WO2017121494A1

WO2017121494A1 - 5'-triphosphated short immunostimulatory nucleotides, oligonucleotides and polynucleotides

Info

Publication number: WO2017121494A1
Application number: PCT/EP2016/050846
Authority: WO
Inventors: Jacques Rohayem
Original assignee: Riboxx Gmbh
Priority date: 2016-01-15
Filing date: 2016-01-15
Publication date: 2017-07-20

Abstract

The present invention relates to the use of 5'-triphosphated nucleotides, oligonucleotides and polynucleotides forming G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures under appropriate conditions for immunostimulation as well as for treating infectious, autoimmune and tumor diseases.

Description

5'-Triphosphated short immunostimulatory nucleotides,

oligonucleotides and polynucleotides

EP-A-1 920 775 (Hartmann et al.) discloses that 5'-triphosphated RNA oligonucleotides being at least 12 nucleotides in length exert induction of interferon (IFN) through RIG-I stimulation. In a later article, the same group published studies according to which efficient RIG-I stimulation is dependent on a 5'-triphosphate attached to double stranded RNA (Schlee et al. (2009) Immunity 31 (1 ), pp. 4-5).

Co-pending International Patent Application No. PCT/EP2012/071641 describes double stranded RNA having a 5'-triphosphate on at least one 5'-end and having multiple G/C pairs at both ends of the double stranded molecule.

Co-pending International Patent Application No. PCT/EP2015/050519 discloses G- quadruplex-forming nucleotides, oligonucleotide and polynucleotides especially for use as delivery-vehicles for introducing cargo molecules into cells. The technical problem underlying the present invention is to provide improved, in particular short, nucleic acid molecules for activation of innate and specific immunity, in particular for immunostimulation and therapy/treatment of infectious, autoimmune and tumor diseases.

The solution to the above technical problem is provided by the embodiments of the present invention as described herein and in the claims. In particular, the present invention provides the use of 5'-triphosphated nucleotides, oligonucleotides and polynucleotides forming G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures (i.e., in general, intermolecular or intramolecular complexes of or within one or more of such 5'-triphosphated nucleotides, oligonucleotides or polynucleotides, typically based on interactions other than Watson-Crick base pairing) under appropriate conditions, for immuostimulation in a subject, preferably in a mammalian subject, more preferably in a human subject. In the context of the present invention the term "forming G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures under appropriate conditions" relates to assemblies of said nucleotides (preferably G, C, U, T, or A or analogues thereof), oligonucleotides or polynucleotides through inter and/or intramolecular interactions between and/or within said species under permissive conditions including buffer, salt components, temperature etc., more preferably under physiological conditions. The conditions required for the formation of said G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures are generally known in the art. Reference is given in this context, e.g. to

Haider et al. "RNA Quadruplexes" in Sigel (ed.): Structural and catalytic roles of metal ions in RNA, RSC Publishing, Cambridge, 201 1 , pp. 125-139) and also to Neidle and

Balasubramanian (eds.): Quadruplex Nucleic Acids, RSC Publishing, 2005. Various sequence species for use in the present invention allow for the formation of such assemblies (see the above reference to Neidle and Balasubramanian (eds.) : Quadruplex Nucleic Acids, RSC Publishing, 2005). Preferred embodiments of the invention are 5'- triphosphated oligonucleotides or polynucleotides that contain or have a sequence of the following formula (I): nGpXm (I) wherein X_n and X_m is any nucleotide sequence of length n or m, respectively, preferably being m = n, and G_p is any number of guanines of length p involved in formation of G- quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures and/or tetrads.

Further preferred embodiments of 5'-triphosphated species of use in the present invention are represented by sequences containing two guanine repeats separated by non-guanine nucleotides, for example 5'-triphosphated oligonucleotides or polynucleotides containing or having a sequence of the following formula (II): X_nGolXpG₀2Xm (II) wherein X_n and X_m is any non-guanine nucleotide sequence of length n or m, respectively, preferably being n=m, G_o1 and G₀2 is any number of guanines of length o1 and o2, respectively, involved in formation of G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures and/or tetrads, preferably being o1 = o2, and X_p is any nucleotide sequence of length p involved in loop formation.

Further preferred embodiments of 5'-triphosphated species of use in the present invention are represented by sequences forming intramolecular G-quadruplexes containing or having a sequence of the following formula (III):

X_nGolXplGo2X 2Go3X 3G_o4Xm (ill) wherein X_n and X_m is any non-guanine nucleotide sequence of length n or m, respectively, preferably being n=m, G₀i , G_o2, G_o3 and G_o4 of length o1 , o2, o3 and o4, respectively, are independently any number of guanines involved in formation of G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures and/or tetrads, preferably being o1 = o2 = o3 = o4, and X_pi , X_p2 and X_p3 are indepedently any nucleotide sequence of length p1 , p2 and p3, respectively, involved in loop formation, preferably being p1 =p2=p3.

According to the invention, not only 5'-triphosphated species comprising four G-repeats (present in one molecule as is the case for above formula (III) or provided by more than one molecule) but any number of G-repeats can be included into a run of guanines and fold into intramolecular and/or intermolecular structures and/or suprastructures and/or tetrads and/or quadruplexes.

Further species for use in the present invention are 5'-triphonsphated species forming self- association of Watson-Crick duplex sequences, G:C tetrads formed, e.g. from (CCC)n:CCG repeats or other tetrad forming sequences such as the human quadruplex structures formed from sequences such as (TAGGGTTAGGGT)₂ (SEQ ID NO: 1 ) repeats and GAGCCAGT.

It is to be understood that, according to the invention, a "5'-triphosphated nucleotide oligonucleotide or polynucleotide" as defined herein has a free (i.e. uncapped) triphosphate group covalently bound to the 5' position of the (or at least one, in case of double stranded molecules) 5'-terminal ribose or deoxyribose, respectively.. In general, the length of the oligonucleotides or polynucleotides as described herein is not critical. Especially preferred 5'-triphosphated nucleic acids, in particular ribonucleic acids, as disclosed herein such as those of the above and the below formulas have a length of up to 1 1 nucleotides. In view of the teaching of the prior art (see EP-A-1 920 775) it is very surprising that oligonucleotides of the invention having less than 12, in particular 2 to 1 1 , i.e. having a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1 , more preferably (ribo)nucleotides, exert a strong activation of immune cells as evidenced by the secretion of TNF-a and/or IL-6.

Without being bound to any specific theory, it is envisaged that the preferred small 5'- triphosphated species of the invention can exert their effect, in particular on effectors of innate immunity such as, but not limited to, retinoic acid inducible gene I (RIG-I) and/or Tolllike Receptor 3 (TLR-3), Toll-like Receptor 7 (TLR-7) and/or melanoma differentiation antigen 5 (MDA-5), since they adopt a larger volume than one would expect for the sequence of the oligonucleotides per se.

Further preferred species of use in the present invention are 5'-triphosphated poly and oligonucleotides, respectively, having the following formula (IV)

PPP(G)_n (IV) wherein n is an integer of at least 2 or at least 3, preferably 2 or 3 to 40, more preferably 2 or 3 to 20, and ppp is a free 5'-triphosphate group. In the following, the molecules of the invention according to formula (I) will be referred to as "oligonucleotides", independent of their length.

The person skilled in the art understands that sequences of formulas (IV), (III) and (II) form sub-groups of the species according to formula (I).

It has been shown according to the invention that oligonucleotides and compositions thereof as used herein exert at least an equal, preferably substantially stronger (e.g. at least twofold, preferably, at least threefold, more preferably at least fourfold) activation of macrophages and dendritic cells (as measured by TNF-a) in comparison to polyinosinic-polycytidylic acid (poly(l:C)) which is still the standard for immunostimulation experiments and therapy. In preferred oligonucleotides of the invention, n in the above formula (IV) is 2, 3, 4, 5 or 6 to 14, particularly preferred 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1 . Further preferred oligonucleotides for use in the invention are those in which n in the above formula (IV) is 6 to 14, most preferred 6 to 1 1 .

As noted above, according to particularly preferred embodiments of the present invention, the nucleotides, poly or oligonucleotides as defined herein form G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures under appropriate conditions, (including buffer, salt components, temperature; see, e.g. the review by Haider et al. "RNA Quadruplexes" in Sigel (ed.): Structural and catalytic roles of metal ions in RNA, Royal Society of Chemistry, Cambridge, 201 1 , pp. 125-139).

Particularly preferred oligonucleotides and polynucleotides for use in the present invention are single stranded and at least comprise ribonucleotides. Mixed ribonucleic

acid/deoxyribonucleic acid (RNA DNA) species are contemplated as well, but especially preferred oligonucleotides are RNA oligonucleotides.

According to further preferred embodiments of the invention compositions of at least two different nucleotides, oligonucleotides or polynucleotides as defined above, more preferably of oligonucleotides according to above formula (I) are used as taught herein. Preferred compositions contain at least two different oligonucleotides selected from the group consisting of ppp(G)₆ , ppp(G)₇, ppp(G)₈, ppp(G)₉, ppp(G)i₀ (SEQ ID NO: 2), ppp(G)n (SEQ ID NO: 3), ppp(G)i2 (SEQ ID NO: 4), ppp(G)i₃ (SEQ ID NO: 5) and ppp(G)i₄ (SEQ ID NO: 6). It is to be understood that all possible combinations of at least two of the above

oligonucleotides may be used according to the present invention. More preferably, the oligonucleotides in such compositions are, or, in other embodiments, comprise ppp(G)₈, ppp(G)₉ and ppp(G)i₀ (SEQ ID NO: 2). In other preferred embodiments, the compositions contain, as oligonucleotide species (or at least as the majority of oligonucleotide species) ppp(G)₆, ppp(G)₇, ppp(G)₈, ppp(G)₉, ppp(G)i₀ (SEQ ID NO: 2), ppp(G)n (SEQ ID NO: 3), ppp(G)i2 (SEQ ID NO: 4), ppp(G)i₃ (SEQ ID NO: 5) and ppp(G)i₄ (SEQ ID NO: 6). According to certain embodiments of the invention the nucleotides, oligonucleotides or polynucleotides are preferably free of chemical modifications or other nucleotide analogues.

In further embodiments of the invention, however, the nucleotides, oligonucleotides or polynucleotides of the present invention can comprise at least one chemically modified and/or labeled nucleotide and/or other nucleotide analogues such as inosine. The chemical modification of the nucleotide analogue in comparison to the natural occurring nucleotide may be at the ribose, phosphate, and/or base moiety. With respect to molecules having an increased stability, especially with respect to RNA degrading enzymes, modifications at the backbone, i.e. the ribose and/or phosphate moieties, are especially preferred.

Preferred examples of ribose-modified ribonucleotides are analogues wherein the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH₂, NHR, NR₂, or CN with R being CrC₆ alkyl, alkenyl or alkynyl and halo being F, CI, Br or I. It is clear for the person skilled in the art that the term "modified ribonucleotide" also includes 2'- deoxyderivatives, such as 2'-0-methyl derivatives, which may at several instances also be termed "deoxynucleotides".

As mentioned before, the at least one modified ribonucleotide may be selected from analogues having a chemical modification at the base moiety. Examples of such analogues include, but are not limited to, 5-aminoallyl-uridine, 6-aza-uridine, 8-aza-adenosine, 5-bromo- uridine, 7-deaza-adenine, 7-deaza-guanine, N⁶-methyl-adenine, 5-methyl-cytidine, pseudo- uridine, and 4-thio-uridine. Examples of backbone-modified ribonucleotides, wherein the phosphoester group between adjacent ribonucleotides is modified, are phosphothioate groups.

The "label" that may be included in the nucleotide analogue can be any chemical entity which enables the detection of the oligonucleotide in question via physical, chemical, and all biological means. Examples of typical labels linked to or integrated into one or more of the nucleotides or added to one end of the molecules as disclosed herein are radioactive labels, chromophores, and fluorophores (e. g. fluorescein, TAM etc.).

In general, the 5'-triphosphated nucleotides, oligonucleotides and polynucleotides of the invention may be prepared chemically (see, e.g., Zlatev et al. (2010) Org. Lett. 12 (10), pp. 2190-2193).

In other embodiments, oligonucleotides and polynucleotides for use in the present invention may be prepared by chemical synthesis methods or enzymatically, or by a combination of chemical and enzymatic steps. Methods for the enzymatic preparation of the oligonucleotides are preferably those that use RNA-dependent RNA polymerases (RdRps) of caliciviruses as disclosed in WO-A-2007/012329. With respect to enzymatic synthesis of chemically modified RNAs using such RdRps the methods referred to in WO-A-2009/150156 are preferred.

Enzymes of this type are also capable of using DNA templates; see WO-A-2012/038448.

Preferably, the above oligonucleotides or polynucleotides for use in the present invention may be prepared by a method comprising the step of incubating a corresponding

oligonucleotide or polynucleotide template with an RNA-dependent RNA polymerase (RdRp) of a calicivirus in the presence of appropriate rNTPs, in particular rGTP, rATP, rCTP, rUTP, and/or modified analogues thereof. In case of the compounds according to above formula (IV) only GTP (and/or modified analogue thereof) is needed.

For preparing preferred compositions for use in the invention, the oligonucleotides as defined in formula (IV), in particular oligonucleotides selected from the group consisting of of ppp(G)_6, ppp(G)₇, ppp(G)₈, ppp(G)₉, ppp(G)i₀ (SEQ ID NO: 2), ppp(G)n (SEQ ID NO: 3), ppp(G)i₂ (SEQ ID NO: 4), ppp(G)_i3 (SEQ ID NO: 5), and ppp(G)_i4 (SEQ ID NO: 6), more preferred a composition containing (as oligonucleotide species) ppp(G)₈, ppp(G)₉ and ppp(G)i₀ (SEQ ID NO: 2), or ppp(G)₆ , ppp(G)₇, ppp(G)₈, ppp(G)₉, ppp(G)i₀ (SEQ ID NO: 2), ppp(G)n (SEQ ID NO: 3), ppp(G)i2 (SEQ ID NO: 4), ppp(G)i₃ (SEQ ID NO: 5) and ppp(G)i₄ (SEQ ID NO: 6), it is preferred to incubate a (C) >₅ template, more preferably a rC5 or dC5, rC6 or dC6 template, with an RdRp of a calicivirus in the presence of rGTP (and/or chemically modified and/or labeled analogue(s) thereof) wherein the template may be RNA, DNA or mixed RNA/DNA.

The result is a mixture of the above preferred oligonucleotides and, depending on the amount of template used, the (C) >₅ template, optionally with minor contributions of the type (C)>₅(G)x (x being an integer of about 1 to 5). The length and mixture of oligonucleotides prepared in this manner can be controlled by the reaction conditions and reagent

concentrations used, in particular the reaction time. Optionally, individual oligonucleotide species for use in the invention can also be isolated from these compositions by gel electrophoresis or chromatography, especially HPLC, in a known manner. Without being bound to a specific theory, the mechanism underlying this preferred manufacturing method for certain oligonucleotide compositions as disclosed herein is that the calicivirus RNA polymerase transcribes the (C)>₅ template into a corresponding homopolymeric polyG and then adds one or several G residues to the 3' end of template and/or the homopolymeric polyG though its terminal transferase activity (see WO-A-2007/012329; WO-A- 2012/038448). Since, according to preferred embodiments of the invention, the template is rather short, the melting temperature of the oligo(G:C) product of the transcription process is rather low so that the strands separate easily and a new round of transcription of the template can start. The co-production of by-products such as, e.g. (C)>₅(G)_X can be avoided by adjusting, preferably lowering, the concentration of the template.

The 5'-triphosphated species and compositions thereof useful in the present invention are particularly for use as agonists of RIG-I. In that function, the oligonucleotide molecules and compositions thereof according to the present invention exert an immunostimulatory effect in cells or organisms. The 5'-triphosphated, preferably short, nucleotides, oligonucleotides and polynucleotides as defined herein as well as their compositions of the present invention are therefore useful as medicaments, in particular immunostimulatory preparations. The 5'- triphosphated species and their compositions for use in the present invention are also contemplated for the manufacture of a medicament for immunostimulation and/or for treatment and/or prevention of diseases disclosed herein. For use according to the present invention the 5'-triphosphated species as defined herein is typically present in a

pharmaceutical composition comprising at least one 5'-triphosphated species as defined herein, or a composition of at least two of such 5'-triphosphated molecules, and at least one pharmaceutically acceptable carrier, excipient, and/or diluent. The preparation of

pharmaceutical compositions in the context of the present invention, their dosages and their routes of administration are known to the skilled person, and general guidance can be found in the latest edition of Remington's Pharmaceutical Sciences (Mack publishing Co., Eastern, PA, USA).

In cases where the 5'-triphosphated molecules or compositions thereof, in particular an RNA oligonucleotide or their combinations, according to the invention are used as

immunostimulatory drugs alone, a topical administration of the appropriate preparation (e.g. a spray or solution) to skin and/or mucosa is preferred. As an "oligonucleotide drug" the 5'- triphosphated molecules of the present invention typically lead to activation of innate immunity, for instance by stimulation of dendritic cells and in particular therapeutic set ups of generation of a CD8+ T cell response. Such application of the inventive oligonucleotides and their combinations is especially useful in the treatment of infectious diseases, e.g. by viruses (such as Herpesvirus, Papillomavirus) or in the treatment of cancer or autoimmune diseases.

The improved immunostimulatory effects the 5'-triphosphated species as well as their combinations according to the present invention are also useful in combination with an antigen directed to a certain disease. Thus, the 5'-triphosphated molecules and their combinations according to the invention may also be used as an "oligonucleotide adjuvant" or "vaccine adjuvant" in prophylactic and/or therapeutic vaccine preparations (or

administered in a distinct preparation together with the vaccine, or sequentially). Accordingly the present invention is also directed to vaccine compositions comprising the nucleotide, oligonucleotide, polynucleotide or compositions thereof in combination with at least one antigen. Such antigen-containing pharmaceutical compositions can also include a further adjuvant known in the art (other than the oligonucleotides and combinations thereof as defined herein). The one or more antigens contained in such vaccine compositions of the invention are preferably selected from tumor antigens, viral antigens, bacterial antigens, fungal antigens and parasitic antigens.

Simultaneous or sequential administration of the vaccine or antigen and the

immunostimulatory 5'-triphosphated nucleotides, oligonucleotides or polynucleotides of the present invention should improve the immune response against the antigen, optionally the antigen of the vaccine, by generating a protective CD8+ T cell response to soluble proteins (antigens), triggering DC activation and induction of type I IFN production. For use in the present invention, the 5'-triphosphated molecules or their pharmaceutical compositions as disclosed herein can also be combined with further immunostimulatory drugs known in the art such as other Toll-like Receptor (TLR) agonists (including, but not limited to, agonists of Toll-like Receptor 5 (TLR-5), Toll-like Receptor 7 (TLR-7), Toll-like Receptor 8 (TLR-8) and/or Toll-like Receptor (TLR-9) and or RIG-l-like Receptors (RLRs) agonists such as agonists of MDA-5 and/or (and preferably) of RIG-I; see also co-pending International Patent Applications No. PCT/EP2012/071640 and No. PCT/EP2012/071641 .

The nucleotides, oligonucleotides and polynucleotides as disclosed herein may also be conjugated with other chemical entities, in particular with chemical groups which can be used to target the nucleotide, oligonucleotide or polynucleotide, respectively, to a cell in which said nucleotide, oligonucleotide or polynucleotide is to exert its immunostimulating property.

Chemical entities may be coupled to the inventive molecule through any kind of bonds such as covalent bonds, hydrogen bonds or Van der Waals bonds. Chemical entities to which the inventive molecule can be coupled include aptamers, polyethylenglycol such as PEG groups having an average molecular weight of from about 500 to about 1000 Da, e.g. about 750 Da, peptides, a palmitoyl group, cholesterol groups, phospholipids, proteins such as antibodies, and partners of non-covalent binding pairs such as biotin or digoxigenin. In the case of biotin, it can serve for associating the construct to a streptavidin group present on an antibody, or the biotin may serve as the ligand of an anti-biotin antibody which may, e.g. in turn have a second affinity to a target cell or tissue such as by having an affinity to a target receptor and/or ligand expressed intracellulary and/or on the surface of the target cell and/or tissue. Antibodies in the context of the present invention may be selected from polyclonal, monoclonal, humanized, chimeric, single-chain antibodies and antibody fragments which antibodies or antibody fragments may be single-specific or bispecific species. The antibody fragment may be a Fab fragment, a F(ab')₂ fragment, or any fragment that retains the antigen-binding specificity of the intact antibody. Especially preferred antibody ligands that may be coupled to the 5'-triphosphated molecules as disclosed herein are selected from antibodies or fragments thereof directed against cancer and/or tumor antigens, cancer- and/or tumor-associated antigens or oncoproteins, or antigens present on non-cancer and/or non-tumoral cells. According to further embodiments of the invention the antibody or fragment thereof may be directed against a viral, bacterial, fungal or parasitic antigen.

In other embodiments of the invention, a 5'-triphosphated molecule as defined herein may be complexed with other entities enabling the entry of the dsRNA into cells such as, e.g., polycationic compounds such as protamine or poly-L-lysine, and liposomes, in particular cationic liposomes.

For use in the present invention, the nucleotide, oligonucleotide or polynucleotide as well as their compositions, and pharmaceutical compositions as disclosed herein can also be combined with further immunostimulatory drugs known in the art such as other TLR agonists (including, but not limited to, agonists of TLR-5, TLR-7, TLR-8 and/or TLR-9) and or RLR agonists such as agonists of MDA-5 and/or (and preferably) of RIG-I.

The nucleotides, oligonucleotides and polynucleotides as well as their combinations as disclosed herein are particularly useful in the treatment of diseases, including autoimmune diseases, infectious diseases caused by infectious agents such as a bacterium, virus, parasite and fungus, and tumors.

The present invention also provides a method for the treatment of a disease as mentioned above, preferably a viral, bacterial, parasitic or fungal infection, a tumor disease or autoimmune disease, comprising administering an effective amount of a pharmaceutical composition as defined above to a preferably mammalian, particularly human, patient or subject, respectively, in need of such treatment.

In cases where the 5'-triphosphated molecule (or multiples thereof) of the invention is (are) used as an immunostimulatory drug alone, a topical administration of the appropriate preparation (e.g. a lotion, a topical cream, a spray or an injectable solution) to skin and/or mucosa is preferred. As an active pharmaceutical ingredient (API) the 5'-triphosphated molecules of the present invention lead to stimulation of antigen-presenting cells such as dendritic cells, monocytes and macrophages and generation of a Th2 and/or Th1 immune response including a CD8+ T cell response, natural killer (NK) cell response and/or B cell response leading to antibody production. Such stimulation of immune and/or non-immune cells typically leads to the induction of cytokine expression such as of type I and/or type II IFN production, and/or of TNF-a and/or ΙΙ_1 -β and/or IL-6 and/or induction of chemokine expression such as of IP-10 and/or MCP-1 and/or RANTES and/or l-TAC and/or GRO-a.

Accordingly, the present invention relates also to methods for induction of

cytokines/chemokines, in particular induction of type I and/or type II IFN production, and/or of TNF-a and/or ΙΙ_1 -β and/or IL-6 and/or IP-10 and/or MCP-1 and/or RANTES and/or l-TAC and/or GRO-a in non-immune cells, preferably in tumor and/or cancer cells, endothelial cells and/or neuronal cells, or in immune cells, especially DCs, macrophages and/or monocytes, in vitro or in vivo, in particular when present in a subject, preferably a mammal, more preferably a human, especially when in need for such induction of such

cytokines/chemokines, by administering an effective amount of one or more of the 5'- triphosphated molecules of the invention and/or a pharmaceutical composition of the invention to a preferably mammalian, particularly human, patient or subject, respectively, in need of such treatment.

The following description of non-limiting examples and appended drawings further illustrate the present invention, wherein the figures show:

Fig. 1. Synthesis of short immunostimulatory oligonucleotides using an RNA polymerase (RdRp) of a calicivirus. An ssRNA template (rC5; 5^'-CCCCC-3') or an ssDNA template (dC6; 5'-CCCCCC-3') was incubated with an RdRp of a calicivirus for 15 min, 45 min or 90 min. After purification of the reaction products using size exclusion

chromatography, the amount of single stranded RNA was measured. (A) Graphical representation of the amount of ssRNA produced using the ssRNA template. (B) Amount of ssRNA produced using the ssDNA template.

Fig. 2. Analysis of oligonucleotide synthesis using an RdRp of a calicivirus and a single stranded RNA (rC5; 5^'-CCCCC-3) as a template. All reactions were performed in a total volume of 100 μΙ at 37°C. The reaction mix contained 8 μg of the template, 10 μΜ RdRp, 2 mM GTP, 20 μΙ reaction buffer (HEPES 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), and RNAse-DNAse free water to a total volume of 100 μΙ. The reaction was stopped at 15 min, 45 min and 90 min by adding 10 μΙ EDTA 0.3 M. The reaction products obtained at these time points were analyzed using ion exchange HPLC and the area under the curve (AUC) of the eluted peak was measured. (A) to (C) Elution profiles of the product of the reaction using the rC5 template by IEX-HPLC at 15 min (A), 45 min (B) and 90 min (C), respectively. (D) Plot of AUC of eluted product of (A) to (C) as a function of time.

Fig. 3. Analysis of produced oligonucleotides. All reactions were performed in a total volume of 100 μΙ at 37°C using a single stranded RNA (rC5; 5^'-CCCCC-3') or a single stranded DNA (dC6; 5^'-CCCCCC-3^') as a template. The reaction mix contained 8 μg of the template, 10 μΜ RdRp, 2 mM GTP, 20 μΙ reaction buffer (HEPES 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), and RNAse-DNAse free water to a total volume of 100 μΙ. The reaction was stopped at 15 min, 30 min, 45 min, 60 min, 90 min and 120 min by adding 10 μΙ EDTA 0.3 M. After purification of the reaction products by size exclusion chromatography using the riboxxPURE kit according to instructions of manufacturer (RiboxX GmbH, Radebeul, Germany), 15 μ I of each reaction was analyzed on 20% PAGE and visualized after staining of nucleic acids. RNA marker corresponds to double stranded RNA of 19 bp, 21 bp and 25 bp, respectively, in length, as well as a single stranded RNA of 24 nt in length, as indicated. (A) Time course of synthesis of reaction products. The products of the reaction are shown. The visible products are isolated single stranded 5'-triphosphated poly(G) and/or single stranded 5'-triphosphated poly(G) forming complexes and G-quadruplexes (PPPrGx) as well as suprastructures. The single stranded RNA template has been used as a marker (rC5; 5^'- CCCCC-3^'). (B) Analysis of the reaction product resulting from a reaction as outlined above using a single stranded RNA (rC5; 5^'-CCCCC-3^') or a single stranded DNA (dC6; 5^'- CCCCCC-3^') as a template, as indicated. The reaction was allowed to proceed for 45 min, then stopped and analyzed by PAGE as described above. In the following, the reaction product using the rC5 template will also be referred to as "Qxxim-rC5", whereas the reaction product using the dC6 template will be as referred to as "Qxxim-dC6".

Fig. 4. Characterisation of the synthesized products according to the reaction as described for Fig. 3B using a single stranded RNA (rC5; 5 -CCCCC-3 ) as a template by HPLC. The HPLC elution profile of the products resulting from the reaction as described in Fig.3B is shown.

Fig. 5. Characterisation of the products synthesized as described above for Fig. 3B by ESI/MS. The single stranded RNA template (rC5; 5^'-CCCCC-3^') was used for the reaction as described above (see Fig. 3B). The product was denatured and characterized by ESI/MS. The elution product of the peak as indicated in panel A was analyzed by ESI/MS, as shown in panel B. It displays a molecular weight (MW) of 1463.22 Da corresponding to the template rC5 (RNA molecule; 5^'-CCCCC-3^').

Fig. 6. Characterisation of the products synthesized as described above for Fig. 3B by ESI/MS. The single stranded RNA template (rC5; 5^'-CCCCC-3^') was used for the reaction as described above (see Fig. 3B). The product was denatured and characterized by ESI/MS. The elution product of the peak as indicated in panel A was analyzed by ESI/MS, as shown in panel B. It displays a MW of 1808.27 Da corresponding to the MW of the product rC5G1 (RNA molecule; 5^'-CCCCCG-3^'). This product is expected to result from a terminal transferase activity of the RdRp on the rC5 template.

Fig. 7. Characterisation of the products synthesized as described above for Fig. 3B by ESI/MS. The single stranded RNA template (rC5; 5^'-CCCCC-3^') was used for the reaction as described above (see Fig. 3B). The product was denatured and characterized by ESI/MS. The elution product of the peak as indicated in panel A was analyzed by ESI/MS, as shown in panel B. It displays a MW of 2499.43 Da corresponding to the MW of the product rC5G3 (i.e. RNA molecule 5^'-CCCCCGGGG-3^'). This product is also expected to result from a terminal transferase activity of the RdRp on the rC5 template. Fig. 8. Characterisation of the products synthesized as described above for Fig. 3B by ESI/MS. The single stranded RNA template (rC5; 5^'-CCCCC-3^') was used for the reaction as described above (see Fig. 3B). The product was denatured and characterized by ESI/MS. The elution products of the peak as indicated in panel A were analyzed by ESI/MS, as shown in panel B. They display MWs of 2249 Da, 2594 Da, 2939 Da, 3284 Da, 3630 Da, 3975 Da, 4320 Da, and 4665 Da, respectively, as indicated, corresponding to the respective MW of the following products:

PPPG6, i.e. RNA molecule 5^'-GGGGGG-3^', with a triphosphate bound to the 5' end of the molecule;

PPPG7, i.e. RNA molecule 5^'-GGGGGGG-3^', with a triphosphate bound to the 5^' end of the molecule;

PPPG8, i.e. RNA molecule 5^'-GGGGGGGG-3^', with a triphosphate bound to the 5^' end of the molecule;

PPPG9, i.e. RNA molecule; 5^'-GGGGGGGGG-3^', with a triphosphate bound to the 5^' end of the molecule;

PPPG10, i.e. RNA molecule 5^'-GGGGGGGGGG-3^' (SEQ ID NO: 2), with a triphosphate bound to the 5^' end of the molecule; PPPG1 1 , i.e. RNA molecule 5^'-GGGGGGGGGGG-3^' (SEQ ID NO: 3), with a triphosphate bound to the 5^' end of the molecule;

PPPG12, i.e. RNA molecule 5^'-GGGGGGGGGGGG-3^' (SEQ ID NO: 4), with a triphosphate bound to the 5^' end of the molecule;

PPPG13, i.e. RNA molecule 5^'-GGGGGGGGGGGGG-3^' (SEQ ID NO: 5), with a

triphosphate bound to the 5^' end of the molecule.

Fig. 9. Characterisation of the products synthesized as described above for Fig. 3B by ESI/MS. The single stranded RNA template (rC5; 5^'-CCCCC-3^') was used for the reaction as described above (see Fig. 3B). The product was denatured and characterized by ESI/MS. The elution products of the peak as indicated in panel A were analyzed by ESI/MS, as shown in panel B. They display MWs of 3630 Da, 3975 Da, 4320 Da, 4665 Da, and 501 1 Da, respectively, as indicated, corresponding to the respective MW of the following products: PPPG10, i.e. RNA molecule 5^'-GGGGGGGGGG-3^' (SEQ ID NO: 2), with a triphosphate bound to the 5^' end of the molecule;

PPPG1 1 , i.e. RNA molecule 5^'-GGGGGGGGGGG-3^' (SEQ ID NO: 3), with a triphosphate bound to the 5^' end of the molecule;

PPPG13, i.e. RNA molecule 5^'-GGGGGGGGGGGGG-3^' (SEQ ID NO: 5), with a

triphosphate bound to the 5^' end of the molecule;

PPPG14, i.e. RNA molecule 5^'-GGGGGGGGGGGGGG-3^' (SEQ ID NO: 6), with a triphosphate bound to the 5^' end of the molecule. Fig. 10. Activation of RAW 264.7 mouse macrophages by short immunostimulatory oligonucleotides produced according to Fig. 3B or poly (l:C) high molecular weight compound (HMW, 1.5-8 kb). Oligonucleotide compositions Qxxim-rC5 and Qxxim-dC6 were synthesized as described in Fig. 3B. Composition Qxxim-rC5 was synthesized using rC5 template, whereas composition Qxxim-dC6 was prepared using the dC6 template.

Compositions Qxxim-rC5 and Qxxim-dC6 or poly(l:C) were incubated with RAW 264.7 macrophages for 16h at 50 μg ml. After 16h, supernatant was harvested and secretion of TNF-a was measured using the ELISArray mouse TNF-a kit according to instructions of manufacturer (Qiagen, Hilden, Germany). The activation of RAW 264.7 is expressed in pg/ml. Values shown are mean +/- SEM of two independent measurements. As a negative control, supernatant of cells incubated with mock was used. For the values shown in the graphical representation, values of negative control have been subtracted from the measured values. A positive control using 1 ng/ml of TNF-a is shown as well. Fig. 11. Activation of JAWS II mouse dendritic cells by short immunostimulatory oligonucleotides produced according to Fig. 3B Oligonucleotide compositions Qxxim-rC5 and Qxxim-dC6 were synthesized as described in Fig. 3B. Composition Qxxim-rC5 was synthesized using rC5 template, whereas composition Qxxim-dC6 was prepared using the dC6 template. Compositions Qxxim-rC5 and Qxxim-dC6 were incubated with JAWS II mouse dendritic cells for 16h at 50 μg ml. After 16h, supernatant was harvested and secretion of IL- 6 was measured using the ELISArray mouse IL-6 kit according to instructions of

manufacturer (Qiagen, Hilden, Germany). The activation of JAWS II is expressed in pg/ml. Values shown are mean +/- SEM of two independent measurements. As a negative control, supernatant of cells incubated with mock was used. For the values shown in the graphical representation, values of negative control have been subtracted from the measured values. A positive control using 1 ng/ml of IL-6 is shown as well.

Fig. 12. Activation of JAWS II mouse dendritic cells by short immunostimulatory oligonucleotides produced according to Fig. 3B in comparison to substances consisting of a mix of single stranded RNA of a defined homopolymeric (rG) or homopolymeric (dG) sequence. Oligonucleotide compositions Qxxim-rC5 and Qxxim-dC6 were synthesized as described in Fig. 3B. Composition Qxxim-rC5 was synthesized using rC5 template, whereas composition Qxxim-dC6 was prepared using the dC6 template.

The homopolymeric riboguanosine (rG) is shown as (rGx)n, and consists of an equimolar mix of:

rG5, i.e. RNA molecule 5^'-GGGGG-3^', without triphosphate bound to the 5^' end of the molecule;

rG8, i.e. RNA molecule 5^'-GGGGGGGG-3^', without triphosphate bound to the 5^' end of the molecule;

rG10, i.e. RNA molecule 5^'-GGGGGGGGGG-3^' (SEQ ID NO: 7), without triphosphate bound to the 5^' end of the molecule;

rG12, i.e. RNA molecule 5^'-GGGGGGGGGGGG-3^' (SEQ ID NO: 8), without triphosphate bound to the 5^' end of the molecule;

rG14, i.e. RNA molecule 5^'-GGGGGGGGGGGGGG-3^' (SEQ ID NO: 9), without triphosphate bound to the 5^' end of the molecule.

The homopolymeric deoxyriboguanosine (dG) is shown as (dGx)n, and consists of an equimolar mix of:

dG6, i.e. DNA molecule 5^'-GGGGGG-3^', without triphosphate bound to the 5^' end of the molecule; dG7, i.e. DNA molecule 5^'-GGGGGGG-3^', without triphosphate bound to the 5^' end of the molecule;

dG8, i.e. DNA molecule 5^'-GGGGGGGG-3^', without triphosphate bound to the 5^' end of the molecule;

dG9, i.e. DNA molecule 5^'-GGGGGGGGG-3^', without triphosphate bound to the 5^' end of the molecule;

dG10, i.e. DNA molecule 5^'-GGGGGGGGGG-3^' (SEQ ID NO: 10), without triphosphate bound to the 5^' end of the molecule;

dG1 1 , i.e. DNA molecule 5^'-GGGGGGGGGGG-3^' (SEQ ID NO: 1 1 ), without triphosphate bound to the 5^' end of the molecule;

dG13, i.e. DNA molecule 5^'-GGGGGGGGGGGGG-3^' (SEQ ID NO: 12), without triphosphate bound to the 5^' end of the molecule;

dG14, i.e. DNA molecule 5^'-GGGGGGGGGGGGGG-3^' (SEQ ID NO: 13), without triphosphate bound to the 5^' end of the molecule;

dG15, i.e. DNA molecule 5^'-GGGGGGGGGGGGGGG-3^' (SEQ ID NO: 14), without triphosphate bound to the 5^' end of the molecule.

Oligonucleotide compositions Qxxim-rC5 and Qxxim-dC6, (rGn)x and (dGn)x or poly(l:C) were incubated with JAWS II mouse dendritic cells for 16h at 50 μg ml. After 16h, supernatant was harvested and secretion of IL-6 was measured using the ELISArray mouse IL-6 kit according to the instructions of the manufacturer (Qiagen, Hilden, Germany). The activation of JAWS II is expressed in pg/ml. Values shown are mean +/- SEM of two independent measurements. As a negative control, supernatant of cells incubated with mock was used. For the values shown in the graphical representation, values of negative control have been subtracted from the measured values. A positive analytical control of 1 ng/ml of IL-6 is shown as well.

Fig. 13. Activation of RAW 264.7 mouse macrophages by short immunostimulatory oligonucleotides in comparison to substances consisting of a mix of single stranded RNA of a defined homopolymeric (rG) or homopolymeric (dG) sequence.

Oligonucleotide compositions Qxxim-rC5 and Qxxim-dC6, (rGn)x and (dGn)x or poly(l:C) as described above for Fig. 12 were incubated with RAW 264.7 macrophages for 16h at 50 μg ml. After 16h, supernatant was harvested and secretion of IL-6 was measured using the ELISArray mouse IL-6 kit according to the instructions of the manufacturer (Qiagen, Hilden, Germany). The activation of RAW 264.7 macrophages is expressed in pg/ml. Values shown are mean +/- SEM of two independent measurements. As a negative control, supernatant of cells incubated with mock was used. For the values shown in the graphical representation, values of negative control have been subtracted from the measured values. A positive analytical control of 1 ng/ml of TNF-a is shown as well.

Example 1 : Synthesis of short immunostimulatory oligonucleotides by RNA- dependent RNA polymerase of a calicivirus using ssRNA or ssDNA template

All reactions were performed in a total volume of 100 μΙ at 37°C using a single stranded RNA (rC₅: 5'-CCCCC-3') or a single stranded DNA (dC₆: 5'-CCCCCC-3') as template. The reaction mix contained 8 μg of the template, 10 μΜ calicivirus RdRp, 2 mM GTP, 20 μΙ reaction buffer (HEPES 250 mM, MnCI₂ 25 mM, DTT 5 mM, pH 7.6), and RNAse-DNAse free water to a total volume of 100 μΙ. The reaction was stopped at 15 min, 45 min and 90 min by adding 10 μΙ EDTA 0.3 M. After purification of the reaction products using size exclusion

chromatography, the amount of single stranded RNA was measured at the indicated time points. The results are shown in Figs. 1 A and B.

In parallel experiments, the reaction products were also analyzed using ion exchange HPLC and the area under the curve (AUC) of the eluted peak was calculated. Results for the reaction using the rC5 template are shown in Fig. 2A (elution profile of reaction stopped after 15 min), Fig. 2B (elution profile of reaction stopped after 45 min) and Fig. 2C (elution profile of reaction stopped after 90 min). Fig. 2D shows the plot of the results of the AUC

measurements.

Example 2: Analysis of reaction products by PAGE

RdRp reactions were carried out as in Example 1 using the rC₅ template or the dC₆ template, and the reactions were stopped at 15 min, 30 min, 45 min, 60 min, 90 min and 120 min by adding 10 μΙ EDTA 0.3 M. After purification of the reaction products by size exclusion chromatography using the riboxxPURE kit according to the instructions of the manufacturer (RiboxX GmbH, Radebeul, Germany), 15 μΙ of each reaction were analyzed by 20% PAGE and visualized through UV transillumination. Result of the time course experiment using the rC5 ssRNA template is shown in Fig. 3A. Fig, 3B shows the products of reactions using the rC₅ or the rC₆ template each allowed to proceed for 45 min. The bands visible correspond to isolated single stranded 5'-triphosphated poly(G) and/or single stranded 5'-triphosphated poly(G) forming complexes and G-quadruplexes (PPPrGx) as well as higher suprastructures. Example 3: Characterization of RdRp reaction products using rC₅ template by HPLC/mass spectroscopy

The reaction product of Example 1 using the rC₅ template was subjected to HPLC. The elution profile is shown in Fig. 4. The peak fractions as indicated in Figs. 5A, 6A, 7A, 8A and 9A were denatured and further analyzed by ESI/MS. The corresponding mass spectra are shown in Fig. 5B, 6B, 7B, 8B and 9B, respectively. The peak(s) in the mass spectra correspond to the molecular weight of the species indicated in Fig. 5B, 6B, 7B, 8B and 9B, respectively.

Example 4: Activation of RAW 264.7 mouse macrophages and JAWS II mouse

dendritic cells by oligonucleotides produced according to Example 1.

JAWS II and RAW 264.7 cell lines were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). JAWS II is an immortalized immature myeloid DC line derived from C57BL/6 mice, which displays a similar phenotypic profile as resting bone-marrow- derived DCs (BMDCs) (Jiang et al. (2008) Infection and Immunity 76 (6), 2392-2401 ). RAW 264.7 is a mouse leukemic monocyte/macrophage cell line which was established from the ascites of a tumor induced in a male mouse by intraperitoneal injection of Abelson Mouse Leukaemia Virus (A-MuLV).

Cells were plated in round-bottomed 96-well plates at 5 x 10⁴ cells/well in DMEM

supplemented with 10% fetal calf serum (FCS) and 1 % penicillin/streptomycin (100 U/ml). Cells were incubated with the compositions Qxxim-rC5, Qxxim-dC6 or poly(l:C) for 16h at 50 μg ml. After 16h, supernatant was harvested and secretion of TNF-a or IL-6 was measured using the ELISArray mouse TNF-a kit and ELISArray IL-6 kit, respectively, according to instructions of manufacturer (Qiagen, Hilden, Germany). As a negative control, supernatant of cells incubated with mock was used. 1 ng/ml of TNF-a or 1 ng/ml IL-6 were used as positive analytical controls.

Results are shown in Figs. 10 to 13.

Claims

A 5'-triphosphated nucleotide, oligonucleotide or polynucleotide forming G_:

quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures under appropriate conditions, in particular under appropriate buffer, salt and temperature conditions, preferably physiological conditions for use in immunostimulation in a subject, preferably a mammal, more preferably a human.

A 5'-triphosphated oligonucleotide or polynucleotide for use of claim 1 comprising or having a sequence of formula (I) nGpXm (I) wherein X_n and X_m is any nucleotide sequence of length n or m, respectively, preferably being m = n, and G_p is any number of guanines of length p involved in formation of G-quadruplexes, and/or intramolecular and/or intermolecular structures and/or superstructures and/or tetrads..

An oligonucleotide or polynucleotide for use of claim 1 or 2 comprising or having a sequence selected from the group consisting of formulas (II), (III) and (IV):

X_nGolXpG₀2Xm (II) wherein X_n and X_m is any non-guanine nucleotide sequence of length n or m, respectively, preferably being n=m, G₀i and G₀2 is any number of guanines of length o1 and o2, respectively, involved in formation of G-quadruplexes, and/or

intramolecular and/or intermolecular structures and/or superstructures and/or tetrads, preferably being o1 = o2, and X_p is any nucleotide sequence of length p involved in loop formation;

X_nGolXpl Go2X 2Go3X 3G₀4Xm (HI) wherein X_n and X_m is any non-guanine nucleotide sequence of length n or m, respectively, preferably being n=m, G₀i , G₀2, G₀3 and G_o4 are independently any number of guanines of length o1 , o2, o3 and o4, respectively, involved in formation of G-quadruplexes, and/or intramolecular and/or intermolecular structures and/or superstructures and/or tetrads, preferably being o1 = o2 = o3 = o4, and X_pi, X_p2 and Xp₃ are indepedently any nucleotide sequence of length p1 , p2 and p3, respectively, involved in loop formation, preferably being p1 =p2=p3;

PPP(G)_n (IV) wherein n is an integer of at least 2 or 3, preferably 3 to 40, more preferably 3 to 20, and ppp is a 5'-triphosphate group.

4. The oligonucleotide or polynucleotide for use of claim 3 wherein n in formula (IV) is an integer of from 2 or 3 to 14, more preferably 6 to 14.

5. The oligonucleotide for use of claim 4 wherein n in formula (IV) is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 .

6. An oligonucleotide or polynucleotide for use according to any one of the preceding claims wherein the oligonucleotide or polynucleotide is single-stranded.

7. An oligonucleotide or polynucleotide for use according to any one of the preceding claims consisting of ribonucleotides.

8. An oligonucleotide or polynucleotide for use according to any one of the preceding claims comprising at least one chemically modified and/or labeled nucleotide.

9. An oligonucleotide or polynucleotide as defined in any one of the preceding claims for use in inducing an anti-viral, anti-bacterial and/or anti-tumor response in a subject, preferably a mammal, more preferably a human.

10. An oligonucleotide or polynucleotide as defined in any one of the preceding claims for use in the treatment and/or prevention of a disease selected from the group consisting of infectious diseases, autoimmune diseases and tumor diseases.

1 1 . A composition containing at least two different nucleotides, oligonucleotide(s) and/or polynucleotide(s) as defined in any one of the preceding claims for use in

immunostimulation in a subject, preferably a mammal, more preferably a human.

12. The composition for use of claim 1 1 wherein the composition contains at least two different oligonucleotides selected from the group consisting of ppp(G)_6, ppp(G)₇, ppp(G)₈, ppp(G)₉, ppp(G)io (SEQ ID NO: 2), ppp(G)n (SEQ ID NO: 3), ppp(G)i₂ (SEQ ID NO: 4), ppp(G)i3 (SEQ ID NO: 5) and ppp(G)i₄ (SEQ ID NO: 6).

13. The composition as defined in claim 12 or 13 for use in inducing an anti-viral, antibacterial and/or anti-tumor response in a subject, preferably a mammal, more preferably a human.

14. The composition as defined in claim 12 or 13 for use in the treatment and/or

prevention of a disease selected from the group consisting of infectious diseases, autoimmune diseases and tumor diseases.

15. A vaccine composition comprising a nucleotide, oligonucleotide or polynucleotide according to any one of claims 1 to 8 or the composition of claim 1 1 or 12 and at least one antigen.

16. The vaccine of claim 15 wherein the antigen is selected from the group consisting of tumor antigens, viral antigens, bacterial antigens, fungal antigen, and parasitical antigens.