer neutralization assays were completed on these 24 aptamers. Eight aptamers that most effectively and consistently neutralized the effect of TcdA at the 500X and 1000X aptamer molar concentrations are described below.

Six independent experiments confirmed that CDA-33 significantly inhibited the cytotoxic effect of TcdA with CDA-33 molar concentrations of 500X, 1000X, 2500X and 5000X (Figure 3E; * = P < 0.05). Analyses (n=4) indicated that

CDA-34 also significantly neutralized the cytotoxic effect of TcdA with 500X and 1000X molar excess of CDA-34 (Figure 3F). Three independent experiments indicated that CDA-37 significantly inhibited the cytotoxic effect of TcdA at 500X, 1000X and 2500X molar concentrations of CDA-37 (Figure 3G). Four

independent experiments confirmed that CDA-53 significantly inhibited the cytotoxic effect of TcdA with CDA-53 molar concentrations of 500X, 1000X, 2500X and 5000X (Figure 3H). Four independent experiments were performed to determine the inhibitory effect of CDA-19 on TcdA cytotoxicity (Figure 3A). All concentrations of CDA-19 inhibited the cytotoxic effects of TcdA, in particular the 500X molar excess of CDA-19 attained significance (P = 0.049; * in Figure 3A). Analysis of aptamer CDA-21 (n = 3) provided a small increase in cell viability (Figure 3B).

Analyses (n=4) indicated CDA-23 effectively neutralized the toxic effect of TcdA (Figure 3C), with significant increases in cell viability identified with 500X, 1000X and 2500X molar concentrations of CDA-23. Analysis of aptamer CDA-29 (n = 5) provided increases in cell viability (Figure 3D).

(iii) In Vitro Aptamer Effect on Cell Proliferation

RAW 264.7 cells were treated with 500X and 1000X concentrations of

phosphorothiolate-modified CDA-34 or CDA-53 without toxin. Cell viability was determined using the XTT viability assayed as described above. No significant changes in cell viability were detected (Figure 4); a slight decrease in cell viability was detected at 1000X concentrations of CDA-53.

(C) CONCLUSIONS

The following conclusions can be made based on the data presented in this analysis:

1. The SELEX method was used to identify 40 potential aptamers against

TcdA.

2. Neutralization assays were used to identify those aptamers that exhibited > 15% increase in cell viability of RAW 264.7 cells treated with TcdA, and eight aptamers were selected for additional neutralization assays.

3. Assays to determine the effect of the selected aptamers on the cytotoxicity of TcdA indicated that six aptamers (CDA-33, CDA-34, CDA-37, CDA-53, CDA-19 and CDA-23) consistently and significantly increased the cell viability of RAW 264.7 cells treated with TcdA, suggesting these aptamers were capable of neutralizing the cytotoxic effects of TcdA.

4. Treatment of RAW 264.7 cells with the phosphorothiolate-modified

aptamers alone (CDA-34 or CDA-53) did not significantly change the viability of the RAW 264.7 cells compared to cells treated with media alone. Example 2: Selection and Characterisation of Highly Neutralising

DNA aptamers against Clostridium difficile Toxin B (TcdB)

The purpose of this study was to identify aptamers that bind to C. difficile TcdB in vitro. Iterative rounds of selection and amplification of ss-DNA aptamers identified 43 potential TcdB aptamers. RAW 264.7 cells were selected to assay the effect of the aptamers on TcdB induced cell cytotoxicity.

(A) MATERIALS AND METHODS

(i) Cell lines and reagents

The cell lines and reagents used in this study may be found in the methodology of Example 1. (ii) Random library and primers

The random library and primers used in this study may be found in the

methodology of Example 1.

(iii) In vitro selection procedure

The in vitro selection procedure was conducted in an analogous procedure to that described in Example 1. Approximately fifty colonies in total from the TcdB pool were picked randomly, PCR was performed to verify aptamer insert (Figure 5), and the plasmid DNA was purified by DNA mini-prep kit and sequenced (Beckman Coulter, Danvers, MA). A total of 45 TcdB unique sequences were obtained and 42 TcdB aptamers were selected for the in vitro neutralization study.

(iv) In vitro TcdB cytotoxicity assay

To test the cytotoxicity of TcdB, RAW 264.7 (mouse macrophage) cells were seeded into 24 well plates at 1.0x10^s cells per well and grown to 80% confluence. These cell lines were cultured in Dulbecco's modified Eagles's Medium (DMEM), supplemented with 100 units/ml penicillin and streptomycin and 10 % FBS for the duration of the experiment. The cells were maintained in a humidified incubator with 5% C0₂ and 95 % air at 37 °C. To determine lethal dose of 50 % (LD₅₀), media alone or TcdB in media was added to the cells at varying concentrations in a volume of 200 μΙ . To assess cell viability, the XTT Cell Proliferation Kit II protocol was performed 24 hours post challenge by combining 56 μΙ_ of electron coupling reagent with 2.8 ml_ of XTT labeling reagent for each 24 well plate. The amount of soluble formazan salt produced by the cleavage of the tetrazolium salt XTT by viable cells was measured using the Synergy HT microplate reader from BIO-TEK (Winooski, VT) at 450 nm. The percent viability for each group was determined by dividing the absorbance of the treated cells by the average of the untreated media control absorbance. Multiple experiments were combined and the average ± standard error of mean was graphed. The standard error of the mean was calculated by dividing the standard deviation by the square root of the number of samples. Significant differences in cell viability were determined using the Student's t test function of Microsoft Excel.

(v) In vitro TcdB aptamer neutralization assay

To analyze the neutralization activity of aptamers against the cytotoxic effects of TcdB, phosphorothioate modified aptamers were synthesised as described in Example 1 and TcdB (280 kDa) were added to DMEM and 200 μΙ_ of the solution was added directly to RAW 264.7 cells. The aptamers were tested at 500, 1000, 2500 and 5000 fold molar excess of the corresponding toxin. The XTT Cell Proliferation assay was utilized as described above to determine percent viability compared to untreated media control.

(vi) Effect of TcdB specific Aptamers on RAW 264.7 Cell Viability

To analyze the effect of aptamer alone on the viability of RAW 264.7 cells, phosphorothioate modified aptamers were added to the media and 200 μΙ_ of the solution was added directly to RAW 264.7 cells. Two aptamers, CDB-5 and CDB- 46, were tested at 500 and 1000 fold (X) molar excess of the corresponding toxin. The XTT Cell Proliferation assay was utilized as described above to determine percent viability compared to untreated media control.

(B) RESULTS AND DISCUSSION (i) TcdB In Vitro Cytotoxicity Assay

Three independent experiments were performed to determine the effect of TcdB on the viability of RAW 264.7 cells, and the average ± SEM were graphed (Figure 6). A concentration of TcdB of 750 ng/ml (64%) exhibited a trend toward decreased viability (P = 0.07). This concentration of TcdB was therefore used for the in vitro characterisation of aptamers described below.

(ii) In vitro Characterization of Aptamers

In vitro Aptamer Neutralization of TcdB.

Forty-three TcdB aptamers (CDB-1 through CDB-49) were initially tested for the ability to neutralize the effect of TcdB on Raw 264.7 cells. Of these 43 aptamers tested, 21 provided at least a 15% increase in viability in the initial analysis. A single aptamer from two repetitive sequences (CDB-12/CDB-30 and CDB- 17/CDB-28) was analyzed . CDB-12 significantly inhibited TcdB cytotoxicity.

Additional aptamer neutralization assays were completed on the 21 aptamers that initially inhibited the cytotoxic effect of TcdB by > 15%. Five aptamers that most effectively and consistently neutralized the effect of TcdB at the 500X and 1000X aptamer molar concentrations are described below. Five independent experiments were performed to determine the inhibitory effect of aptamer CDB-4 on TcdB cytotoxicity (Figure 7A). All concentrations of aptamer CDB-4, except 5000X, inhibited the cytotoxic effects of TcdB. Although inhibition of the cytotoxic effects of TcdB failed to attain significance, 500X, 1000X and 2500X trended toward significance (P = 0.06, 0.08, and 0.06, respectively).

Experiments indicated that the 500X concentration of CDB-5 (n = 3, Figure 7B) significantly inhibited the cytotoxic effects of TcdB (P = 0.01); both the 1000X and 2500X concentrations trended toward significance. Both the 500X and 2500X concentrations of CDB-12 (n = 6) significantly inhibited the cytotoxic effects of TcdB (P = 0.006 and 0.002, respectively; Figure 7C); the 1000X concentration of CDB-12 trended toward significance (P = 0.06). Four independent experiments indicated that the 500X molar concentration of aptamer CDB-34 (Figure 7D) significantly inhibited the cytotoxic effects of TcdB. Experiments (n = 5) also indicated that both the 500X and 1000X molar concentrations of aptamer CDB-46 (Figure 7E) significantly inhibited the cytotoxic effects of TcdB. (iii) In Vitro Aptamer Effect on Cell Proliferation

To verify the aptamers were not toxic alone, RAW 264.7 cells were treated with 500X and 1000X concentrations of phosphorothiolate-modified CDB-5 or CDB- 46. Cell viability was determined using the XTT viability assayed as described above. There was no toxicity associated with treatment of RAW 264.7 cells with aptamer alone (Figure 8).

(C) CONCLUSIONS

The following conclusions can be made based on the data presented in this study:

(i) The SELEX method was used to identify 43 potential aptamers against TcdB.

(ii) Neutralization assays were used to identify those aptamers that exhibited > 15% increase in cell viability of RAW 264.7 cells treated with TcdB, and five aptamers were selected for additional neutralization assays.

(iii) Four aptamers consistently and significantly increased the cell viability of RAW 264.7 cells treated with TcdB (CDB-5, CDB-12, CDB-34 and CDB-46), suggesting these aptamers were capable of neutralizing the cytotoxic effects of TcdB. A fifth aptamer (CDB-4) also increased cell viability of RAW 264.7 cells treated with TcdB.

(iv) The phosphorothiolate-modified aptamers alone did not exhibit any cytotoxic effects on RAW 264.7 cells at the concentrations tested .

Example 3: Neutralizing Activity of L-Rhamnose Conjugated

Aptamers against Clostridium difficile Toxin A and B (TcdA and TcdB)

The purpose of this study was to demonstrate the neutralizing capabilities of L- rhamnose conjugated, C. difficile toxin specific aptamers to culture supernatants from C. difficile strains that were knocked out for TcdA or TcdB in the presence of human serum.

(A) MATERIALS AND METHODS

(i) Cell culture medium

Unless stated otherwise, all tissue culture reagents were purchased from Sigma. Vero cells were cultured in DMEM - high glucose with 10% vol/vol fetal bovine serum and 5% vol/vol penicillin-streptomycin solution. Cells were cultured at 37°C with 5% C0₂ to a confluence level of 90%.

(ii) Synthesis of L-rhamnose conjugated aptamers

Aptamers were synthesized by Biosearch Technologies (Novato, CA) with a 5' amine and fully monophosphorothioate backbone. Carboxyl (COOH)

functionalized L-rhamnose was conjugated at the 5' of the aptamer post synthesis via standard carboxy-amine coupling . The aptamers were purified by HPLC and the conjugation was verified by LC-MS. L-rhamnose conjugated aptamers were dissolved in water to ΙΟΟμΜ for use.

(iii) Clostridium difficile strains and preparation of toxin supernatant

Clostridium difficile mutant TcdA and TcdB expressing strains, 630Aerm A+B-, and 630Aerm A-B+ (respectively) have been described previously (Kuehne et al. 2010, Nature 467(7316), 711-713). These were used to generate the toxin supernatants following the ClosTron method (Heap et a/. 2007, J Microbiol Methods 70(3), 452-464; Heap et a/. 2010, J Microbiol Methods 80(1), 49-55); they were grown for 72 hours in RPMI 1640 containing 0.1% wt/vol L-Cysteine and 2% wt/vol Bacto Casamino Acids (Beckton Dickinson, Cat. No : 223050). These culture supernatants were harvested, titred for the presence of toxin and used in the subsequent assays as the source of TcdB and TcdB toxin.

(iv) Hamster Anti-L-Rhamnose sera

Anti-rhamnose sera was obtained from hamsters following subcutaneous immunization using Freund's complete adjuvant and 30ug Ovalbumin-L rhamnose conjugate on 4 separate occasions, as previously described (Chen et al. 2011, ACS Chem Biol 6(2), 185-191). The anti-rhamnose titre of immunized animals was determined using an ELISA-based assay, as described previously (Chen et a/. 2011, supra).

(v) Neutralization assay

Vero cells were seeded into a 96 well plate in ΙΟΟμΙ volumes (lxlO⁵ cells/well) and incubated at 37°C with 5% C0₂, for 48 hours to allow the cells to reach a confluence level of 90%. To test efficacy of L-rhamnose conjugated aptamers, 20μΙ 1 :4 dilution of toxin supernatant, 20μΙ at lOOuM aptamer (CDA-34T with A+B- and CDB-05T with A-B+) and 20μΙ PBS were mixed. The aptamer concentration was 33μΜ. To test efficacy of the rhamnose-conjugated aptamers in the presence of anti-rhamnose antiserum, 20μΙ of aptamer was mixed with 20μΙ of the relevant supernatant diluted 1 :4 in PBS and 20μΙ of 1/50 or 1/100 dilution of either the serum from a hamster with a high titre (1/6400 serum dilution) of anti-rhamnose antibody or the serum from a low titre (1/100 serum dilution) animal. Controls containing 20μΙ 1 : 4 dilution of toxin supernatant and 40μΙ PBS

(Positive control), or 20μΙ RPMI medium diluted 1 :4 with PBS and 40μΙ PBS (negative control) were included in each assay. 20μΙ of each mixture was then added to triplicate microtitre wells containing the Vero cells. The plates were incubated overnight and cells were viewed by light microscopy and neutralization scored by eye, as described previously by N. Minton and co-workers (Kuehne et a\. 2010, supra).

(B) RESULTS AND DISCUSSION

The results of the in vitro analysis using conjugated aptamers and hamster anti- rhamnose sera are shown in Table 1 :

Table 1: Neutralising Activity of Rhamnose Conjugated Aptamers

Note : N/T = not tested = full toxicity observed, no signs of neutralization as observed by cells displaying uniform rounded morphology (Figure 9).

-+ = signs of neutralisation observed, the cells are not all showing the uniform rounding seen with full toxicity (Figure 10). - + + = greater signs of neutralisation observed, fewer cells showing a less round appearance.

Serum 1 - taken from a hamster with low anti-rhamnose titre

Serum 2 - taken from a hamster with high anti-rhamnose titre.

Sera were tested twice at a dilution of 1/100 and a third time at a dilution of 1/50 with similar results.

The results shown in Table 1 demonstrate that in the absence of anti-rhamnose serum, the L-rhamnose conjugated TcdA and TcdB-specific aptamers

demonstrate low toxin neutralization activity. The presence of serum in the assay increases the neutralizing efficacy of the aptamers and the extent of neutralization appears to correlate with the anti-rhamnose titre of the sera.

(C) CONCLUSIONS

1. There is low toxin neutralization from aptamers alone without anti-L- rhamnose antibodies and immune cells present in hamster serum.

2. The presence of serum increases the neutralizing efficacy of the aptamers.

Claims

1. An immunity linker molecule comprising at least one first binding site that binds to an immune system molecule and at least one second binding site that is an aptamer which binds to Clostridium difficile or a Clostridium difficile toxin.

2. An immunity linker molecule as defined in claim 1, wherein the aptamer binds to a Clostridium difficile toxin.

3. The immunity linker molecule as defined in claim 1 or claim 2, wherein the Clostridium difficile toxin is toxin A.

4. The immunity linker molecule as defined in claim 3, wherein the aptamer comprises any one of the following aptamer sequences: CDA-19 (SEQ ID NO: 1), CDA-21 (SEQ ID NO: 2), CDA-23 (SEQ ID NO: 3), CDA-29 (SEQ ID NO: 4), CDA-33 (SEQ ID NO: 5), CDA-34 (SEQ ID NO: 6), CDA-37 (SEQ ID NO: 7) or CDA-53 (SEQ ID NO: 8), such as CDA-34 (SEQ ID NO: 6).

5. The immunity linker molecule as defined in claim 1 or claim 2, wherein the Clostridium difficile toxin is toxin B.

6. The immunity linker molecule as defined in claim 5, wherein the aptamer comprises any one of the following aptamer sequences: CDB-4 (SEQ ID NO: 9), CDB-5 (SEQ ID NO: 10), CDB-12 (SEQ ID NO: 11), CDB-34 (SEQ ID NO: 12) or CDB-46 (SEQ ID NO: 13), such as CDB-5 (SEQ ID NO: 10).

7. The immunity linker molecule as defined in any one of claims 1 to 6, wherein the first site binds to an antibody.

8. The immunity linker molecule as defined in claim 7 wherein the antibody is from a human or animal that has been preimmunized against the first site.

9. A pharmaceutical composition comprising an immunity linker molecule as defined in any one of claims 1 to 8 and one or more pharmaceutically acceptable excipients.

10. The pharmaceutical composition as defined in claim 9, which comprises at least one immunity linker molecule wherein the aptamer binds to Clostridium difficile toxin A in combination with at least one immunity linker molecule wherein the aptamer binds to Clostridium difficile toxin B.

11. A method of prophylaxis and/or treatment of disease or infection from Clostridium difficile which comprises administering to a subject in need thereof an immunity linker molecule as defined in any one of claims 1 to 8 or a pharmaceutical composition as defined in claim 9 or claim 10.

12. The method as defined in claim 11, which additionally comprises administration of an antibacterial agent, such as an antibiotic, in particular vancomycin or metronidazole.

13. A method of immunizing a human or animal against Clostridium difficile or a Clostridium difficile toxin comprising administering to the human or animal an immunity linker molecule, the immunity linker molecule containing at least one first site that binds to an immune system molecule, and a second site that is an aptamer which binds to Clostridium difficile or the Clostridium difficile toxin.

14. The method of claim 13, wherein the human or animal has been preimmunized against the first site on the immunity linker molecule.

15. A method of diverting an immune response to Clostridium difficile or a Clostridium difficile toxin in an individual comprising, administering to the individual an effective amount of a composition comprising one or more immunity linkers, wherein the linker molecules comprise at least one first binding site and at least one second binding site, wherein the individual has a pre-existing immune response to the first binding site, and wherein the first binding site binds to an immune system molecule and the second binding site is an aptamer which binds to Clostridium difficile or the Clostridium difficile toxin.

16. The method of any one of claims 11 to 15, wherein the individual is a human and the first binding site comprises an alpha-galactosyl epitope.

17. The method of any one of claims 11 to 16, wherein the immune system molecule is an antibody.

18. The method as defined in any one of claims 11 to 17, wherein the aptamer binds to a Clostridium difficile toxin.

19. The method as defined in any one of claims 11 to 18, wherein the Clostridium difficile toxin is toxin A.

20. The method as defined in claim 19, wherein the aptamer comprises any one of the following aptamer sequences: CDA-19 (SEQ ID NO: 1), CDA-21 (SEQ ID NO: 2), CDA-23 (SEQ ID NO: 3), CDA-29 (SEQ ID NO: 4), CDA-33 (SEQ ID NO: 5), CDA-34 (SEQ ID NO: 6), CDA-37 (SEQ ID NO: 7) or CDA-53 (SEQ ID NO: 8).

21. The method as defined in any one of claims 11 to 18, wherein the Clostridium difficile toxin is toxin B.

22. The method as defined in claim 21, wherein the aptamer comprises any one of the following aptamer sequences: CDB-4 (SEQ ID NO: 9), CDB-5 (SEQ ID NO: 10), CDB-12 (SEQ ID NO: 11), CDB-34 (SEQ ID NO: 12) or CDB-46 (SEQ ID NO: 13).