RU2338750C2 - IMMUNOSTIMULATING PHOSPHOROTHIOATE CpG-OLIGONUCLEOTIDES, CONTAINING PHOSPHODIESTER LINKS, METHOD OF IMMUNOMODULATION, METHOD OF IMMUNE RESPONSE STIMULATION - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention concerns to immunostimulating phosphorothioate CpG-oligonucleotides which can be used for stimulation of the immune response.

EFFECT: obtaining of CpG-oligonucleotides.

49 cl, 76 fig, 19 tbl, 34 ex

Description

FIELD OF THE INVENTION

In general terms, the present invention relates to immunostimulatory nucleic acids, as well as to immunostimulatory oligonucleotides having a reduced inflammatory effect on the kidneys, to their compositions and to methods of using these immunostimulatory nucleic acids.

State of the art

Bacterial DNA has an immunostimulating effect aimed at activating B cells and natural killer cells, while vertebrate DNA does not have a similar effect (Tokunaga T., et al., 1988. Jpn. J. Cancer Res. 79: 682-686; Tokunaga, T., et al., 1984, JNCI 72: 955-962; Messina JP, et al., 1991, J. Immunol. 147: 1759-1764; see also Kreig, 1998, Applied Oligonucleotide Technology, S. A. Stein and AM Kreig (Eds.), John Wiley and Sons, Inc., New York, NY, pp. 431-448). It is now known that these immunostimulatory effects of bacterial DNA are the result of the presence of unmethylated CpG dinucleotides with a specific set of bases (CpG motifs) that are widely represented in bacterial DNA but are not well represented in vertebrate DNA where they are methylated (Krieg et ai., 1995, Nature 374: 546-549; Krieg 1999, Biochim. Biophys. Acia 93321: 1-10). The immunostimulatory effects of bacterial DNA can be mimicked by synthetic oligodeoxynucleotides (ODNs) containing the aforementioned CpG motifs. Such CpG-ODNs have a strong stimulatory effect on human and murine white blood cells, including proliferation of B cells; secretion of cytokines and immunoglobulins; the cytolytic activity of natural killers (NK) and the secretion of IFN-γ; activation of dendritic cells (DC) and antigen-presenting cells for the expression of co-stimulating molecules and secretion of cytokines, and in particular Th1-like cytokines, which play an important role in stimulating the production of Th1-like T-cell responses. These immunostimulatory effects of the native phosphodiester backbone of CpG-ODN are highly CpG-specific in the sense that they sharply decrease when the specified CpG motif is methylated or replaced by GpC or otherwise removed or modified (Krieg et al., 1995, Nature 374: 546-549; Hartmann et al., 1999, Proc. Natl. Acad. Sci. USA 96: 9305-10).

Early studies have shown that an immunostimulatory CpG motif has the formula purine-purine-CpG-pyrimidine-pyrimidine (Krieg et al., 1995, Nature 374: 546-549; Pisetsky, 1996, J. Immunol. 156: 421-423 ; Hacker et al., 1998, EMBO J. 17: 6230-6240; Lipford et al, 1998 Trends in Microbiol. 6: 496-500). However, it is now apparent that murine lymphocytes respond quite well to phosphodiester CpG motifs that do not fit this “formula” (Yi et al., 1998, J. Immunol. 160: 5898-5906), and this is true for human B -cells and dendritic cells (Hartmann et al., 1999, Proc. Natl. Acad. Sci. USA 96: 9305-10; Liang, 1996, J. Clin. Invest. 98: 1119-1129).

Recently, several different classes of nucleic acids containing CpG have been described. One class contains strong activators of B cells, but relatively weak inducers of activation of IFN-α and NK cells, and this class was called the B-class. Class B CpG-containing nucleic acids are typically fully stabilized and contain the unmethylated CpG dinucleotide with a certain preferred set of bases. See, for example, US Patent Nos. 6194388, 6207646, 6214806, 6218371, 6239116 and 6339068. CpG-containing nucleic acids of a different class activate B cells and NK cells and induce IFN-α; and this class was called the C-class. CpG-containing C-class nucleic acids, as first defined, are usually fully stabilized and contain a sequence type such as a B-class sequence and a GC-rich palindrome or a sequence close to the palindromic sequence. This class was described in the simultaneously pending provisional application for US patent No. 60/313273, filed August 17, 2001, and in the application for US patent No. 10/224523, filed August 19, 2002, as well as in the related patent application PCT, PCT / US02 / 26468, published under the number of International publication WO 03/015711.

SUMMARY OF THE INVENTION

It has been unexpectedly discovered that the immunostimulatory properties of CpG-containing B-class and C-class nucleic acids and other stabilized immunostimulatory nucleic acids can be maintained or even improved by selectively incorporating one or more unstabilized bonds between specific nucleotides. Such unstabilized bonds are preferably natural bonds, i.e. phosphodiester bonds or phosphodiester-like bonds. An unstable bond is usually, but not necessarily, relatively sensitive to nuclease cleavage. The immunostimulatory nucleic acids of the present invention contain at least one unstabilized bond located between 5'-pyrimidine (Y) and adjacent 3'-purine (Z), and preferably guanine (G), where 5'Y and 3 'Z are internal nucleotides.

Like the fully stabilized immunostimulatory nucleic acids, the immunostimulatory nucleic acids of the present invention can be used to induce a Th1-like immune response. Accordingly, the immunostimulatory nucleic acids of the present invention can be used as adjuvants for vaccination, and they can also be used to treat diseases, including cancer, infectious disease, allergies and asthma. Obviously, these nucleic acids can be particularly effective for the treatment of any conditions that require continuous or repeated administration of an immunostimulatory nucleic acid, regardless of the purpose of its administration.

Although fully stabilized immunostimulatory nucleic acids are intended for multipurpose use, however, in some embodiments, the immunostimulatory nucleic acids of the present invention, in contrast to the above fully stabilized immunostimulatory nucleic acids, have advantages such as increased efficiency and reduced toxicity.

The present invention relates in part to immunostimulatory CpG-containing oligonucleotides. In one of its aspects, the present invention relates to an oligonucleotide having the formula: 5'T * C * G * T * CGTTTTGAN ₁ CGN ₂ * T * T3 '(SEQ ID NO: 296). In oligonucleotide N ₁ consists of 0-6 nucleotides, and in N ₂ consists of 0-7 nucleotides. The symbol * means the presence of a stabilized internucleotide bond. Internucleotide bonds not marked with an asterisk * may be unstabilized or stabilized, provided that the oligonucleotide contains at least 2 phosphodiester internucleotide bonds. The stabilized internucleotide bond may be a phosphorothioate bond. In some embodiments, N ₁ consists of 0-2 nucleotides. This oligonucleotide is preferably 16-24 nucleotides in length.

In some embodiments, the oligonucleotide has one of the following structures:

The symbol _ indicates the presence of a phosphodiester internucleotide bond.

A preferred oligonucleotide is:

In other aspects, the present invention relates to an oligonucleotide containing 5'T * C * G * (T * / A *) TN ₃ CGTTTTN ₄ CGN ₅ * T * T3 '(SEQ ID NO: 301). N ₃ consists of 0-4 nucleotides. N ₄ consists of 1-5 nucleotides. N ₅ consists of 0-7 nucleotides. The symbol * means the presence of a stabilized internucleotide bond. Internucleotide bonds not marked with an asterisk * may be unstabilized or stabilized, provided that the oligonucleotide contains at least 2 phosphodiester internucleotide bonds. The stabilized internucleotide bond may be a phosphorothioate bond. In some embodiments, N ₄ consists of 1-2 nucleotides. A preferred oligonucleotide is an oligonucleotide of 16-24 nucleotides in length.

In some embodiments, the oligonucleotide has one of the following structures:

A preferred oligonucleotide is:

In accordance with its other aspects, the present invention relates to an oligonucleotide: 5'T * C * G * T * C * GNNNCGNCGNNNC * G * N * C * G * T * T3 '(SEQ ID NO: 306). N represents any nucleotide. The symbol * means the presence of a stabilized internucleotide bond. Internucleotide bonds not marked with an asterisk * may be unstabilized or stabilized, provided that the oligonucleotide contains at least 3 phosphodiester internucleotide bonds. The stabilized internucleotide bond may be a phosphorothioate bond. In some embodiments, the oligonucleotide contains 5 phosphodiester internucleotide bonds. A preferred oligonucleotide has a length of 16-24 nucleotides.

In some embodiments, the oligonucleotide has one of the following structures:

In one embodiment, the oligonucleotide is: 5'T * C * G * T * C * G * T * T * A * C_G_T_C_G_T * T * A * C * G * T * C * G * T * T3 '(SEQ ID NO: 310). The symbol _ indicates the presence of a phosphodiester internucleotide bond.

In other embodiments, the oligonucleotide comprises at least one C_G motif with a phosphodiester internucleotide linkage. In yet another embodiment, the oligonucleotide does not contain any C_G motifs with a phosphodiester internucleotide linkage.

In other aspects, the present invention relates to an oligonucleotide having the structure 5'T * C_G (N ₆ C_GN ₇ ) _2-3 T * C_G * T * T3 '(SEQ ID NO: 311-312). N ₆ and N ₇ , independently, have a length of 1-5 nucleotides, and the oligonucleotide has a length of 16-40 nucleotides.

In some embodiments, N ₆ is a single nucleotide, for example, N ₆ can be T or A. In some embodiments, N ₇ is five nucleotides, for example, N ₇ can be five pyrimidines or TTTTG.

In some embodiments, the oligonucleotide has the structure:

In other aspects, the present invention relates to an oligonucleotide having the structure 5'T * CGCGN ₈ CGCGC * GN ₉ 3 '(SEQ ID NO: 315). N ₈ has a length of 4-10 nucleotides and contains at least 1 C_G motif. N ₉ has a length of 0-3 nucleotide. The oligonucleotide has a length of 15-40 nucleotides.

In some embodiments, N ₈ contains at least 2 or 3 CG motifs. In other embodiments, N ₈ is PuCGPyPyCG or PuCGPyPyCGCG. N _{8 is} optionally ACGTTCG. N ₉ may contain at least one CG motif, such as CCG.

In some embodiments, the oligonucleotide has the structure:

In another aspect, the present invention relates to an oligonucleotide having the formula: 5'T * T * GX ₁ X ₂ TGX ₃ X ₄ T * T * T * T * N ₁₀ T * T * T * T * T * T * T3 '(SEQ ID NO: 318). N ₁₀ has a length of 4-8 nucleotides and contains at least 1 motive C_G. X ₁ , X ₂ , X ₃ and X ₄ independently represent C or G. The oligonucleotide has a length of 24-40 nucleotides.

In some embodiments, N ₁₀ contains at least 2 or 3 CG motifs. In other embodiments, the oligonucleotide has one of the following structures:

In other embodiments, the oligonucleotide has the structure: 5'T * C * G * C_G * A * C * G * T * T * C_G * G * C * G * C_G * C * G * C * C * G3 '(SEQ ID NO: 321).

In some aspects of the present invention, an ODN is an oligonucleotide having a sequence selected from the group consisting of

In another aspect, the present invention relates to an oligonucleotide comprising an octameric sequence comprising at least one YZ dinucleotide having a phosphodiester or phosphodiester-like internucleotide bond, and at least 4 T nucleotides, where Y is pyrimidine or a modified pyrimidine, and Z is guanosine or a modified guanosine and wherein the oligonucleotide contains at least one stabilized internucleotide linkage.

Y may be unmethylated C. Z may be guanosine. In some embodiments, Y is cytosine or modified cytosine bases selected from the group consisting of 5-methylcytosine, 5-methylisocytosine, 5-hydroxycytosine, 5-halogenocytosine, uracil, N4-ethylcytosine, 5-fluorouracil and hydrogen. In other embodiments, Z is guanine or a modified guanine base selected from the group consisting of 7-deazaguanine, 7-deaz-7-substituted guanine (such as 7-dease-7- (C2-C6) alkynylguanine), 7 -dease-8-substituted guanine, hypoxanthine, 2,6-diaminopurine, 2-aminopurine, purine, 8-substituted guanine such as 8-hydroxyguanine and 6-thioguanine, 2-aminopurine and hydrogen.

In some embodiments, the octameric sequence comprises a TTTT motif. In other embodiments, the octameric sequence comprises two YZ dinucleotides. Both YZ dinucleotides, but not necessarily, have a phosphodiester or phosphodiester-like internucleotide linkage.

In some embodiments, the octameric sequence is selected from the group consisting of:

where * means the presence of a stabilized internucleotide linkage and where _ means the presence of a phosphodiester internucleotide linkage.

In other embodiments, the oligonucleotide has a length of 8-40 nucleotides.

The phosphodiester-like bond may be borane phosphonate or diastereomerically pure Rp phosphorothioate. Stable internucleotide bonds are, but not necessarily, phosphorothioate, phosphordithioate, methylphosphonate, methylphosphorothioate, or any combination thereof.

The oligonucleotide may have a 3'-3'-bond with one or two available 5'-ends. In some preferred embodiments, the oligonucleotide has two available 5'-ends, each of which is 5'TCG.

In another aspect, the present invention relates to an oligonucleotide comprising: 5'TCGTCGTTTTTGACGTTTTGTCGTT3 '(SEQ ID NO: 368). At least one CG dinucleotide has a phosphodiester or phosphodiester-like internucleotide bond, and the oligonucleotide contains at least one stabilized internucleotide bond.

In other aspects, the present invention relates to an oligonucleotide containing 5'GNC3 ', where N is a nucleic acid sequence with a length of 4-10 nucleotides; which at least 50% consists of T and does not contain a CG dinucleotide, and the oligonucleotide contains at least one stabilized internucleotide linkage. In one embodiment, N contains a TTTT motif. In other embodiments, the oligonucleotide is selected from the group consisting of G * T * T * T * T * G * T * C and G * T * T * T * T * G * A * C, where * means the presence of a stabilized internucleotide communication.

In another aspect, the present invention relates to an immunostimulatory nucleic acid molecule having at least one internal pyrimidine-purine (YZ) dinucleotide and optionally pyrimidine-guanosine (YG) dinucleotide, and a chimeric backbone, where at least one internal YZ dinucleotide has a phosphodiester or phosphodiester-like internucleotide bond, where each additional internal YZ dinucleotide has, but is not necessarily, a phosphodiester, phosphodiester-like or stabilized internucleotide bond and where all other internucleotide linkages are stabilized. In one embodiment, the immunostimulatory nucleic acid comprises a plurality of internal YZ dinucleotides, each of which has a phosphodiester or phosphodiester-like internucleotide linkage. In one embodiment, each internal YZ dinucleotide has a phosphodiester or phosphodiester-like internucleotide linkage.

In one embodiment, the immunostimulatory nucleic acid molecule is selected from the group consisting of:

where * means phosphorothioate, _ means phosphodiester, U means 2'-deoxyuracil, and 7 means 7-deazaguanine.

In one embodiment, the immunostimulatory nucleic acid molecule is selected from the group consisting of:

where * means phosphorothioate, and _ means phosphodiester.

In another aspect, the present invention relates to an immunostimulatory nucleic acid molecule containing a chimeric backbone and at least one N ₁ YGN ₂ sequence, where in each N ₁ YGN ₂ YG sequence independently represents an internal pyrimidine-guanosine dinucleotide ( YG), N ₁ and N ₂ independently represent any nucleotide and where, in at least one N ₁ YGN ₂ sequence and optionally in each additional N ₁ YGN ₂ sequence: the YG dinucleotide has a phosphodiester or phosphodiester a good internucleotide bond, wherein N ₁ and Y are connected by a phosphodiester or phosphodiester-like internucleotide bond if N ₁ is an internal nucleotide, and G and N ₂ are connected by a phosphodiester or phosphodiester-like internucleotide bond if N ₂ is an internal nucleotide, or N ₁ and Y linked by a phosphodiester or phosphodiester-like internucleotide bond if N ₁ is an internal nucleotide and G and N ₂ are linked by a phosphodiester or phosphodiester-like internucleotide bond if N ₂ is an internal The lower nucleotide, where all other internucleotide bonds are stabilized.

In one embodiment, the immunostimulatory nucleic acid comprises a plurality of N ₁ YGN ₂ sequences, where in each sequence N ₁ YGN ₂ : the YG dinucleotide has a phosphodiester or phosphodiester-like internucleotide bond, wherein N ₁ and Y are linked by a phosphodiester or phosphodiester-like internucleotide if ₁ represents an internal nucleotide, and G and N ₂ are connected by a phosphodiester or phosphodiester-like internucleotide bond, if N ₂ is an internal nucleotide, or N ₁ and Y are linked they are phosphodiester or phosphodiester-like internucleotide linkages if N ₁ is an internal nucleotide and G and N ₂ are linked by a phosphodiester or phosphodiester-like internucleotide link if N ₂ is an internal nucleotide.

In one embodiment, the immunostimulatory nucleic acid molecule is selected from the group consisting of:

where * means phosphorothioate, and _ means phosphodiester.

In one embodiment, the immunostimulatory nucleic acid molecule is selected from the group consisting of:

where * means phosphorothioate, and _ means phosphodiester.

In one embodiment, the immunostimulatory nucleic acid molecule is selected from the group consisting of:

where * means phosphorothioate, and _ means phosphodiester.

In one embodiment, the immunostimulatory nucleic acid molecule is selected from the group consisting of:

where * means phosphorothioate, and _ means phosphodiester.

In one embodiment, the at least one internal YG dinucleotide having a phosphodiester or phosphodiester-like internucleotide bond is CG. In one embodiment, the at least one internal YG dinucleotide having a phosphodiester or phosphodiester-like internucleotide bond is TG.

In one embodiment, the phosphodiester or phosphodiester-like internucleotide bond is a phosphodiester. In one embodiment, the phosphodiester-like bond is borane phosphonate or diastereomerically pure Rp phosphorothioate.

In one embodiment of the invention, stabilized internucleotide bonds are selected from the group consisting of phosphorothioate, phosphordithioate, methylphosphonate, methylphosphorothioate, and any combination thereof. In one embodiment, the stabilized internucleotide linkage is a phosphorothioate linkage.

In one embodiment, the immunostimulatory nucleic acid molecule is a B-class immunostimulatory nucleic acid molecule. In one embodiment of the invention, said immunostimulatory nucleic acid molecule is a C-class immunostimulatory nucleic acid molecule.

In one embodiment, the immunostimulatory nucleic acid molecule has a length of 4-100 nucleotides. In one embodiment of the invention, said immunostimulatory nucleic acid molecule has a length of 6-40 nucleotides. In one embodiment of the invention, said immunostimulatory nucleic acid molecule has a length of 6-19 nucleotides.

In one embodiment, the immunostimulatory nucleic acid molecule is not an antisense oligonucleotide; triple helix oligonucleotide; or ribozyme.

In another aspect, the present invention relates to an oligonucleotide that contains:

N ₁ -C_G-N ₂ -C_G-N ₃

where each of N ₁ and N ₃ independently represents a nucleic acid sequence with a length of 1-20 nucleotides and where _ means an internal phosphodiester or phosphodiester-like internucleotide bond, N ₂ independently represents a nucleic acid sequence with a length of 0-20 nucleotides, and GN ₂ -C contains 1 or 2 stabilized bonds.

In another aspect, the present invention relates to an oligonucleotide that contains:

N ₁ -C_G-N ₂ -C_G-N ₃

where each of N ₁ and N ₃ independently represents a nucleic acid sequence with a length of 1-20 nucleotides and where _ means an internal phosphodiester or phosphodiester-like internucleotide bond, N ₂ independently represents a nucleic acid sequence with a length of 4-20 nucleotides, and GN ₂ -C contains at least 5 stabilized bonds.

In another aspect, the present invention relates to an oligonucleotide that contains:

N ₁ -C_G-N ₂ -C_G-N ₃

where each of N ₁ , N ₂ and N ₃ independently represents a nucleic acid sequence of 0-20 nucleotides in length, where _ denotes an internal phosphodiester or phosphodiester-like internucleotide bond, and where said oligonucleotide is not an antisense oligonucleotide, an oligonucleotide forming a triple helix, or ribozyme.

In another aspect, the present invention relates to an oligonucleotide that contains:

X ₁ -N ₁ - (GTCGTT) _n -N ₂ -X ₂ (SEQ ID NO: 18-20 and 57)

where each of N ₁ and N ₂ independently represents a nucleic acid sequence with a length of 0-20 nucleotides, where n = 2 or n = 4-6, and each of X ₁ and X ₂ independently represents a nucleic acid sequence having phosphorothioate internucleotide bonds and having a length of 3-10 nucleotides; and where N ₁ - (GTCGTT) _n -N ₂ contains at least one phosphodiester internucleotide bond, and the 3'- and 5'-nucleotides of said oligonucleotide do not contain poly-G-, poly-A-, poly-T- , or poly-C sequence.

In one embodiment of the invention, said nucleic acid has a backbone comprising deoxyribose or ribose.

In one embodiment, said oligonucleotide has a backbone comprising deoxyribose or ribose.

In one embodiment of the invention, said oligonucleotide is in a pharmaceutical composition optionally comprising a pharmaceutically acceptable carrier.

In one embodiment of the invention, said oligonucleotide further comprises an adjuvant or cytokine.

In one embodiment of the invention, said oligonucleotide further comprises an antigen, wherein said oligonucleotide is an adjuvant vaccine.

In one embodiment of the invention, said antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, and a tumor antigen. In one embodiment of the invention, said antigen is encoded by vector nucleic acid. In one embodiment of the invention, said antigen is a peptide antigen. In one embodiment of the invention, said antigen is covalently linked to an oligonucleotide or an immunostimulatory nucleic acid molecule. In another embodiment of the invention, said antigen is not covalently linked to an oligonucleotide or to an immunostimulatory nucleic acid molecule.

In another aspect, the present invention relates to a method for identifying the relative efficacy or toxicity of a test immunostimulatory nucleic acid molecule. The specified method involves the selection of a known immunostimulatory nucleic acid having a known sequence, a stabilized backbone and having a known immunostimulating activity or toxicity; selection of a test immunostimulatory nucleic acid having a known sequence, a phosphodiester or phosphodiester-like bond instead of a stabilized bond between Y and N of at least one internal YN dinucleotide in a known sequence, where Y is pyrimidine and N is any nucleotide and has a test immunostimulatory activity or toxicity; and comparing the test immunostimulatory activity or toxicity with known immunostimulatory activity or toxicity to identify the relative activity or toxicity of the test immunostimulatory nucleic acid molecule.

In one embodiment, the test immunostimulatory nucleic acid is a stronger inducer of activity directed to the transmission of the TLR9 signal than the known immunostimulatory nucleic acid.

In one embodiment, the test immunostimulatory nucleic acid is a stronger inducer of type 1 interferon than the known immunostimulatory nucleic acid.

In one embodiment, the test immunostimulatory nucleic acid is a stronger IP-10 inducer than the known immunostimulatory nucleic acid.

In one embodiment, YN is YG. In one embodiment, the at least one internal YG dinucleotide is CG. In one embodiment, the at least one internal YG dinucleotide is TG.

In one embodiment of the invention, the test immunostimulatory nucleic acid contains many internal YG dinucleotides, each of which has a phosphodiester or phosphodiester-like internucleotide bond. In one embodiment, the at least one internal YG dinucleotide is each internal YG dinucleotide.

In one embodiment, the phosphodiester or phosphodiester-like internucleotide bond is a phosphodiester. In one embodiment, the phosphodiester-like bond is borane phosphonate or diastereomerically pure Rp phosphorothioate.

In one embodiment of the invention, stabilized internucleotide bonds are selected from the group consisting of phosphorothioate, phosphordithioate, methylphosphonate, methylphosphorothioate, and any combination thereof. In one embodiment of the invention, the stabilized backbone contains many phosphorothioate internucleotide bonds.

In one embodiment, a known immunostimulatory nucleic acid molecule is a B-class immunostimulatory nucleic acid molecule. In one embodiment of the invention, a known immunostimulatory nucleic acid molecule is a C-class immunostimulatory nucleic acid molecule.

In one embodiment, a known immunostimulatory nucleic acid molecule has a length of 4-100 nucleotides. In one embodiment of the invention, a known immunostimulatory nucleic acid molecule has a length of 6-40 nucleotides. In one embodiment, a known immunostimulatory nucleic acid molecule has a length of 6-19 nucleotides.

In another aspect, the present invention relates to a method for constructing a stabilized immunostimulatory nucleic acid molecule having a length of less than 20 nucleotides. The specified method involves the selection of a sequence of 6-19 nucleotides in length, where the specified sequence contains at least one internal CG dinucleotide; selection of a phosphodiester or phosphodiester-like bond between C and G of at least one internal CG dinucleotide; independent selection of a phosphodiester, phosphodiester-like or stabilized bond between C and G of each attached internal CG dinucleotide; and selection of a stabilized bond for all other internucleotide bonds.

In another aspect, the present invention relates to a method for the treatment or prevention of allergies or asthma. The specified method is carried out by introducing to the subject described here immunostimulatory CpG-oligonucleotide in an amount effective for the treatment or prevention of allergies or asthma. In one embodiment, the oligonucleotide is delivered to the surface of the mucosa. In other embodiments, the oligonucleotide is administered as an aerosol composition. Said oligonucleotide may be administered intranasally.

In another aspect, the present invention relates to a method for inducing the production of cytokines. The method is carried out by administering to a subject an immunostimulatory CpG oligonucleotide described herein in an amount effective to induce a cytokine selected from the group consisting of IL-6, IL-8, IL-12, IL-18, TNF, IFN-α, chemokines and IFN-γ.

In another aspect, the present invention relates to a composition comprising the immunostimulatory CpG oligonucleotides described herein in combination with an antigen or with another therapeutic compound, such as an antimicrobial agent. The specified antimicrobial agent may be, for example, an antiviral agent, an antiparasitic agent, an antibacterial agent or an antifungal agent.

In another aspect, the present invention relates to a composition made in the form of a sustained release preparation comprising the immunostimulatory CpG oligonucleotides described herein.

Said composition may, but not necessarily, comprise a pharmaceutical carrier, and / or it may be formulated for delivery. In some embodiments, the delivery preparation is selected from the group consisting of cationic lipids; proteins that penetrate the cell, and drugs of prolonged action. In one embodiment, the sustained release preparation is a biodegradable polymer or microparticle.

In another aspect, the present invention relates to a method for stimulating an immune response. This method involves administering to a subject an immunostimulatory CpG oligonucleotide in an amount effective to induce an immune response in the subject. An immunostimulatory CpG oligonucleotide is preferably administered orally, topically, as a sustained release preparation, through the mucosa, systemically, parenterally or intramuscularly. If an immunostimulatory CpG oligonucleotide is introduced onto the surface of the mucosa, then it can be delivered in an amount effective to induce an immune response in the mucosa or a systemic immune response. In preferred embodiments, the mucosal surface is selected from the group consisting of the surface of the oral cavity, nose, rectum, vagina, and eye.

In some embodiments, the method comprises administering to the subject an antigen, wherein the produced immune response is an angigen-specific immune response. In some embodiments, said antigen is selected from the group consisting of a tumor antigen, a viral antigen, a bacterial antigen, a parasitic antigen, and a peptide antigen.

Immunostimulatory CpG oligonucleotides may have the ability to stimulate broad-spectrum immune responses. For example, these immunostimulatory CpG oligonucleotides can be used to reorient the Th2 immune response to the Th1 immune response. Immunostimulatory CpG oligonucleotides can also be used to activate immune cells, such as a lymphocyte (e.g., B and T cells), a dendritic cell, and an NK cell. Such activation can be carried out in vivo, in vitro or ex vivo, i.e., by isolating an immune cell from a subject, contacting said immune cell with an immunostimulatory CpG oligonucleotide in an amount effective to activate the immune cell, and then administering the activated immune cell to said subject. In some embodiments, the dendritic cell is a cancer antigen. Said dendritic cell may be exposed ex vivo to a cancer antigen.

The immune response produced by immunostimulatory CpG oligonucleotides can also lead to the induction of cytokine production, for example, the production of IL-6, IL-8, IL-12, IL-16, TNF, IFN-α, chemokines and IFN-γ.

In yet another embodiment, immunostimulatory CpG oligonucleotides can be used to treat a malignant tumor. In accordance with other aspects of the present invention, immunostimulatory CpG oligonucleotides can also be used to prevent a malignant tumor (e.g., to reduce the risk of developing a malignant tumor) in a subject at risk of developing a malignant tumor. A malignant tumor may be selected from the group consisting of bile duct cancer; breast cancer; cervical cancer; choriocarcinomas; colon and rectal cancer; endometrial cancer; stomach cancer; intraepithelial neoplasm; lymphomas liver cancer; lung cancer (e.g., small cell and non-small cell); melanomas; neuroblastomas; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; colorectal cancer; sarcomas; thyroid cancer and kidney cancer, as well as other carcinomas and sarcomas. In some preferred embodiments, the cancer is selected from the group consisting of bone cancer; brain cancer and central nervous system; connective tissue cancer; esophageal cancer; eye cancer; Hodgkin's lymphomas; laryngeal cancer; oral cancer; skin cancer and testicular cancer.

Immunostimulatory CpG oligonucleotides can also be used to increase the susceptibility of malignant cells to the treatment of a malignant tumor (e.g., anti-cancer therapy), optionally by administering an immunostimulating CpG oligonucleotide in combination with anti-cancer therapy. Such anti-cancer therapy may be chemotherapy, vaccine therapy (for example, using a vaccine based on in vitro primed dendritic cells or a vaccine based on cancer antigen) or therapy using antibodies. Antibody therapy may also include the administration of an antibody specific for a cell surface antigen, such as a cancer cell, where the immune response leads to the production of antibody-dependent cell cytotoxicity (ADCC). In one of the embodiments of the invention, the antibody can be selected from the group consisting of ributaxin, herceptin, quadramet, panorex, IDEC-Y2B8, BEC2, C225, oncolim, SMART M195, ATRAGEN, ovarex, bexar, LDP-03, ior t6, MDX -210, MDX-11, MDX-22, OV103, 3622W94, antibodies against VEGF, zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMab-G2, TNT, Gliomab-H , GHI-250, EMD-72000, LymphoCide, СМА-676, monopharm-C, 4B5, ior egf.r3, ior c5, BABS, antibodies against FLK-2, MDX-260, antibodies against ANA, antibodies against SMART 1D10, antibodies against SMART ABL364 and ImmuRAIT-CEA.

Thus, in accordance with some aspects of the present invention, a subject suffering from a malignant tumor or a subject at risk of developing a malignant tumor is administered an immunostimulatory CpG oligonucleotide in combination with anti-cancer therapy. In some embodiments, the anti-cancer therapy is selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, and an anti-cancer vaccine.

In yet another embodiment of methods aimed at preventing or treating a malignant tumor, interferon-α may be further administered to said subject.

In other aspects, the present invention relates to methods for preventing a disease in a subject. This method involves the regular administration to a subject of an immunostimulatory CpG oligonucleotide in order to stimulate the susceptibility of the subject's immune system to prevent this disease. Examples of diseases or conditions for the prevention of the prophylactic methods of the present invention are directed are microbial infections (for example, sexually transmitted diseases) and anaphylactic shock caused by food allergies.

In other aspects, the present invention relates to a method for inducing a natural immune response by administering to a subject an immunostimulatory CpG oligonucleotide in an amount effective to activate a natural immune response.

In another aspect, the present invention relates to a method for treating or preventing a viral or retroviral infection. The method comprises administering to a subject suffering from a viral or retroviral infection, or to a subject at risk of a viral or retroviral infection, any of the compositions of the present invention in an amount effective to treat or prevent a viral or retroviral infection. In some embodiments of the invention, such a viral infection is caused by a hepatitis B virus, for example, hepatitis B virus, hepatitis C virus, HIV, herpesvirus or papillomavirus.

In another aspect, the present invention relates to a method for treating or preventing a bacterial infection. This method comprises administering to a subject suffering from a bacterial infection, or to a subject at risk of developing a bacterial infection, any of the compositions of the present invention in an amount effective to treat or prevent a bacterial infection. In one embodiment of the invention, such a bacterial infection is caused by intracellular bacteria.

In another aspect, the present invention relates to a method for treating or preventing a parasitic infection, comprising administering to a subject suffering from a parasitic infection or a subject at risk of causing a parasitic infection any of the compositions of the present invention in an amount effective to treat or prevent a parasitic infection. In one embodiment of the invention, such a bacterial infection is caused by intracellular parasites. In another embodiment, said parasitic infection is caused by parasites other than helminths.

In some embodiments, the subject is a human, and in other embodiments, a non-human vertebrate and selected from the group consisting of a dog, cat, horse, cow, pig, turkey, goat, fish, monkey, chicken, rat, mouse and sheep.

In another aspect, the present invention relates to a method for inducing an immune TH1 response, comprising administering to said subject any of the compositions of the present invention in an amount effective to produce an immune Th1 response.

In another aspect, the present invention relates to a method for inducing an immune response, comprising administering to a subject in need thereof an effective amount of an immunostimulatory oligonucleotide 5'T * C * G * T * X ₁ * T * T3 ', where X ₁ has a length of 3 -30 nucleotides, * means the presence of a stabilized internucleotide linkage, and said oligonucleotide contains at least two phosphodiester internucleotide linkages.

In another aspect, the present invention relates to a method for treating an autoimmune disease, comprising administering to a subject suffering from an autoimmune disease, or a subject at risk of developing an autoimmune disease, any of the compositions of the present invention in an amount effective to treat or prevent an autoimmune disease.

In other embodiments, the oligonucleotide is administered to a subject in an amount effective to induce cytokine expression. Such a cytokine is selected, but not necessarily, from the group consisting of IL-6, TNF-α, IFN-α, IFN-γ and IP-10. In other embodiments, the oligonucleotide is administered to a subject in an amount effective to switch the Th2 immune response to the Th1 response.

In another aspect, the present invention relates to a method for treating airway remodeling, the method comprising administering to a subject an oligonucleotide containing a CG dinucleotide in an amount effective to treat airway remodeling. In one embodiment of the invention, such a subject is a subject suffering from asthma or chronic obstructive pulmonary disease, or a smoker. In another embodiment, the subject does not show asthma symptoms.

In one aspect, the present invention also relates to the use of the oligonucleotide of the present invention to stimulate an immune response.

In addition, the present invention relates to a method for producing an oligonucleotide to stimulate an immune response.

In another aspect, the present invention relates to a method for stimulating an immune response by administering to a subject an oligonucleotide of at least 5 nucleotides in length in an amount effective to stimulate an immune response, wherein said oligonucleotide contains at least one immunostimulatory dinucleotide motif, wherein the internucleotide linkage between the nucleotides of said dinucleotide has R-chirality, and where at least 70% of the other internucleotide linkages of said oligonucleotide have S-chirality.

Each of the limitations of the present invention may encompass various embodiments. It follows that each of the limitations of the present invention, containing any one element or combination of elements, may be included in each aspect of the present invention.

A brief description of the graphic material

. Тестируемыми олигонуклеотидами, показанными на фиг. 1А, являются SEQ ID NO:322, SEQ ID NO:323 и SEQ ID NO:324, а олигонуклеотидом позитивного контроля является SEQ ID NO:242. Тестируемыми олигонуклеотидами, показанными на фиг.1В, являются SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327 и SEQ ID NO:328, а олигонуклеотидом позитивного контроля является 5'TCG TCG TTT TGA CGT TTT GTC GTT 3' (SEQ ID NO:329). Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ). Указанные данные представляют собой среднее, полученное от шести доноров. Ниже графиков указаны уровни интерферона-альфа (пг/мл), секретируемого клетками, обработанными негативным контролем (средой), для каждого эксперимента.Figure 1 presents a series of graphs illustrating the levels of interferon-alpha (pg / ml) secreted from human PBMCs after treatment of these cells with oligonucleotides, which are indicated by numbers along the X axis of this graph and indicated by ▲, compared with the level secreted after treatment with oligonucleotide positive control indicated

. The test oligonucleotides shown in FIG. 1A are SEQ ID NO: 322, SEQ ID NO: 323, and SEQ ID NO: 324, and the positive control oligonucleotide is SEQ ID NO: 242. The test oligonucleotides shown in FIG. 1B are SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327 and SEQ ID NO: 328, and the positive control oligonucleotide is 5'TCG TCG TTT TGA CGT TTT GTC GTT 3 '(SEQ ID NO: 329). The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM). These data are the average obtained from six donors. The graphs below show the levels of interferon-alpha (pg / ml) secreted by cells treated with a negative control (medium) for each experiment.

. Тестируемыми олигонуклеотидами, показанными на фиг.2А, являются SEQ ID NO:322, SEQ ID NO:323 и SEQ ID NO:324, а олигонуклеотидом позитивного контроля является SEQ ID NO:242. Тестируемыми олигонуклеотидами, показанными на фиг.2В, являются SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327 и SEQ ID NO:328, а олигонуклеотидом позитивного контроля является SEQ ID NO:329. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ). Указанные данные представляют собой среднее, полученное от шести доноров. Ниже графиков указаны уровни IL-10 (пг/мл), секретируемого клетками, обработанными негативным контролем (средой), для каждого эксперимента.Figure 2 presents a series of graphs illustrating the levels of IL-10 (pg / ml) secreted from human PBMCs after treatment of these cells with oligonucleotides, which are indicated by numbers along the X axis of this graph and indicated by ▲, compared with the level secreted after treatment with the oligonucleotide positive control indicated

. The test oligonucleotides shown in FIG. 2A are SEQ ID NO: 322, SEQ ID NO: 323 and SEQ ID NO: 324, and the positive control oligonucleotide is SEQ ID NO: 242. The test oligonucleotides shown in FIG. 2B are SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327 and SEQ ID NO: 328, and the positive control oligonucleotide is SEQ ID NO: 329. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM). These data are the average obtained from six donors. The graphs below show the levels of IL-10 (pg / ml) secreted by cells treated with the negative control (medium) for each experiment.

. Тестируемыми олигонуклеотидами, показанными на фиг.3А, являются SEQ ID NO:322, SEQ ID NO:323 и SEQ ID NO:324, а олигонуклеотидом позитивного контроля является SEQ ID NO:329. Тестируемыми олигонуклеотидами, показанными на фиг.3В, являются SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327 и SEQ ID NO:328, а олигонуклеотидом позитивного контроля является SEQ ID NO:329. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ). Указанные данные представляют собой среднее, полученное от трех доноров. Ниже графиков указаны уровни TNF-альфа (пг/мл), секретируемого клетками, обработанными негативным контролем (средой) и ЛПС, для каждого эксперимента.Figure 3 presents a series of graphs illustrating the levels of TNF-alpha (pg / ml) secreted from human PBMCs after treatment of these cells with oligonucleotides, which are indicated by numbers along the X axis of this graph and indicated by ▲, compared to the level secreted after treatment with oligonucleotide positive control indicated

. The oligonucleotides tested shown in FIG. 3A are SEQ ID NO: 322, SEQ ID NO: 323 and SEQ ID NO: 324, and the positive control oligonucleotide is SEQ ID NO: 329. The test oligonucleotides shown in FIG. 3B are SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327 and SEQ ID NO: 328, and the positive control oligonucleotide is SEQ ID NO: 329. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM). These data are the average obtained from three donors. The graphs below show the levels of TNF-alpha (pg / ml) secreted by cells treated with the negative control (medium) and LPS for each experiment.

. Тестируемыми олигонуклеотидами, показанными на фиг. 4А, являются SEQ ID NO:322, SEQ ID NO:323 и SEQ ID NO:324, а олигонуклеотидом позитивного контроля является SEQ ID NO:329 (с полноразмерной основной цепью, модифицированной фосфортиоатом). Тестируемыми олигонуклеотидами, показанными на фиг. 4В, являются SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327 и SEQ ID NO:328, а олигонуклеотидом позитивного контроля является SEQ ID NO:329. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ). Указанные данные представляют собой среднее, полученное от трех доноров. Ниже графиков указаны уровни IL-6 (пг/мл), секретируемого клетками, обработанными негативным контролем (средой) и ЛПС, для каждого эксперимента.Figure 4 presents a series of graphs illustrating the levels of IL-6 (pg / ml) secreted from human PBMCs after treatment of these cells with oligonucleotides, which are indicated by numbers along the X axis of this graph and indicated by ▲, compared with the level secreted after treatment with the oligonucleotide positive control indicated

. The test oligonucleotides shown in FIG. 4A are SEQ ID NO: 322, SEQ ID NO: 323, and SEQ ID NO: 324, and the positive control oligonucleotide is SEQ ID NO: 329 (with a full length phosphorothioate modified backbone). The test oligonucleotides shown in FIG. 4B are SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, and SEQ ID NO: 328, and the positive control oligonucleotide is SEQ ID NO: 329. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM). These data are the average obtained from three donors. The graphs below show the levels of IL-6 (pg / ml) secreted by cells treated with the negative control (medium) and LPS for each experiment.

. Тестируемыми олигонуклеотидами, показанными на фиг.5А, являются SEQ ID NO:322, SEQ ID NO:323 и SEQ ID NO:324, а олигонуклеотидом позитивного контроля является SEQ ID NO:329. Тестируемыми олигонуклеотидами, показанными на фиг.5В, являются SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327 и SEQ ID NO:328, а олигонуклеотидом позитивного контроля является SEQ ID NO:329. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ). Указанные данные представляют собой среднее, полученное от трех доноров. Ниже графиков указаны уровни интерферона-гамма (пг/мл), секретируемого клетками, обработанными негативным контролем (средой) и ЛПС, для каждого эксперимента.5 is a series of graphs illustrating interferon-gamma levels (pg / ml) secreted from human PBMCs after treatment of these cells with oligonucleotides, which are indicated by numbers along the X axis of this graph and indicated by ▲, compared to levels secreted after treatment with the oligonucleotide positive control indicated

. The test oligonucleotides shown in FIG. 5A are SEQ ID NO: 322, SEQ ID NO: 323 and SEQ ID NO: 324, and the positive control oligonucleotide is SEQ ID NO: 329. The test oligonucleotides shown in FIG. 5B are SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327 and SEQ ID NO: 328, and the positive control oligonucleotide is SEQ ID NO: 329. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM). These data are the average obtained from three donors. The graphs below show the levels of interferon-gamma (pg / ml) secreted by cells treated with the negative control (medium) and LPS for each experiment.

. Тестируемыми олигонуклеотидами, показанными на фиг. 6А, являются SEQ ID NO:322, SEQ ID NO:323 и SEQ ID NO:324, а олигонуклеотидом позитивного контроля является SEQ ID NO:329. Тестируемыми олигонуклеотидами, показанными на фиг.6В, являются SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327 и SEQ ID NO:328, а олигонуклеотидом позитивного контроля является SEQ ID NO:329. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ). Указанные данные представляют собой среднее, полученное от трех доноров. Ниже графиков указаны уровни экспрессии CD69 на NK-клетках, обработанных негативным контролем (средой) и ЛПС, для каждого эксперимента.6 is a series of graphs illustrating expression levels of CD69 (MFI) on NK cells, which are indicators of NK cell activation after treatment of these cells with oligonucleotides, which are indicated by numbers along the X axis of this graph and indicated by ▲, and a positive control oligonucleotide, designated

. The test oligonucleotides shown in FIG. 6A are SEQ ID NO: 322, SEQ ID NO: 323, and SEQ ID NO: 324, and the positive control oligonucleotide is SEQ ID NO: 329. The test oligonucleotides shown in FIG. 6B are SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327 and SEQ ID NO: 328, and the positive control oligonucleotide is SEQ ID NO: 329. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM). These data are the average obtained from three donors. The graphs below show expression levels of CD69 on NK cells treated with negative control (medium) and LPS for each experiment.

, по сравнению с уровнями, полученными с применением олигонуклеотида позитивного контроля SEQ ID NO:242, обозначенного ●. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ).7 is a series of graphs illustrating the levels of interferon alpha (IFN-α) (7A) and IL-10 (7B) produced by human PBMCs after treatment of these cells with oligonucleotide SEQ ID NO: 313, indicated

compared to levels obtained using the positive control oligonucleotide SEQ ID NO: 242, indicated by ●. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

, по сравнению с уровнями, полученными с применением олигонуклеотида позитивного контроля SEQ ID NO:242, обозначенным ●. ODN негативного контроля представляет собой SEQ ID NO:330: tccaggacttctctcaggtt. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ).On Fig presents a series of graphs illustrating the levels of interferon-alpha (IFN-α) (8A) and IL-10 (8B) produced by human PBMC after treatment of these cells with the oligonucleotide SEQ ID NO: 314, indicated

compared to levels obtained using the positive control oligonucleotide of SEQ ID NO: 242, indicated by ●. The negative control ODN is SEQ ID NO: 330: tccaggacttctctctcaggtt. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

, по сравнению с уровнями, полученными с применением олигонуклеотида позитивного контроля SEQ ID NO:242, обозначенным ●. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ).Figure 9 presents a series of graphs illustrating the levels of interferon alpha (IFN-α) (9A) and IL-10 (9B) produced by human PBMCs after treatment of these cells with the oligonucleotide SEQ ID NO: 319, indicated

compared to levels obtained using the positive control oligonucleotide of SEQ ID NO: 242, indicated by ●. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

, по сравнению с уровнями, полученными с применением олигонуклеотида позитивного контроля SEQ ID NO:242, обозначенным ●. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ).Figure 10 presents a series of graphs illustrating the levels of interferon-alpha (IFN-α) (10A) and IL-10 (10B) produced by human PBMCs after treatment of these cells with oligonucleotide SEQ ID NO: 316, indicated

compared to levels obtained using the positive control oligonucleotide of SEQ ID NO: 242, indicated by ●. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

, по сравнению с уровнями, полученными с применением олигонуклеотида позитивного контроля SEQ ID NO:242, обозначенным ●. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ).11 is a series of graphs illustrating the levels of interferon alpha (IFN-α) (11A) and IL-10 (11B) produced by human PBMCs after treatment of these cells with the oligonucleotide SEQ ID NO: 317, indicated

compared to levels obtained using the positive control oligonucleotide of SEQ ID NO: 242, indicated by ●. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

, по сравнению с уровнями, полученными с применением олигонуклеотида позитивного контроля SEQ ID NO:242, обозначенным ●. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ).12 is a series of graphs illustrating the levels of interferon alpha (IFN-α) (12A) and IL-10 (12B) produced by human PBMCs after treatment of these cells with oligonucleotide SEQ ID NO: 320, indicated

compared to levels obtained using the positive control oligonucleotide of SEQ ID NO: 242, indicated by ●. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

, по сравнению с уровнями, полученными с применением олигонуклеотида позитивного контроля SEQ ID NO:242, обозначенным ●. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ).On Fig presents a series of graphs illustrating the levels of expression of CD86 on b cells (13A) and expression of CD80 on monocytes (13B) after treatment of these cells with the oligonucleotide SEQ ID NO: 313, indicated

compared to levels obtained using the positive control oligonucleotide of SEQ ID NO: 242, indicated by ●. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

, по сравнению с уровнями, полученными с применением олигонуклеотида позитивного контроля SEQ ID NO:242, обозначенным ●. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ).On Fig presents a series of graphs illustrating the levels of expression of CD86 on b cells (14A) and expression of CD80 on monocytes (14B) after treatment of these cells with the oligonucleotide SEQ ID NO: 314, indicated

compared to levels obtained using the positive control oligonucleotide of SEQ ID NO: 242, indicated by ●. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

, по сравнению с уровнями, полученными с применением олигонуклеотида позитивного контроля SEQ ID NO:242, обозначенным ●. Концентрация олигонуклеотида, используемого для получения экспериментальных точек, отложена по оси Х (мкМ).On Fig presents a series of graphs illustrating the levels of expression of CD86 on b cells (15A) and expression of CD80 on monocytes (15B) after treatment of these cells with the oligonucleotide SEQ ID NO: 319, indicated

compared to levels obtained using the positive control oligonucleotide of SEQ ID NO: 242, indicated by ●. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

On Fig presents a series of graphs illustrating the levels of expression of CD86 on b cells (16A) and expression of CD80 on monocytes (16B) after treatment of these cells with the oligonucleotide SEQ ID NO: 316, compared with levels obtained after treatment with the oligonucleotide positive control SEQ ID NO: 242 and oligonucleotide 5 'TCC AGG ACT TCT CTC AGG TT 3' (SEQ ID NO: 330). The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

On Fig presents a series of graphs illustrating the levels of interferon-alpha (IFN-α) (17A) and IL-10 (17B) produced by human PBMC cells after treatment of these cells with the oligonucleotide SEQ ID NO: 321, compared with the levels obtained after treatment with the control oligonucleotide SEQ ID NO: 242 and the oligonucleotide SEQ ID NO: 330. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

On Fig presents a series of graphs illustrating the levels of expression of CD86 on b cells (18A) and expression of CD80 on monocytes (18B) after treatment of these cells with the oligonucleotide SEQ ID NO: 321, compared with the levels obtained after treatment with the oligonucleotide positive control SEQ ID NO: 242 and oligonucleotide SEQ ID NO: 330. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

On Fig presents a series of graphs illustrating the levels of expression of CD86 on b cells (19A) and expression of CD80 on monocytes (19B) after treatment of these cells with oligonucleotide SEQ ID NO: 317, compared with the levels obtained after treatment with oligonucleotide positive control SEQ ID NO: 242 and oligonucleotide SEQ ID NO: 330. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

On Fig presents a series of graphs illustrating the levels of expression of CD86 on b cells (20A) and expression of CD80 on monocytes (20B) after treatment of these cells with oligonucleotide SEQ ID NO: 320, compared with levels obtained after treatment with oligonucleotide positive control SEQ ID NO: 242 and oligonucleotide SEQ ID NO: 330. The concentration of the oligonucleotide used to obtain the experimental points is plotted along the X axis (μM).

21 is a schematic representation of a portion of a nucleic acid molecule illustrating structural features, including bases (B), sugars, and a phosphodiester bond backbone (indicated by a circle) between 5′-cytidine and 3′-guanosine, and adjacent phosphorothioate bonds.

On Fig presents a histogram showing the relative amounts of phosphorothioate (SEQ ID NO: 242), soft oligonucleotide (SEQ ID NO: 294) and semi-soft oligonucleotide (SEQ ID NO: 241) in the tissues of the kidneys, spleen and liver after 48 hours after subcutaneous injection to mice. Oligonucleotides SEQ ID NO: 242 and SEQ ID NO: 241 have identical nucleotide sequences and differ in the composition of their main chain.

23 illustrates in vitro stimulation of human immune cells by inducing the cytokines IL-6, IL-10, IFN-α, and IP-10.

On Fig illustrates the stimulation of murine splenocytes in vitro by increasing the efficiency and / or activity of TLR9-associated cytokines IL-6, IL-10, IL-12p40, IFN-α, TNF-α and IP-10, used as inducers, without detectable secretion of IL-1, IL-2, IL-4, IL-5 or GM-CSF.

On Fig illustrates the expression of TLR9-associated genes (IL-6, TNF-α, IFN-α, IFN-γ and IP-10) in the lungs induced by ODN of the present invention (SEQ ID NO: 313).

FIG. 26 illustrates the effect of CpG-ODN on antigen-induced lymph node formation in mice in vivo.

On Fig demonstrated that CpG-ODN suppresses the Th2 response to sensitization by antigen.

FIG. 28 illustrates the effect on antigen-induced IgE production in mice in vivo.

On Fig demonstrated that antigenic stimulation causes an increase in the total number of leukocytes, mainly eosinophils, in the lumen of the respiratory tract.

In FIG. 30 and 32 demonstrate that antigenic stimulation causes an increase in the total number of leukocytes, mainly eosinophils, in the airway lumen, and that this stimulation is suppressed by the ODN of the present invention (SEQ ID NO: 313) depending on the dose.

In FIG. 31 and 32 demonstrate that antigenic stimulation causes hyperactivity of the airways and that this stimulation is suppressed by the ODN of the present invention (SEQ ID NO: 313) in a dose-dependent manner.

On Fig shows the concentration of ODN in plasma of rats after i.v. and i.t.-administration at 5 mg / kg. Data obtained for plasma showed that SEQ ID NO: 313 is excreted from plasma faster than SEQ ID NO: 329 after i.v. and i.t.-introduction.

On Fig shows the concentration of ODN in rat lungs after i.v. and i.t.-administration at 5 mg / kg. After i.v. administration at the same doses, the concentrations of SEQ ID NO: 313 in the lungs were lower than the concentrations of SEQ ID NO: 329. After i.t.-administration, these differences were less noticeable. Data for SEQ ID NO: 329 in the lungs were obtained only 48 hours after dosing.

On Fig shows the concentration of ODN in the kidneys of rats after i.v. and i.t.-administration at 5 mg / kg. Data obtained for the kidneys showed that the absolute levels of SEQ ID NO: 313 in the kidneys were lower than the corresponding concentrations of SEQ ID NO: 329 after i.v. and i.t.-introduction.

Treatment of the kidneys with the oligonucleotide SEQ ID NO: 313 after i.t.-administration showed, in particular, a marked decrease in the levels of this oligonucleotide compared to the levels observed after treatment with the same dose of SEQ ID NO: 329.

36 shows ODN concentrations in rat kidneys after i.v. administration at 5 mg / kg.

On Fig shows the concentration of ODN in the kidneys of rats after i.t.-administration at 5 mg / kg

On Fig shows the concentration of SEQ ID NO: 313 and its 8-dimensional (s) metabolite (s) in the kidneys of rats after i.v.-introduction of SEQ ID NO: 313 at 5 mg / kg

On Fig shows the concentration of SEQ ID NO: 313 and its 8-dimensional (s) metabolite (s) in the kidneys of rats after i.t.-administration of SEQ ID NO: 313 at 5 mg / kg

40 is a graph illustrating the stimulation index of a series of semi-soft ODNs compared to fully phosphorothioate ODNs having the same sequence.

41 is a bar graph illustrating the induction of cytokines A and B (IP-10), C (IFN) and D and E (TNF) in response to the administration of mild ODN (SEQ ID NO: 294), semi-soft ODN (SEQ ID NO : 241) and a fully phosphorothioate ODN (SEQ ID NO: 242).

On Fig presents a series of graphs illustrating the activity of antibodies and cytotoxic T-lymphocytes in response to the introduction of soft ODN (SEQ ID NO: 294), semi-soft ODN (SEQ ID NO: 241) and fully phosphorothioate ODN (SEQ ID NO: 242) .

43 is a series of graphs illustrating anti-tumor therapy in mice using a semi-soft ODN (SEQ ID NO: 241) and fully phosphorothioate ODN (SEQ ID NO: 242). In FIG. 43A and B show the results obtained in a renal cell carcinoma model. In FIG. 43C and D show the results obtained in a mouse neuroblastoma model. In FIG. 43E and F show the results obtained in a mouse model of non-small cell lung cancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to soft and semi-soft immunostimulatory nucleic acids. In some embodiments of the present invention, the immunostimulatory oligonucleotides described herein have improved properties, including similar or increased activity, reduced systemic effects on the kidneys, liver and spleen, and may have reduced reactivity at the injection site. Although the applicant does not intend to be limited to the described mechanism, he suggests that these improved properties are associated with the strategic location of phosphodiester or phosphodiester-like "internucleotide bonds" in immunostimulatory oligonucleotides. As used herein, the term “internucleotide linkage” means a covalent backbone linkage connecting two adjacent nucleotides in a nucleic acid molecule. Such a covalent backbone bond is typically a modified or unmodified phosphate bond, but other modifications are possible. Thus, a linear oligonucleotide that has a length of n nucleotides contains a total number of internucleotide bonds of n-1. In accordance with the description of the present invention, said covalent backbone bonds in immunostimulatory oligonucleotides may be modified or unmodified.

In particular, phosphodiester or phosphodiester-like internucleotide bonds are included in “internal dinucleotides”. The term "internal dinucleotide" in general terms means any pair of adjacent nucleotides connected by an internucleotide bond in which none of this pair of nucleotides is a terminal nucleotide, that is, none of this pair of nucleotides is a nucleotide that defines 5'- or 3 ' -end of this oligonucleotide. Thus, a linear oligonucleotide that has a length of n nucleotides contains a total of n-1 dinucleotides and only n-3 internal dinucleotides. Each internucleotide link in the internal dinucleotide is an internal internucleotide link. Thus, a linear oligonucleotide that has a length of n nucleotides contains a total of n-1 internucleotide bonds and only n-3 internal internucleotide bonds. Therefore, “strategically located phosphodiester or phosphodiester-like internucleotide bonds” means phosphodiester or phosphodiester-like internucleotide bonds located between any pair of nucleotides in a nucleic acid sequence. In some embodiments, there are no phosphodiester or phosphodiester-like internucleotide bonds between any pair of nucleotides closest to the 5 ′ or 3 ′ end.

In some of its aspects, the present invention is based on the unexpected discovery of the fact that the soft and semi-soft nucleic acids described herein have at least the same, or in many cases, greater immunostimulatory activity than the corresponding fully stabilized immunostimulatory oligonucleotides having the same nucleotide sequence . This fact was unexpected, since there is a widespread opinion that phosphorothioate oligonucleotides generally have greater immunostimulating activity than unstabilized oligonucleotides. These results were unexpected, since it was assumed that if the “softening” bond is located between important immunostimulating motifs, that is, CG, then this nucleic acid may have reduced activity, since such a nucleic acid should easily decompose to form fragments that do not contain CG, in vivo. Contrary to these expectations, many of these nucleic acids actually had equivalent or increased in vitro and in vivo activity. Obviously, soft and semi-soft oligonucleotides are as effective, if not more effective, as their fully stabilized analogues; however, the total immunostimulating effect of soft and semi-soft oligonucleotides is a balance between activity and stability. At high concentrations, this equilibrium is disturbed in favor of activity, that is, their activity becomes higher. At low concentrations, this equilibrium is disturbed in favor of stability, that is, the relative instability associated with nuclease susceptibility predominates.

In one of its aspects, the present invention relates to soft oligonucleotides. A soft oligonucleotide is an immunostimulatory oligonucleotide having a partially stabilized backbone in which phosphodiester or phosphodiester-like internucleotide bonds are present in at least one pyrimidine-purine (YZ) internal dinucleotide, or are at least as indicated internal dinucleotide. Preferred YZ is YG, the pyrimidine-guanosine dinucleotide (YG). This at least one internal YZ dinucleotide itself has a phosphodiester or phosphodiester-like internucleotide bond. Such a phosphodiester or phosphodiester-like internucleotide bond that is directly adjacent to at least one internal YZ dinucleotide may be a bond located on the 5 ′, 3 ′ or 5 ′ and 3 ′ ends with respect to at least to one internal dinucleotide YZ. Preferably, a phosphodiester or phosphodiester-like internucleotide linkage that is directly adjacent to at least one internal YZ dinucleotide is itself an internal internucleotide linkage. So, for example, for the sequence N ₁ YZN ₂ , where each of N ₁ and N ₂ independently represents any one nucleotide; the YZ dinucleotide has a phosphodiester or phosphodiester-like internucleotide bond, and in addition (a) N ₁ and Y are linked by a phosphodiester or phosphodiester-like internucleotide bond, if N ₁ is an internal nucleotide, (b) Z and N ₂ are linked by a phosphodiester or phosphodiester, if phosphodiester or phosphodiester, N ₂ is an internal nucleotide, or (c) N ₁ and Y are linked by a phosphodiester or fosfodiefiropodobnoy internucleotide linkage when N ₁ is an internal nucleotide and Z and N ₂ are linked by a phosphodiester or fosfodiefirop It is convenient internucleotide linkage when N ₂ is an internal nucleotide.

Non-limiting examples of soft oligonucleotides are oligonucleotides represented by SEQ ID NO: 105-231, SEQ ID NO: 232-234, SEQ ID NO: 235-237 and SEQ ID NO: 238-240.

Obviously, the soft oligonucleotides of the present invention are relatively susceptible to nuclease digestion compared to fully stabilized oligonucleotides. Without pretending to any particular theory or mechanism, the authors suggest that the soft oligonucleotides of the present invention can be cleaved to form fragments with reduced immunostimulatory activity, or no such activity, compared to full-sized soft oligonucleotides. It is obvious that the introduction of at least one internucleotide linkage sensitive to nuclease, in particular, almost in the middle of the oligonucleotide, leads to its “disconnection” and thereby to a change in the pharmacokinetics of this oligonucleotide, so that the duration of the maximum immunostimulatory activity decreases the specified oligonucleotide. This property can be especially important in tissues and in clinical applications where it is necessary to avoid damage associated with chronic local inflammation or immunostimulation, for example, in the kidneys.

In another aspect, the present invention relates to semi-soft oligonucleotides. A semi-soft oligonucleotide is an immunostimulatory oligonucleotide having a partially stabilized backbone in which phosphodiester or phosphodiester-like internucleotide bonds are present in at least one internal pyrimidine-purine dinucleotide (YZ). Semi-soft oligonucleotides usually have increased immunostimulatory activity compared to the corresponding fully stabilized immunostimulatory oligonucleotides. For example, the immunostimulatory activity of the semi-soft oligonucleotide SEQ ID NO: 241 is 2-5 times higher than the activity of the fully phosphorothioate oligonucleotide SEQ ID NO: 242, where these two oligonucleotides have the same nucleotide sequence and differ only in the internal internucleotide bonds of YZ, as shown below, where * means phosphorothioate, and _ means phosphodiester:

The oligonucleotide SEQ ID NO: 241 introduces internal phosphodiester internucleotide bonds, resulting in the formation of CG and TG dinucleotides (both YZ). Due to the higher activity of semi-soft oligonucleotides, these semi-soft oligonucleotides can be used at lower effective concentrations and have lower effective doses to achieve the desired biological effect than standard fully stabilized immunostimulatory oligonucleotides.

Despite the fact that fully stabilized immunostimulatory oligonucleotides can give the maximum dose-dependent response, dose-response curves for semi-soft oligonucleotides of the present invention exhibit a monotonic increase (as was analyzed by TLR9 stimulation), in which there is an increase in the effect with increasing concentrations exceeding the optimal concentration for the corresponding fully stabilized immunostimulatory oligonucleotides. Thus, it is obvious that the semi-soft oligonucleotides of the present invention can induce a higher degree of immunostimulation than fully stabilized immunostimulatory oligonucleotides.

In accordance with the present invention, it was found that the immunostimulatory activity of fully stabilized oligonucleotides with weak immunostimulatory activity can be increased by introducing at least one internal YZ dinucleotide with a phosphodiester or phosphodiester-like internucleotide linkage. Thus, one can start with an oligonucleotide with weak immunostimulating activity, having a fully stabilized backbone, and increase its immunostimulating activity by replacing the stabilized internucleotide linkage of at least one internal YG dinucleotide with a phosphodiester or phosphodiester-like internucleotide linkage. For example, it was found that the oligonucleotide SEQ ID NO: 243 has a greater immunostimulating activity than its fully stabilized analogue of SEQ ID NO: 244, where the oligonucleotide SEQ ID NO: 244 is a relatively weak immunostimulatory oligonucleotide compared to SEQ ID NO: 242 :

Although fully stabilized immunostimulatory nucleic acids having a length of less than 20 nucleotides may have moderate immunostimulatory activity compared to longer (for example, 24 nucleotides) fully stabilized oligonucleotides, it was found that semi-soft oligonucleotides having a length of at least at least 16 nucleotides, have an immunostimulating activity of at least equal to the immunostimulating activity of fully stabilized oligonucleotides, guides over the length of 20 nucleotides. So, for example, oligonucleotides of SEQ ID NO: 245 and 5602 (which are both 16-measures with sequences partially similar to SEQ ID NO: 242) have immunostimulatory activity comparable to the activity of SEQ ID NO: 242 (24-measure).

It was found that in some cases where the 6-dimensional phosphorothioate oligonucleotide does not have immunostimulating activity, replacing the phosphorothioate bond with even one phosphodiester internal internucleotide linkage YZ leads to the formation of the corresponding 6-measure with immunostimulating activity.

It is also apparent that the aforementioned properties of semi-soft oligonucleotides usually improve with an increase in the “number” of phosphodiester or phosphodiester-like internucleotide bonds present in the internal YZ dinucleotides. Thus, it is obvious that, mainly for a given oligonucleotide sequence with five internal YZ dinucleotides, an oligonucleotide with five internal phosphodiester or phosphodiester-like internucleotide bonds YZ has better immunostimulating properties than an oligonucleotide with four internal phosphodiester, or phosphodiester possesses better immunostimulating properties than an oligonucleotide with three internal phosphodiester or phosphodiester ester-like internucleotide bonds of YZ, which, in turn, has better immunostimulating properties than an oligonucleotide with two internal phosphodiester or phosphodiester-like internucleotide bonds of YZ, which, in turn, has better immunostimulatory properties than an oligonucleotide-olipheonodipheidophosphodiester . It is important to note that the content of even one internal phosphodiester or phosphodiester-like internucleotide linkage YZ may be preferable in comparison with the absence of an internal phosphodiester or phosphodiester-like internucleotide linkage YZ. In addition to the number of phosphodiester or phosphodiester-like internucleotide bonds, the position of these bonds along the entire length of the nucleic acid can also influence efficiency.

Non-limiting examples of soft oligonucleotides are oligonucleotides represented by SEQ ID NOS: 1-99 and 241 and SEQ ID NO: 100-104.

The immunostimulatory oligonucleotides of the present invention are usually protected from their rapid degradation in serum. The immunostimulatory oligonucleotides of the present invention are also usually protected from rapid degradation in most tissues, with the exception of tissues with specific or excessive nuclease activity, in which degradation of immunostimulatory oligonucleotides can occur. This leads to a decrease in the level of immunostimulatory oligonucleotides in these specific tissues, the accumulation of which can somehow lead to undesirable effects during long-term treatment with the use of degradation-resistant oligonucleotides. The oligonucleotides of the present invention, in addition to phosphodiester or phosphodiester-like internucleotide bonds in preferred internal positions, usually contain 5'- and 3'-ends, which are resistant to degradation. Such degradation resistant ends, as compared to the corresponding unmodified ends, can provide any suitable modification that leads to an increase in resistance to exonuclease cleavage. So, for example, the 5'- and 3'-ends can be stabilized by introducing at least one phosphate modification into the main chain. In a preferred embodiment of the present invention, the at least one phosphate modification in the main chain at each end is independently phosphorothioate, phosphorodioate, methylphosphonate or methylphosphorothioate internucleotide bonds. In another embodiment of the invention, the degradation resistant end comprises one or more nucleotide units connected by peptide or amide bonds at the 3'-end. Other stabilized ends also fall within the scope of the present invention, including but not limited to the ends described below.

As described above, the oligonucleotides of the present invention contain phosphodiester or phosphodiester-like bonds present in and optionally adjacent to internal YG dinucleotides. Such YG dinucleotides are often part of immunostimulatory motifs. However, it is not necessary that the oligonucleotide contains phosphodiester or phosphodiester-like bonds in each immunostimulating motif. An example is an oligonucleotide such as T * C * G * T * C * G * T * T * T * T * G * T * C * G * T * T * T * T * G * T * C * G * T * T (SEQ ID NO: 242), where four CpG dinucleotides can have phosphodiester bonds between C and G of the second, third or fourth CpG dinucleotide and any combination thereof. For faster decomposition in the kidneys of certain “stabilized oligonucleotides”, additional phosphodiester or phosphodiester-like bonds may also be preserved. Thus, for example, the oligonucleotide SEQ ID NO: 242 furthermore contains two internal TG dinucleotides, each of which are either individually or in combination with each other, or a combination of internal CG dinucleotides, and may have phosphodiester or phosphodiester-like internucleotide bonds.

The phosphodiester internucleotide bond is a type of bond characteristic of natural nucleic acids. As shown in FIG. 20, the phosphodiester internucleotide bond contains a phosphorus atom flanked by two bridged oxygen atoms and also bound by two additional oxygen atoms, one of which is charged and the other uncharged. A phosphodiester internucleotide bond is especially preferred if it plays an important role in decreasing the half-life of the oligonucleotide in the tissue.

A phosphodiester-like internucleotide bond is a phosphorus-containing bridging group that is chemically and / or diastereomeric similar to phosphodiester. A measure of similarity to the phosphodiester is sensitivity to nuclease cleavage and the ability to activate RNase N. So, for example, phosphodiester and not phosphorioate oligonucleotides are sensitive to nuclease cleavage, whereas RNAse H activate both phosphodiester and phosphorothioate oligonucleotides. In a preferred embodiment, the phosphodiester-like internucleotide bond is a borane phosphate (or equivalently borane phosphonate) bond. See US patent No. 5177198, US patent No. 5859231, US patent No. 6160109, US patent No. 6207819; Sergueev et al., (1998) J. Am. Chem. Soc. 120: 9417-27. In another preferred embodiment, the phosphodiester-like internucleotide bond is diastereomerically pure Rp phosphorothioate. Obviously, diastereomerically pure phosphoriate Rp is more sensitive to nuclease digestion and activates RNase H better than mixed or diastereomerically pure phosphorothioate Sp. The stereoisomers of CpG oligonucleotides are the subject of a co-pending application for US patent 09/361575, filed July 27, 1999, and published application PCT, PCT / US99 / 17100 (WO 00/06588). It should be noted that for the purposes of the present invention, the term "phosphodiester-like internucleotide linkage", in particular, does not contain phosphordithioate and methylphosphonate internucleotide linkages.

The immunostimulatory nucleic acid molecules of the present invention have a chimeric backbone. For the purposes of the present invention, the term “chimeric backbone” means a partially stabilized backbone, where at least one internucleotide bond is a phosphodiester or phosphodiester-like bond and where at least one other internucleotide bond is a stabilized internucleotide bond, at least at least one phosphodiester or phosphodiester-like bond, and at least one stabilized bond are different. Since boranephosphonate bonds have been reported to be stabilized compared to phosphodiester bonds, in order to determine the chimeric nature of the backbone, boranephosphonate bonds can be classified either as phosphodiester-like bonds or as stabilized bonds, as the case may be. So, for example, in one embodiment of the invention, the chimeric backbone of the present invention may contain at least one phosphodiester (phosphodiester or phosphorodioether) bond and at least one boranophosphonate (stabilized) bond. In another embodiment, the chimeric backbone of the present invention may contain boranophosphonate (phosphodiester or phosphodiester-like) and phosphorothioate (stabilized) bonds. The term “stabilized internucleotide linkage” means an internucleotide linkage that is relatively resistant to in vivo degradation (e.g., degradation by exonuclease or endonuclease) compared to a phosphodiester internucleotide linkage. Preferred stabilized internucleotide bonds include, but are not limited to, phosphorothioate, phosphordithioate, methylphosphonate and methylphosphorothioate bonds. Other stabilized internucleotide bonds include, but are not limited to, peptide bonds, alkyl bonds, dephospho bonds, and other bonds described above.

Modified backbones, such as phosphorothioates, can be synthesized using automated equipment using either phosphoramidate or H-phosphonate chemical synthesis methods. Aryl and alkyl phosphonates can be obtained, for example, as described in US Pat. No. 4,469,863, and alkyl phosphotriethers (in which the charged oxygen group is alkyl, as described in US Pat. No. 5023243 and European Patent Application No. 092574), can be obtained by automatic solid phase synthesis using commercially available reagents. Methods for introducing other modifications and substitutions into the DNA backbone have been described. Uhlmann, E. et al., (1990) Chem. Rev. 90: 544; Goodchild J. (1990) Bioconjugate Chem. 1: 165. Methods for producing chimeric oligonucleotides are also known in the art. Such methods are described, for example, in patents issued by Uhlmann et al.

A modified backbone ODN can be synthesized using a commercially available DNA synthesizer and standard phosphoramidite chemical synthesis methods (FE Eckstein, "Oligonucleotides and Analogs - A Practical Approach" IRL Press, Oxford, UK, 1991, and MD Matteucci & Caruthers MH , Tetrahedron Lett. 21: 719 (1980)). After attachment, PS bonds are introduced by sulfonation using a Bocage reagent (RP Iyer, W. Egan, JB Regan & SL Beaucage, J. Am. Chem. Soc., 112, 1253 (1990)) (0.075 M in acetonitrile) or phenylacetyl disulfide (PADS) followed by capping with acetic anhydride, 2,6-lutidine in tetrahydrofuran (1: 1: 8, vol: vol: vol) and N-methylimidazole (16% in tetrahydrofuran). This capping step is carried out after the sulfonation reaction in order to minimize the formation of undesired phosphodiester (PO) bonds at the positions where the phosphorothioate bond should be localized. In the case of introducing a phosphodiester bond, for example, into a CpG dinucleotide, intermediate phosphorus-III is oxidized by treatment with a solution of iodine in a water / pyridine mixture. After cleavage from the solid support and final deprotection by treatment with concentrated ammonia (15 hours at 50 ° C), the ODNs were analyzed by HPLC on a Gen-Pak Fax column (Millipore-Waters) using a NaCl gradient (e.g. buffer A: 10 mM NaH ₂ PO ₄ in acetonitrile / water = 1: 4 / v: v, pH 6.8; buffer B: 10 mM NaH ₂ PO ₄ , 1.5 M NaCl in acetonitrile / water = 1: 4 / v .: vol., 5-60% B, 30 minutes, 1 ml / min) or by electrophoresis in capillary gel. ODN can be purified by HPLC or HPLC on a high resolution Source High Performance column (Amersham Pharmacia). Homogeneous HPLC fractions are combined and desalted either on a C18 column or by ultrafiltration. ODNs are analyzed by MALDI-TOF mass spectrometry to confirm the calculated mass.

Nucleic acids of the present invention may also contain other modifications. Such modifications are nonionic DNA analogues, such as alkyl and aryl phosphates (in which the charged oxygen of the phosphonate is replaced by an alkyl or aryl group), phosphodiester and alkyl phosphotriesters in which the charged oxygen radical is alkyl. It has also been shown that nucleic acids containing a diol, such as tetraethylene glycol or hexaethylene glycol, at either or both ends are generally resistant to nuclease digestion.

The size (i.e., the number of nucleotide residues in a given nucleic acid) of an immunostimulatory oligonucleotide may also affect the stimulatory activity of a given oligonucleotide. To facilitate the penetration of immunostimulatory oligonucleotides into cells, they should preferably have a minimum length of 6 nucleotide residues. In accordance with the present invention, nucleic acids of any size exceeding 6 nucleotides (even a large number of kb) are capable of inducing an immune response if a sufficient number of immunostimulatory motifs are present, since a greater number of nucleic acids decompose within the cells. The authors of the present invention believe that semi-soft oligonucleotides containing at least 4 nucleotides may also have an immunostimulating effect if they can be delivered inside the cell. In some preferred embodiments, the immunostimulatory oligonucleotides have a length of 4-100 nucleotides. In typical embodiments, immunostimulatory oligonucleotides have a length of 6-40 nucleotides. In some preferred embodiments, the immunostimulatory oligonucleotides have a length of 6-19 nucleotides.

It was found that the oligonucleotides of the present invention are nucleic acids that contain specific sequences necessary for generating an immune response. These specific sequences that induce an immune response are called "immunostimulatory motifs", and oligonucleotides containing immunostimulatory motifs are called "immunostimulatory nucleic acid molecules" and similarly "immunostimulatory nucleic acids" or "immunostimulatory oligonucleotides". Immunostimulatory oligonucleotides of the present invention contain at least one immunostimulatory motif. In a preferred embodiment, the immunostimulating motive is an “internal immunostimulating motive”. The term “internal immunostimulatory motif” means that this motif sequence is located within a longer nucleic acid sequence that is at least one nucleotide longer than the sequence of the motif attached to both the 5'- and 3'-end of the immunostimulatory sequence given motive.

In some embodiments, immunostimulatory oligonucleotides comprise immunostimulatory motifs that are “CpG dinucleotides”. The CpG dinucleotide may be methylated or unmethylated. An immunostimulatory nucleic acid containing at least one unmethylated CpG dinucleotide is a nucleic acid molecule that contains the unmethylated cytosine guanine dinucleotide sequence (that is, unmethylated 5'-cytidine is followed by 3'-guanosine that are bound by a phosphate bond ) and which activates the immune system; moreover, such an immunostimulatory nucleic acid is a CpG-containing nucleic acid. CpG-containing nucleic acids have been described in various issued patents, in published patent applications and in other publications, including US Patent Nos. 6194388, 6207646, 6214806, 6218371, 6239116 and 6339068. An immunostimulatory nucleic acid containing at least one methylated CpG dinucleotide is a nucleic acid that contains the methylated cytosine-guanine dinucleotide sequence (that is, the methylated 5'-cytidine is followed by 3'-guanosine, which are bound by a phosphate bond) and which activates the immune This system. In other embodiments, the immunostimulatory oligonucleotides do not contain CpG dinucleotides. Such oligonucleotides that do not contain CpG dinucleotides are called non-CpG oligonucleotides and have immunostimulatory motifs that do not contain CpG. Therefore, the present invention also relates to nucleic acids with immunostimulatory motifs of other types, which may be methylated or unmethylated. In addition, the immunostimulatory oligonucleotides of the present invention may contain any combination of methylated and unmethylated immunostimulatory CpG and non-CpG motifs.

Regarding CpG-containing nucleic acids, it has recently been reported that there are various classes of CpG-containing nucleic acids. One class contains strong activators of B cells, but relatively weak inducers of activation of IFN-α and NK cells, and this class was called the B-class. B-class CpG-containing nucleic acids are typically fully stabilized and contain the unmethylated CpG dinucleotide with a certain preferred set of bases. See, for example, US Patent Nos. 6194388, 6207646, 6214806, 6218371, 6239116 and 6339068. Another class contains strong inducers of IFN-α and NK-cells activation, but relatively weak B-cell stimulants, and this class was called A-class . A-class CpG-containing nucleic acids typically have stabilized poly-G sequences at the 5'- and 3'-ends and a palindromic phosphodiester sequence containing a CpG dinucleotide of at least 6 nucleotides. See, for example, published patent application PCT / US00 / 26527 (WO 01/22990). Another class of CpG-containing nucleic acids activate B cells and NK cells and induce IFN-α; and this class was called the C-class. CpG-containing C-class nucleic acids, as first defined, are usually fully stabilized and contain a sequence type such as a B-class sequence and a GC-rich palindrome or a sequence close to the palindromic sequence. This class was described in the simultaneously pending provisional application for US patent No. 60/313273, filed August 17, 2001, and in US10 / 224523, filed August 19, 2002, which in its entirety are incorporated into this description by reference. Some non-limiting examples of C-class nucleic acids are:

Thus, one aspect of the present invention is based on the finding that specific subclasses of immunostimulatory CpG oligonucleotides having chimeric backbones are highly effective in mediating immunostimulatory effects. These CpG-containing nucleic acids can be used as therapeutic and prophylactic agents to stimulate the immune system to treat cancer, infectious diseases, allergies, asthma, autoimmune diseases and other disorders, and to protect the body from opportunistic infections after anti-cancer chemotherapy . Strong and, in addition, balanced cellular and humoral immune responses to CpG stimulation are the body's natural defense system against invading pathogens and cancer cells.

In one of its aspects, the present invention is based on the discovery of the fact that a subseries of immunostimulatory CpG oligonucleotides has improved immunostimulatory properties and has a reduced inflammatory effect on the kidneys. In some cases, inflammation of the kidneys has been observed in subjects who have been administered a fully phosphorothioate oligonucleotide. Obviously, the chimeric nucleic acids described herein have a weaker inflammatory effect on the kidneys than fully phosphorothioate oligonucleotides. In addition, these oligonucleotides are highly effective stimulants of the immune response. Thus, the phosphodiester region of this molecule does not reduce its effectiveness.

Preferred immunostimulatory CpG oligonucleotides have one of the following 6 general formulas:

In these formulas, N represents any nucleotide; N ₁ has 0-6 nucleotides, N ₂ has 0-7 nucleotides, N ₃ has 0-4 nucleotides, N ₄ has 1-5 nucleotides, N ₅ has 0-7 nucleotides, N ₆ and N ₇ independently have a length of 1 -5 nucleotides, N ₈ has a length of 4-10 nucleotides, N ₉ has a length of 0-3 nucleotides, and N ₁₀ has a length of 4-8 nucleotides. X ₁ , X ₂ , X _3, and X _{4 are} independently C or G. These formulas define subseries of the class of CpG oligonucleotides that exhibit superior immunostimulatory properties and are even more sensitive to degradation in the body than fully phosphorothioate oligonucleotides. In this formula, 5 'is the free 5'-end of the oligonucleotide, and 3' is the free 3'-end of the oligonucleotide.

The * symbol used in these formulas means the presence of a stabilized internucleotide bond. Internucleotide bonds not marked with an asterisk * may be stabilized or unstabilized, provided that the oligonucleotide contains at least 2-3 phosphodiester internucleotide bonds. In some embodiments, it is preferred that such oligonucleotides contain 3-6 phosphodiester bonds. In some cases, the bonds between CG motifs are phosphodiester, and in other cases, they are phosphorthioate or other stabilized bonds.

Other formulas are 5'TCGTCGTTTTGACGTTTTGTCGTT3 '(SEQ ID NO: 368), where at least one CG dinucleotide has a phosphodiester or phosphodiester-like internucleotide linkage and said oligonucleotide contains at least one stabilized C3 N represents a nucleic acid sequence of 4-10 nucleotides in length, which at least 50% consists of T and does not contain a CG dinucleotide; wherein said oligonucleotide contains at least one stabilized internucleotide linkage.

In some embodiments, the oligonucleotide has one of the following structures:

In these structures, the symbol _ means the presence of a phosphodiester internucleotide bond.

Some preferred examples of these structures are:

Immunostimulatory oligonucleotides typically have a length ranging from 4 to 100 nucleotides, and in some embodiments, from 10 to 40 nucleotides. The length of the oligonucleotide may be in the range from 16 to 24 nucleotides.

The terms "nucleic acid" and "oligonucleotide" also contain nucleic acids or oligonucleotides with substitutions or modifications, for example, in bases and / or sugars. For example, they contain nucleic acids having sugars in their backbone that are covalently linked to low molecular weight organic groups that are not a hydroxyl group at the 2'-position and are not a phosphate group or a hydroxy group at the 5'-position. Nucleic acids modified in this way may contain a 2'-O-alkylated ribose group. In addition, modified nucleic acids may contain sugars, for example arabinose or 2'-fluoroarabinose, instead of ribose. So, for example, in the composition of their backbone, nucleic acids can be heterogeneous, that is, they can contain any possible combination of polymer units linked together, such as “peptide-nucleic acids” (which have an amino acid backbone linked to nucleic acid bases )

Nucleic acids also contain substituted purines and pyrimidines, such as bases modified with C-5-propynpyrimidine and 7-dease-7-substituted purine. Wagner R.W. et al. (1996) Nat. Biotechnol. 14: 840-4. Purines and pyrimidines include, but are not limited to, adenine, cytosine, guanine, thymine, 5-methylcytosine, 5-hydroxycytosine, 5-fluorocytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine and other natural and unnatural nucleotide bases, substituted and unsubstituted aromatic groups. Other such modifications are well known to those skilled in the art.

The immunostimulatory oligonucleotides of the present invention, unlike natural RNAs and DNAs, can contain various chemical modifications and substitutions, including a phosphodiester internucleotide bridge, a β-D ribose unit and / or a natural nucleoside base (adenine, guanine, cytosine, thymine, uracil). Examples of chemical modifications are known to those skilled in the art and are described, for example, by Uhlmann E. et al. (1990) Chem. Rev. 90: 543; "Protocols for Oligonucleotides and Analogs" Synthesis and Properties & Synthesis and Analytical Techniques, S. Agrawal, Ed., Humana Press, Totowa, USA 1993; Crooke S.T. et al., (1996) Annu. Rev. Pharmacol Toxicol. 36: 107-129 & Hunziker J. et al. (1995) Mod. Synth. Methods 7: 331-417. The oligonucleotide of the present invention may have one or more modifications, where each modification is located in a specific phosphodiester internucleotide bridge, and / or in a specific β-D ribose unit, and / or in a specific position of the natural nucleotide base, in contrast to an oligonucleotide with the same a sequence that consists of natural DNA or RNA.

So, for example, the present invention relates to an oligonucleotide, which may contain one or more modifications, where each modification is independently selected from:

a) replacing the phosphodiester internucleotide linkage located at the 3 ′ and / or 5 ′ end of the nucleotide with a modified internucleotide bridge,

b) replacing the phosphodiester bridge located at the 3 ′ and / or 5 ′ end of the nucleotide with a dephospho bridge;

c) replacing a sugar phosphate link in the sugar phosphate backbone with another link;

d) replacing the β-D ribose unit with a modified sugar unit; and

e) replacing the natural nucleotide base with a modified nucleotide base.

More detailed examples of chemical modifications of the oligonucleotide are described below.

The phosphodiester internucleotide bond located at the 3 ′ and / or 5 ′ end of the nucleotide can be replaced with a modified internucleotide bond, wherein said modified internucleotide bond is selected, for example, from phosphorothioate, phosphordithioate, NR ¹ R ² -phosphoramidate, borane -hydroxybenzylphosphonate, phosphate- (C ₁ -C ₂₁ ) -O-alkyl ether, phosphate - [(C ₆ -C ₁₂ ) aryl- (C ₁ -C ₂₁ ) -O-alkyl] ether, (C ₁ -C ₈ ) alkylphosphonate and / or (C ₆ -C ₁₂ ) arylphosphonate bridges, (C ₇ -C ₁₂ ) -α-hydroxymethylaryl (for example, described in WO 95/01363), where (C ₆ -C ₁₂ ) aryl, (C ₆ -C _{2 0} ) aryl and (C ₆ -C ₁₄ ) aryl are optionally substituted with halogen, alkyl, alkoxy, nitro, cyano, and where R ¹ and R ^{2 are} independently hydrogen, (C ₁ -C ₁₈ ) alkyl, (C ₆ -C ₂₀ ) aryl, (C ₆ -C ₁₄ ) aryl- (C ₁ -C ₈ ) alkyl, and preferably hydrogen, (C ₁ -C ₈ ) alkyl, and preferably (C ₁ -C ₄ ) alkyl and / or methoxyethyl, or, R ¹ and R ² taken together with the nitrogen atom to which they are bonded form a 5-6 membered heterocyclic ring, which, in addition, may contain an additional heteroatom selected from O, S and N.

The phosphodiester bridge located at the 3 ′ and / or 5 ′ end of the nucleotide is replaced by a dephospho bridge (dephospho bridges are described, for example, in Uhlmann E. and Peymann A., “Methods in Molecular Biology”, Vol.20 , "Protocols for Oligonucleotides and Analogs", S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp. 355 ff), where the dephospho-bridge is selected, for example, from the group consisting of dephospho-bridge formalcetals, 3 '-thioformacetal, methylhydroxylamine, oxime, methylene dimethylhydrazo, dimethyl sulfonic and / or silyl groups.

The sugar-phosphate unit (i.e., β-D-ribose and the phosphodiester internucleotide bridge, together forming the sugar-phosphate unit) in the sugar-phosphate backbone (i.e. in the sugar-phosphate backbone consisting of sugar-phosphate units) can be replaced another link where this other link is, for example, suitable for constructing a morpholino derivative oligomer (as described, for example, by Stirchak EP et al. (1989) Nucleic Acids Res. 17: 6129-41), for example, by substitutions for the "morpholino derivative" link, or for the construction of a polyamide nucleino acid ("PNA", as described, for example, Nielsen P.E. et al. (1994) Bioconjug Chem. 5: 3-7), for example, by replacing a PNA backbone with a link, for example, 2-aminoethyl glycine.

The β-ribose unit or β-D-2'-deoxyribose unit can be replaced with a modified sugar unit, where the specified modified sugar unit is selected, for example, from β-D-ribose, α-D-2'-deoxyribose, L-2 ' -deoxyribose, 2'-F-2'-deoxyribose, 2'-F-arabinose, 2'-O- (C ₁ -C ₆ ) alkylribose, where 2'-O- (C ₁ -C ₆ ) alkylribose is preferably 2'-O-methylribose;2'-O- (C ₂ -C ₆ ) alkenyl ribose, 2 '- [O- (C ₁ -C ₆ ) alkyl-O- (C ₁ -C ₆ ) alkyl] ribose, 2'-NH ₂ -2' -deoxyribose, β-D-xylofuranose, α-arabinofuranose, 2,4-dideoxy-β-D-erythrohexopyranose and carbocyclic analogues (described, for example, Froehler J. (1992) Am. Chem. Soc. 114: 8320) and / or analogs of open-chain sugars (described, for example, by Vandendriessche et al. (1993) Tetrahedron 49: 7223) and / or analogues of bicyclo-sugars (described, for example, Tarkov M. et al. (1993) Helv. Chim. Acta 76: 481 )

In some preferred embodiments, said sugar is 2'-O-methylribose, in particular for one or both nucleotides linked by a phosphodiester or phosphodiester-like internucleotide linkage.

Nucleic acids also contain substituted purines and pyrimidines, such as bases modified with C-5-propynpyrimide and 7-dease-7-substituted purine. Wagner R.W. et al. (1996) Nat. Biotechnol. 14: 840-4. Purines and pyrimidines include, but are not limited to, adenine, cytosine, guanine, thymine, and other natural and unnatural nucleotide bases and substituted or unsubstituted aromatic groups.

A modified base is any base that is chemically different from the natural bases commonly found in DNA and RNA, such as T, C, G, A and U, but whose chemical structure is similar to the natural structure of these bases. Such a modified nucleotide base may be selected, for example, from hypoxanthine, uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5- (C ₁ -C ₆ ) alkylluracil, 5- (C ₂ -C ₆ ) alkenyluracil, 5- (C ₂ -C ₆ ) alkynyluracil, 5- (hydroxymethyl) uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5- (C ₁ -C ₆ ) alkylcytosine, 5- (C ₂ -C ₆ ) alkenylcytosine, 5- (C ₂ -C ₆ ) alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N ² -dimethylguanine, 2,4-diaminopurine, 8-azapurine, substituted 7- deazapurine, preferred o 7-dease-7-substituted and / or 7-dease-8-substituted purine, 5-hydroxymethylcytosine, N4-alkylcytosine, for example N4-ethylcytosine, 5-hydroxydeoxycytidine, 5-hydroxymethyldeoxycytidine, for example N4-alkyldeoxycycididine, for example 6-thiodeoxyguanosine and deoxyribonucleotides of nitropyrrole, C5-propynylpyrimidine and diaminopurine, for example 2,6-diaminopurine, inosine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, hypoxanthine or other modifications of natural nucleotides. This list is for illustrative purposes only and should not be construed as limiting the invention.

The specific formulas described herein define a sequence of modified bases. So, for example, the letter Y used here means a nucleotide containing cytosine or modified cytosine. As used herein, the term “modified cytosine” means a natural or non-natural analog of a pyrimidine base, cytosine, which can be replaced with this base without adversely affecting the immunostimulatory activity of said oligonucleotide. Modified cytosines include, but are not limited to, 5-substituted cytosines (e.g., 5-methylcytosine, 5-fluorocytosine, 5-chlorocytosine, 5-bromocytosine, 5-iodocytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-difluoromethyl cytosine substituted 5-alkynylcytosine), 6-substituted cytosines, N4-substituted cytosines (e.g. N4-ethylcytosine), 5-azacytosine, 2-mercaptocytosine, isocytosine, pseudoisocytosine, cytosine analogues with condensed ring systems (e.g., N, N, N-cytosine cytosine or phenoxazine) and uracil and its production s (e.g., 5-fluorouracil, 5-bromouracil, 5-bromviniluratsil, 4-thiouracil, 5-gidroksiuratsil, 5-propynyluracil). Some preferred cytosines are 5-methylcytosine, 5-fluorocytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine and N4-ethylcytosine. In another embodiment, the cytosine base is replaced by a universal base (e.g., 3-nitropyrrole, P-base), an aromatic ring system (e.g., fluorobenzene or difluorobenzene), or a hydrogen atom (d-spacer).

The letter Z as used herein means a guanine or modified guanine base. As used herein, the term “modified guanine” means a natural or non-natural analogue of a purine base, guanine, which can replace this guanine base without any negative effect on the immunostimulatory activity of said oligonucleotide. Modified guanines are, but are not limited to, 7-deazaguanine, 7-dease-7-substituted guanine (such as 7-dease-7- (C2-C6) alkynylguanine), 7-dease-8-substituted guanine, hypoxanthine, N2 -substituted guanines (e.g. N2-methylguanine), 5-amino-3-methyl-3H, 6H-thiazolo [4,5-d] pyrimidin-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine , indole, adenine, substituted adenines (e.g., N6-methyladenine, 8-oxoadenine), 8-substituted guanine (e.g., 8-hydroxyguanine and 8-bromo-guanine) and 6-thioguanine. In another embodiment, the guanine base is substituted with a universal base (eg, 4-methylindole, 5-nitroindole and K-base), an aromatic ring system (eg, benzimidazole or dichlorobenzimidazole, amide 1-methyl-1H- [1,2,4] triazole-3-carboxylic acid) or a hydrogen atom (d-spacer).

Oligonucleotides may have one or more available 5'-ends. In this case, modified oligonucleotides having two such 5'-ends can be obtained. This can be achieved by attaching two oligonucleotides via a 3'-3'-bond to form an oligonucleotide having one or two accessible 5'-ends. The 3'-3'-bond may be a phosphodiester, phosphorothioate or other modified internucleotide bridge. Methods for forming such bonds are known to those skilled in the art. For example, such relationships are described in Seliger H. et al., Oligonucleotide analogs with terminal 3'-3'-and 5'-5'-internucleotidic linkages as antisense inhibitors of viral gene expression, Nucleotides & Nucleotides (1991), 10 (1-3), 469-77 and Jiang et al., Pseudo-cyclic oligonucleotides: in vitro and in vivo properties, Bioorganic & Medicinal Chemistry (1999), 7 (12), 2727-2735.

In addition, 3'3'-linked nucleic acids, where the link between the 3'-terminal nucleotides is not a phosphodiester, phosphorothioate or other modified bridge, can be obtained using an additional spacer, such as a tri- or tetraethylene glycosphate group (Durand M. et al., Triple-helix formation by an oligonucleotide containing one (dA) 12 and two (dT) 12 sequences bridged by two hexaethylene glycol chains, Biochemistry (1992), 31 (38), 9197-204, US Patent No. 5658738 and US Patent US No. 5668265). Alternatively, the non-nucleotide linker can be obtained from ethanediol, propanediol or a basic deoxyribose unit (d-spacer) (Fontanel, Marie Laurence et al., Sterical recognition by T4 polynucleotide kinase of non-nucleosidic moieties 5'-attached to oligonucleotides; Nucleic Acids Research ( 1994), 22 (11), 2022-7) by the standard phosphoramidite method of chemical synthesis. Non-nucleotide linkers can be included all at once or in several stages, or they can be combined with each other, which allows you to provide any desired distance between the 3'-ends of two connected ODNs.

It has recently been reported that the immunostimulatory effect of CpG oligonucleotides is obviously achieved through their interaction with trap receptor 9 (TLR9). Hemmi H et al. (2000) Nature 408: 740-5. Thus, the activity of a TLR9 signal in response to the action of a CpG oligonucleotide or other immunostimulatory nucleic acid can be measured by detecting NF-kB, NF-kB-associated signals and the corresponding events and intermediate events that occur before the transmission of the NF-kB signal.

The oligonucleotides used in the present invention can be synthesized de novo using a number of procedures well known to those skilled in the art, for example, the β-cyanoethylphosphoramidite method (Beaucage SL & Caruthers MH (1981) Tet. Lett. 22: 1859) and the nucleoside H-phosphonate method (Garegg et al., Tet. Lett. 27: 4051-4054, 1986; Froehler et al., Nucl. Acid. Res. 14: 5399-5407, 1986; Garegg et al., Tet. Lett. 27: 4055-4058, 1986 ; Gaffney et al., Tet. Let. 29: 2619-2622, 1988). These chemical methods can be carried out using a variety of automated nucleic acid synthesizers commercially available. Such oligonucleotides are called synthetic oligonucleotides. The term "isolated oligonucleotide" usually means an oligonucleotide that has been separated from the components with which it is usually associated in nature. Thus, for example, an isolated oligonucleotide may be an oligonucleotide isolated from a cell, nucleus, mitochondria or chromatin.

These oligonucleotides are partially resistant to degradation (for example, are stabilized). The term “stabilized oligonucleotide molecule” means an oligonucleotide that is relatively resistant to in vivo degradation (for example, by exo or endonuclease). Nucleic acid stabilization can be accomplished by making modifications to its backbone. Oligonucleotides having phosphorothioate bonds have maximum activity, and these bonds protect the oligonucleotide from degradation by intracellular exo and endonucleases. Other modified oligonucleotides are phosphodiester-modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acids and nucleic acids modified with methylphosphonate, methylphosphorothioate, phosphordithioate, p-ethoxy, and combinations thereof.

Modified backbones, such as phosphorothioates, can be synthesized using an automated technique using phosphoramidate or H-phosphonate chemical synthesis methods. Aryl and alkyl phosphonates can be prepared, for example, as described in US Pat. No. 4,469,863, and alkyl phosphotriethers (in which the charged oxygen group is alkyl, as described in US Pat. No. 5023243 and European Patent No. 092574) can be prepared by automated solid phase synthesis methods. using commercially available reagents. Methods for making other modifications and substitutions to the DNA backbone are described, for example, by Uhlmann E. & Peyman A., Chem. Rev. 90: 544, 1990; Goodchild J. Bioconjugate Chem. 1: 165, 1990.

Other stabilized oligonucleotides are: nonionic DNA analogues, such as alkyl and aryl phosphates (in which the charged oxygen of the phosphonate is replaced by an alkyl or aryl group), a phosphodiester and alkyl phosphotriethers in which the charged oxygen group is alkyl. It has also been shown that nucleic acids containing a diol, such as tetraethylene glycol or hexaethylene glycol, at one or both ends are generally resistant to nuclease digestion.

Although the CpG effects in mice are well characterized, information regarding the human system is limited. Phosphorothioate CpG oligonucleotides with strong stimulating activity in the mouse system exhibit lower activity in the immune cells of humans and other non-rodent animals. The examples describe the preparation of a highly active human CpG motif and the characterization of its effects and mechanisms of action on human PCMCs, for example B cells and NK cells. DNA containing these CpG motifs and partially modified backbones has a strong stimulating effect on human peripheral blood cells, followed by the production of IL-6, IL-10, IP-10, TNF-α, IFN-α and IFN-γ. IFN-γ levels were increased compared to control levels. NK cells and T cells were also induced with an increase in CD69 expression levels.

In accordance with the present invention, it has been found that subseries of immunostimulatory CpG oligonucleotides have a strong immunostimulatory effect on human cells, such as NK cells, suggesting that these immunostimulatory CpG oligonucleotides are effective therapeutic agents for human vaccination and can be used for immunotherapy of a malignant tumor, asthma immunotherapy, general enhancement of immune function, increase in the number of hematopoietic cells after irradiation, or chemotherapy, for the treatment of autoimmune disease and for immunomodulation used for other purposes.

Thus, in some aspects of the present invention, immunostimulatory CpG oligonucleotides can be used as a vaccine for treating a subject at risk of developing an allergy or asthma, developing an infection caused by an infectious microorganism, or developing a malignant tumor in which a specific cancer antigen has been identified. Immunostimulatory CpG oligonucleotides can also be used without antigen or allergen to protect against infection, allergies or malignant tumors, in which case repeated administration of a dose can provide protection of the body for a longer time. As used herein, the term “subject at risk” refers to a subject who is at risk of contracting an infection caused by a pathogen, or a risk of developing a malignant tumor, or a risk of exposure to an allergen, or a risk of developing a malignant tumor. So, for example, a subject with a risk of disease can be a subject who plans to travel to the region where a certain type of infectious agent was detected; a person who, in his lifestyle or as prescribed by a doctor, takes water procedures that may contain infectious microorganisms, or a person who can be directly exposed to microorganisms, or it can be any person living in the region where the infectious microorganism has been identified or allergen. Subjects at risk of developing an infection are also indigenous people, to whom a medical institution recommends vaccination with specific infectious microorganisms-antigens. If such an antigen is an allergen and the subject develops allergic reactions to this particular antigen, as well as if such a subject is exposed to the action of this antigen, that is, during the seasonal maturation of pollen, then such a subject is at risk of exposure to this antigen. Subjects at risk of developing allergies or asthma are subjects who have identified an allergy or asthma but who do not suffer from an active disease during treatment with an immunostimulating CpG oligonucleotide, as well as subjects who are considered to be susceptible to the development of diseases caused by genetic or external factors.

A subject with a risk of developing a malignant tumor is a subject that has a high probability of developing a malignant tumor. Such subjects are, for example, subjects with genetic abnormalities whose presence has been shown to correlate with an increased likelihood of developing a malignant tumor, and subjects exposed to carcinogens such as tobacco, asbestos and other chemical toxins, or a subject who has previously undergone a course anti-cancer therapy, and a subject in remission. If a subject with a risk of developing a malignant tumor was exposed to an antigen specific to the type of malignant tumor that the subject is at risk of being exposed to and an immunostimulating CpG oligonucleotide, then the indicated malignant cells can be destroyed as they develop. If a tumor begins to develop in a given subject, then a specific immune response against the tumor antigen will be generated in such a subject.

In addition to using immunostimulatory CpG oligonucleotides for prophylactic treatment, the present invention also relates to the use of immunostimulatory CpG oligonucleotides for treating a subject suffering from infection, allergy, asthma, or cancer.

A subject having an infection is a subject that has been exposed to an infectious pathogen and has detectable levels of acute or chronic infection caused by the pathogen in its body. Immunostimulatory CpG oligonucleotides can be used alone or in combination with an antigen to produce an antigen-specific systemic immune response or an immune response produced by a mucosa that can reduce the level of an infectious pathogen or completely eradicate a given infectious pathogen. As used herein, the term "infectious disease" means a disease resulting from the presence of a foreign microorganism in the body of a subject. In this case, it is especially important to develop an effective vaccination and treatment strategy to protect the surfaces of the mucous membrane of the body, which is the main site serving as the entrance gate for the pathogen.

A subject suffering from an allergy is a subject who develops an allergic reaction to the action of an allergen, or a subject at risk of developing such a reaction. The term "allergy" means acquired hypersensitivity to a specific substance (allergen). Allergic conditions include, but are not limited to, eczema, allergic rhinitis or acute rhinitis, hay fever, conjunctivitis, bronchial asthma, urticaria (rash) and food allergies, as well as other atopic disorders.

Allergies are usually caused by the production of IgE antibodies against safe allergens. Cytokines that are induced by systemic or mucosal administration of immunostimulatory CpG oligonucleotides are predominantly cytokines of the class called Th1 (examples are IL-12, IP-10, IFN-α and IFN-γ), and these cytokines induce both humoral and cellular immune responses. Another major type of immune response associated with the production of IL-4 and IL-5 cytokines is the Th2 immune response. In general terms, it is apparent that allergic diseases are mediated by Th2-type immune responses. Since immunostimulatory CpG oligonucleotides have the ability to shift the immune response in a subject mainly from the Th2 response (which is associated with IgE antibody production and allergy) towards a balanced Th2 / Th1 response (which is a protective response to allergic reactions), for treatment or for the prevention of asthma and allergies in a subject, a dose of an immunostimulatory CpG oligonucleotide that is effective for inducing an immune response can be administered to him.

Thus, immunostimulatory CpG oligonucleotides have significant therapeutic efficacy in the treatment of allergic and non-allergic conditions, such as asthma. The level of Th2 cytokines, especially IL-4 and IL-5, increases in the respiratory tract of subjects with asthma. These cytokines stimulate important aspects of the asthmatic inflammatory response, including IgE isotype switching, eosinophil chemotaxis, and mast cell activation and growth. Th1 cytokines, especially IFN-γ and IL-12, can inhibit the formation of Th2 clones and the production of Th2 cytokines. The term "asthma" means a respiratory system disorder characterized by inflammation, narrowing of the airways and increased airway reactivity to inhaled agents. Asthma is often, although not always, associated with atopic or allergic symptoms.

"Subject suffering from a malignant tumor" is a subject in which malignant cells are detected. The tumor may be malignant or non-malignant. Cancer or tumors are, but are not limited to, bile duct cancer; brain cancer; mammary cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer, esophageal cancer; stomach cancer; intraepithelial neoplasm; lymphomas liver cancer; lung cancer (e.g. small cell and non-small cell); melanomas; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; kidney cancer, as well as other carcinomas and sarcomas. In one embodiment, the malignant tumor is leukemic reticuloendotheliosis, chronic myelogenous leukemia, T-cell leukemia of the skin, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma of the carcinoma of the breast, carcinoma of the breast, carcinoma of the breast, .

The term “subject” refers to a human or vertebrate animal, including, but not limited to, a dog, cat, horse, cow, pig, sheep, goat, turkey, chicken, primate, such as a monkey, and fish (as a species of aquaculture), for example salmon. Thus, the present invention can also be used for the treatment of malignant tumors and tumors, infections and allergies / asthma in non-human subjects. A malignant tumor is one of the main causes of death in pets (i.e. cats and dogs).

As used herein, the terms “treat,” “treat,” or “treatment,” referring to a disorder, such as an infectious disease, cancer, allergy, or asthma, means prophylactic treatment that enhances the subject's resistance to the development of the disease (e.g., infection, caused by a pathogen), or, in other words, helps to reduce the likelihood of a subject developing a disease (for example, infection by a pathogen), as well as treating a subject after developing a disease for him diseases with the specified disease (for example, to weaken or completely destroy the infection) or to prevent the progression of this disease.

If the specified CpG oligonucleotide is administered together with the antigen, then the specified subject may be exposed to such an antigen. As used herein, the term “exposed” refers to either the active step of contacting a subject with an antigen, or the passive action of an antigen on a subject in vivo. Active antigen exposure methods to a subject are well known in the art. In general terms, an antigen is administered directly to a subject by any means, such as intravenous, intramuscular, oral, transdermal, transmucosal, intranasal, intratracheal or subcutaneous administration. The antigen may be administered systemically or topically. Methods for administering an antigen and an immunostimulatory CpG oligonucleotide are described in more detail below. It is believed that a subject is exposed to a passive antigen if the antigen becomes available for interaction with immune cells in the body. A subject may be exposed to a passive antigen, for example, by entering a foreign pathogen into the body or by developing a tumor cell expressing a foreign antigen on its surface.

The methods used for passive exposure of an antigen to a subject may, in particular, depend on the time of administration of the immunostimulatory CpG oligonucleotide. So, for example, a subject with a risk of developing a malignant tumor, or an infectious disease, or an allergic or asthmatic reaction can be regularly introduced immunostimulating CpG oligonucleotides if this risk is highest, that is, during a seasonal allergic reaction or after exposure to an agent that causes a malignant tumor. In addition, an immunostimulatory CpG oligonucleotide can be administered to tourists before they visit other countries in which they may be infected by infectious agents. Similarly, an immunostimulatory CpG oligonucleotide can be administered to soldiers or civilians at risk due to hostilities using bacteriological weapons to induce a systemic immune response or mucosal immune response to this antigen after administration.

As used herein, the term “antigen” means a molecule capable of stimulating an immune response. Antigens include, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and non-peptide mimetics of polysaccharides and other molecules, small molecules, lipids, glycolipids, carbohydrates, viruses and viral extracts, and multicellular organisms, such as parasites and allergens. In a broad sense, the term "antigen" contains a molecule of any type that is recognized by the host immune system as foreign. Antigens include, but are not limited to, cancer antigens, microbial antigens, and allergens.

As used herein, the term “cancer antigen” means a compound, such as a peptide or protein, associated with the cell surface of a tumor or cancer cell and capable of stimulating an immune response when expressed on the surface of an antigen-presenting cell in the presence of a corticosteroids molecule. Cancer antigens can be obtained from cancer cells or from untreated extracts of cancer cells, for example, as described by Cohen P.A. et al. (1994) Cancer Research 54: 1055, either by partial purification of antigens using recombinant DNA techniques, or by synthesizing known de novo antigens. Cancer antigens include, but are not limited to, recombinantly expressed antigens, their immunogenic portion, or the entire tumor or malignant tumor. Such antigens can be isolated or obtained by recombinant methods or any other known methods.

As used herein, the term “microbial antigen” means an antigen of a microorganism, and such antigens include, but are not limited to, virus, bacteria, parasites and fungi. Such antigens are intact microorganisms, their natural isolates, fragments or derivatives, as well as synthetic compounds that are identical or similar to the natural antigens of the microorganism and induce an immune response specific to such a microorganism. A compound is analogous to a natural antigen of a microorganism if it induces an immune response (humoral and / or cellular response) to the action of a natural antigen of the microorganism. Such antigens are widely used and are well known in the art.

Examples of viruses that have been found in humans include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 (also referred to as HDTV-III, LAVE, or HTLV-III / LAV or HIV-III; and other isolates, such as HIV-LP); Picornaviridae (e.g. polioviruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, ECHO viruses); Calciviridae (e.g. strains causing gastroenteritis); Togaviridae (e.g. horse encephalitis viruses measles rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow l viruses fever); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabiviruses); Filoviridae (e.g., Ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytia virus e.g. influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunyaviruses, phleboviruses and nairoviruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, poliomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, chickenpox virus, cytomegalovirus (CMV), herpes viruses); Poxviridae (smallpox viruses, vaccinia viruses, poxviruses) and Iridoviridae (e.g. swine fever virus) and unclassified viruses (e.g. hepatitis delta causing agent (presumably defective hepatitis B satellite satellite), non-A causing agents non-B hepatitis (class 1 = transmitted internally; class 2 = transmitted parenterally (i.e. hepatitis C); Norwalk viruses and related viruses and astroviruses).

Gram-negative and gram-positive bacteria can serve as antigens in invertebrates. Such gram-positive bacteria are, but are not limited to, bacteria of the species Pasteurella, Staphylococcus and Streptococcus. Gram-negative bacteria include, but are not limited to, bacteria of the species Escherichia coli, Pseudomonas, and Salmonella. Specific examples of infectious bacteria include, but are not limited to: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (group A streptococci), Streptococcus agalactiae (group B streptococci), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovisocecocococcus, Streptocococcus, Streptococococcus, Streptocococcus, Streptocococcus, Streptocococcus, Streptocococcus, Streptococococcus, Streptococococcus, Streptocococcus Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pastureact mult s sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia and Actinomyces israelli.

Examples of fungi are: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamidia trachomatis, Candida albicans.

Other infectious microorganisms (i.e. protists) are Plasmodium spp. Such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax and Toxoplasma gondii. Blood parasites and / or tissue parasites are Plasmodium spp., Babesia microti, Babesia divergens, Leishmania tropica, Leishmania spp., Leishmania braziliensis, Leishmania donovani, Trypanosoma gambiense and Trypanosoma rhodesiense (African Squamosa disease) .

Other medically important microorganisms are widely described in the literature; see, for example, C.G.A. Thomas, Medical Microbiology, Bailliere Tindall, Great Britain, 1983, which is incorporated herein by reference in its entirety.

The term “allergen” means a substance (antigen) that can induce an allergic or asthmatic response in a susceptible subject. The list of allergens is huge and can contain pollen, insect poisons, animal dander, dust, fungal spores, and medicines (like penicillin). Examples of natural animal and plant allergens are, but are not limited to, proteins specific to the following species: Canine (Canis familiaris); Dermatophagoides (e.g., Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinoasa); Betula (Betula verrucosa); Quercus (Quercus alba) Olea europa); Artemisia {Artemisia vulgaris); Plantago (e.g., Plantago lanceolata); Parietaria (e.g., Parietaria officinalis or Parietaria judaica); Blattella (e.g., Blattella germanica); Apis (e.g., Apis multiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei); Thuya (e.g., Thuya orientalis); Chamaecyparis (e.g. Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana); Agropyron (e.g., Agropyron repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticum aestivum); Dactylis (e.g., Dactylis glomerata); Festuca (e.g., Festuca elatior); Poa (e.g. Poa pratensis or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g., Holcus lanatus); Anthoxanthum (e.g., Anthoxanthum odoratum); Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g., Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalaris arundinacea); Paspalum (e.g., Paspalum notatium); Sorghum (e.g. Sorghum halepensis); and Bromus (e.g., Bromus inermis).

As used herein, the term “substantially purified” refers to a polypeptide that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is usually associated in nature. Each specialist can isolate viral or bacterial polypeptides using standard protein purification methods. Basically, a pure polypeptide often gives one main band on a non-reducing polyacrylamide gel. In the case of partially glycosylated polypeptides or polypeptides having several start codons, they can give several bands on a non-reducing polyacrylamide gel, but they will give a picture characteristic of this polypeptide. The purity of the viral or bacterial polypeptide can also be determined by analysis of the amino-terminal amino acid sequence. The present invention also contains other types of antigens that are not encoded by the nucleic acid vector, such as polysaccharides, small molecules, mimetics, and the like.

The oligonucleotides of the present invention can be administered to a subject together with an antimicrobial agent. As used herein, the term “antimicrobial agent” means a natural or synthetic compound capable of killing or inhibiting infectious microorganisms. The type of antimicrobial agent suitable for use in the present invention depends on the type of microorganism that the subject is infected with or at risk of being infected with the subject. Antimicrobial agents include, but are not limited to, antibacterial agents, antiviral agents, antifungal agents, and antiparasitic agents. Terms such as "anti-infective agent", "anti-bacterial agent", "antiviral agent", "antifungal agent", "antiparasitic agent" and "parasitocide" have meanings well known to those skilled in the art and as defined in the generally available medical literature. In short, antibacterial agents that kill or inhibit bacteria are antibiotics, as well as other synthetic or natural compounds that have similar functions. Antibiotics are low molecular weight compounds produced as secondary metabolites by cells, such as microorganisms. In general terms, antibiotics inhibit one or more bacterial functions or structures that are specific for a given microorganism and which are absent in host cells. Antiviral agents can be isolated from natural sources or synthesized and can be used to kill or inhibit viruses. Antifungal agents are used to treat superficial fungal infections, as well as opportunistic and primary systemic fungal infections. Antiparasitic agents kill parasites or inhibit their development.

Examples of antiparasitic agents, also referred to as parasitocides suitable for administration to humans, include, but are not limited to, albendazole, amphotericin B, benznidazole, bithionol, chloroquine-HCl, chloroquine phosphate, clindamycin, dehydroemethine, diethylcarbamazine, furofluorodinoxidine, glurofenoxidine, furofluorodinoxidine, glurofenoxide, furodoloxidonite , halofantrine, iodhinol, ivermectin, mebendazole, mefloquine, meglumine antimoniate, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquin, paramomycin, pentamidine isethionate, piperazine, praz quantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethanine sulfonamides, pyrimethanine sulfadoxine, quinacrin-HCl, quinine sulfate, quinidine gluconate, spiramycin, sodium stibogluconate (sodium stibogluconate), trisinolimene, trimerizin tetraminocin trimerizin tetrazin tetrazin tetrazin tetrazin tetrazin tetracyne Sulfamethoxazole and triparsamide, some of which are used alone or in combination with each other.

Antibacterial agents kill or inhibit the growth or function of bacteria. A large class of antibacterial agents are antibiotics. Antibiotics that are effective in killing or inhibiting a wide variety of bacteria are called broad-spectrum antibiotics. Other types of antibiotics are antibiotics, the action of which is directed mainly against bacteria belonging to the class of gram-positive or gram-negative bacteria. Antibiotics of this type are called narrow-spectrum antibiotics. Antibiotics of a different type, which are effective against one microorganism or disease, but ineffective against bacteria of another type, are called antibiotics of a limited spectrum of action. Antibacterial agents are sometimes classified according to their basic mechanism of action. In general terms, antibacterial agents are cell wall synthesis inhibitors, cell membrane synthesis inhibitors, protein synthesis inhibitors, nucleic acid synthesis inhibitors, or functional inhibitors and competitive inhibitors.

Antiviral agents are compounds that prevent the infection of cells by viruses or the replication of the virus in this cell. There are far fewer antiviral drugs than antibacterial drugs, because the virus replication process is so closely associated with DNA replication in the host cell that non-specific antiviral agents are in most cases toxic to the host. The virus infection process has several stages that can be blocked or inhibited by antiviral agents. Such stages are the attachment of the virus to the host cell (immunoglobulin or binding peptides), the loss of the envelope by the virus (e.g., amantadine), the synthesis or translation of viral mRNA (e.g., interferon), the replication of viral RNA or DNA (e.g., nucleotide analogues), the maturation of new viral proteins (eg, protease inhibitors), as well as "badging" and the release of the virus.

Nucleotide analogues are synthetic compounds that are similar to nucleotides but have an incomplete or abnormal deoxyribose or ribose group. After nucleotide analogues are incorporated into the cell, they are phosphorylated to form triphosphate, which competes with normal nucleotides for incorporation into viral DNA or RNA. After the triphosphate form of the nucleotide analogue is included in the growing nucleic acid chain, irreversible binding to viral polymerase occurs, as a result of which the chain growth stops. Nucleotide analogues include, but are not limited to, acyclovir (used to treat a disease caused by herpes simplex virus and chickenpox), ganciclovir (used to treat a disease caused by cytomegalovirus), idoxuridine, ribavirin (used to treat a disease caused by respiratory syncytial virus ), dideoxyinosine, dideoxycytidine and zidovudine (azidothymidine), imichimod and resimichimod.

Interferons are cytokines that are secreted by virus-infected cells, as well as immune cells. Interferons function by binding to specific receptors on cells adjacent to infected cells, which leads to changes in the cells that provide them with protection against virus infection. α- and β-interferons also induce the expression of class I and class II MHC molecules on the surface of infected cells, which leads to an increase in the level of presentation of the antigen for recognition by immune host cells. α- and β-interferons are available in recombinant forms and are used to treat chronic infectious hepatitis B and C. But at doses effective for antiviral therapy, interferons give serious side effects such as fever, general malaise, and weight loss.

Antiviral agents that can be used in the present invention include, but are not limited to, immunoglobulins, amantadine, interferons, nucleotide analogues and protease inhibitors. Specific examples of antiviral agents include, but are not limited to, acemannan, acyclovir, acyclovir-sodium, adefovir, alovudine, alficept-sudotox, amantadine hydrochloride, aranotine, aryldon, atevirdine mesylate, hydrochloride, cidofovirdecyl delivide, cidofovirilovirus, delvidilovirus, delvidovilida, tsipovyrilovida, tsipovyrilovida, tsipovyrilovida , didanoxine, disoxaril, edoxudine, enviradine, eniroxime, famciclovir, famotine hydrochloride, fiacacytabine, phialuridine, fosarylate, foscarnet sodium, phosphonet sodium, ganciclovir, ganciclovir sodium, idoxuridine, kdoxuridine, , lobucavir, memotine hydrochloride, metizazone, nevirapine, penciclovir, pyrodavir, ribavirin, sahinavir mesylate, somantadine hydrochloride, sorivudine, statulon, stavudine, tilorone hydrochloride, trifloridin phosphate, hydrochloride of the species, triluridin phosphate hydrochloride, trichloridin phosphate hydrochloride, hydrochloride phosphate , zidovudine and zinviroksim.

Antifungal agents can be used to treat and prevent infectious fungal diseases. Antifungal agents are sometimes classified by their mechanism of action. Some antifungal agents function as inhibitors of cell wall synthesis by inhibiting glucose synthase. Such agents include, but are not limited to, Baziungin / ERUs. Other antifungal agents function by destabilizing membrane integrity. Such agents include, but are not limited to, imidazoles such as clotrimazole, sertaconazole, fluconazole, itraconazole, ketoconazole, miconazole and voriconazole, as well as FK 463, amphotericin B, BAY 38-9502, MK 991, pradimycin, UK 292, butene terbinafine. Other antifungal agents function through the destruction of chitin (e.g. chitinase) or immunosuppression (501 cream).

Immunostimulatory CpG oligonucleotides can be combined with other therapeutic agents, such as adjuvants, to enhance immune responses. An immunostimulatory CpG oligonucleotide and another therapeutic agent may be administered simultaneously or sequentially. If other therapeutic agents are administered at the same time, they can be administered in the same composition or in separate compositions, but at the same time. Other therapeutic agents are administered sequentially with another therapeutic agent and with an immunostimulatory CpG oligonucleotide, if the administration of other therapeutic agents and an immunostimulatory CpG oligonucleotide does not coincide in time. The time period between the administration of these compounds may be several minutes or more. Other therapeutic agents include, but are not limited to, adjuvants, cytokines, antibodies, antigens, and the like.

The compositions of the present invention can also be administered in combination with non-nucleic acid adjuvants. The non-nucleic acid adjuvant may be any molecule or compound, with the exception of the immunostimulatory CpG oligonucleotides described herein, which can stimulate a humoral and / or cellular immune response. Non-nucleic acid adjuvants may be, for example, adjuvants producing a depot effect, immunostimulating adjuvants and adjuvants producing a depot effect and stimulating the immune system.

Immunostimulatory CpG oligonucleotides can also be used as mucosal adjuvants. It was previously found that a systemic immune response and an immune response generated in the mucosa are induced by introducing CpG-containing nucleic acids into the mucosa. Thus, these oligonucleotides can be administered in combination with other mucosal adjuvants.

Immune responses can also be induced or enhanced by co-administration or co-expression of cytokines (Bueler & Mulligan, 1996; Chow et al., 1997; Geissler et al., 1997, Iwasaki et al., 1997; Kim et al., 1997) or co-stimulating B-7 molecules (Iwasaki et al., 1997; Tsuji et al., 1997) with immunostimulatory CpG oligonucleotides. As used herein, the term “cytokine” means the generic name of another group of soluble proteins and peptides that act as regulators of the humoral response in nanopicomolar concentrations and which under normal or pathological conditions modulate the functional activity of individual cells and tissues. These proteins also mediate direct interaction between cells and regulate processes that occur outside the cell. Examples of cytokines include, but are not limited to, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), interferon-γ (γ-IFN), IFN-α, tumor necrosis factor (TNF), TGF-β, ligand FLT-3 and ligand CD40 .

Oligonucleotides can also be used to reorient the immune response from the Th2 response to the Th1 response. This leads to the generation of a relatively balanced Th1 / Th2 response. The reorientation of the Th2 immune response to the Th1 response can be determined by measuring the levels of cytokines produced in response to a nucleic acid (for example, by inducing monocytic cells and other cells so that they produce Th1 cytokines, including IL-12, IFN-γ γ and GM-CSF). Reorienting or shifting the equilibrium from the Th2 immune response to the Th1 response is especially effective for treating or preventing asthma. For example, an effective amount for treating asthma may be an amount that is effective for reorienting the Th2 type immune response that is associated with asthma to a Th1 type response or a balanced Th1 / Th2 response. Levels of Th2 cytokines, and especially IL-4 and IL-5, are increased in the airways of subjects with asthma. The immunostimulatory CpG oligonucleotides of the present invention cause an increase in the levels of Th1 cytokines, which helps to shift the balance in the immune system and prevent or attenuate side effects associated with the prevailing Th2 immune response.

The oligonucleotides of the present invention can also be used to treat airway remodeling. Airway remodeling is the result of smooth muscle cell proliferation and / or thickening of the submucosal airways, which ultimately leads to a narrowing of the airways and thereby limiting air intake. The oligonucleotides of the present invention can prevent subsequent remodeling and can even inhibit tissue formation as a result of the remodeling process.

These oligonucleotides can also be used to stimulate the survival, differentiation, activation and maturation of dendritic cells. Immunostimulatory CpG oligonucleotides have a unique ability to stimulate the survival, differentiation, activation and maturation of dendritic cells.

Immunostimulatory CpG oligonucleotides also contribute to an increase in the cytolytic activity of natural killer cells and antibody-dependent cellular cytotoxicity (ADCC). ADCC can be achieved using an immunostimulatory CpG oligonucleotide in combination with an antibody specific for a cell target, such as a malignant cell. When an immunostimulatory CpG oligonucleotide is administered to a subject in combination with an antibody, the subject's immune system induces the death of a tumor cell. Antibodies used in the ADCC procedure are antibodies that interact with a cell in the body. Many such antibodies against cell targets have been described in the literature, and many of them are commercially available.

Immunostimulatory CpG oligonucleotides can also be administered in combination with anticancer therapy. Anticancer therapy includes the administration of anticancer drugs, radiation therapy, and surgery. As used herein, the term “anti-cancer drug” means an agent that is administered to a subject to treat a cancer. As used herein, the term “treatment of a malignant tumor” means preventing the development of a malignant tumor, alleviating the symptoms of the malignant tumor and / or inhibiting the growth of a developing malignant tumor. In other aspects of the present invention, anticancer drugs are administered to a subject at risk of developing a malignant tumor in order to reduce such a risk. Various types of drugs for the treatment of a malignant tumor are described herein. For the purpose of describing the present invention, anti-cancer drugs are classified as chemotherapeutic agents, immunotherapeutic agents, anti-cancer vaccines, hormone therapy and biological response modifiers.

In addition, the present invention relates to methods comprising the use of more than one anticancer agent together with immunostimulatory CpG oligonucleotides. As examples, where necessary, immunostimulatory CpG oligonucleotides can be administered together with a chemotherapeutic agent and with an immunotherapeutic agent. Alternatively, the term “anti-cancer drug” may contain an immunotherapeutic agent and an anti-cancer vaccine, or a chemotherapeutic agent and an anti-cancer vaccine, or an immunotherapeutic agent and an anti-cancer vaccine, all of which are administered to one subject in order to treat a subject suffering from a malignant tumor or a subject at risk of developing malignant tumors.

The chemotherapeutic agent may be selected from the group consisting of methotrexate, vincristine, adriamycin, cisplatin, sugar-free chloroethylnitrosourea, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, flagilin, meglaminamine II, melaminosamine IIA, glamicamine glaamine IIA , BAY 12-9566, RAS famezyltransferase inhibitor, famesyltransferase inhibitor, MMP, MTA / LY231514, LY264618 / lometexole, glamolec, CI-994, TNP-470, hikamtin / topotecan, PKC412, valsparantone / metrospantra novsparotra / prospantra novsparadra / prospantra novprospara , bathimastat, E 7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel / VX-710, VX-853, ZD0101, ISI641, ODN698, TA2516 / Marmistat, BB2516 / Marmistat, CDP845, D2163, PD183805, DX8951f Lemon DP2202, FK317, Pitsibanyl / OK-432, AD32 / Valrubitsin, Metastron / Strontium Derivative, Temodal / Temozolomide, Evaceta / Liposomal Doxorubicin, Iutaxan / Paclitaxel, Taxol / Paclitaxel, Xeload / Capecitabacin Daclofluceloflucelofluceloflucelofluceloflucelofluceltoflucelofluceloflucelofluceltofluceloflucelofluceltoflucelofluceloflucelofluceltofluceloflucelofluceloflucelun oral taxoid, SPU-077 / cisplatin, HMR1275 / flavopiridol, CP-358 (774) / EGFR, CP-609 (754) / RAS oncogen inhibitor, BMS-182751 / oral platinum, UFT (tegaflura / uracil), ergam ash / levamisole, eniluracil / 776C85 / 5FU enhancer, campto / levamisole, camptosar / irinotecan, tumodex / ralitrexed, leistatin / cladribine, paxex / paclitaxel, doxil / liposomal doxorubicin, caulixorubicarubicarubicorubicum / lipocarubicarubicorubicum / lipocarubicorudicuba , ZD1839, LU79553 / bis-naphthalimide, LU103793 / dolastatin, caetics / liposomal doxorubicin, gemzar / gemcitabine, ZD0473 / anormed, YM116, seed crystals of iodine, CDK4 and CDK2 inhibitors, Dimexidase D4, DI4 inhibitors, vumona / teniposide, paraple atine / carboplatin, plantinol / cisplatin, vepezid / etoposide, ZD9331, taxotere / docetaxel, prodrugs of arabinoside guanine, taxane analogue, nitrosoureas, alkylating agents such as melfelan and cyclophosphamide; aminoglutethimide, asparaginase, busulfan, carboplatin, chlororubucil, citrabin-HCl, dactinomycin, daunorubicin-HCl, estramustine sodium phosphate, etoposide (VR16-213), phloxuridine, fluorouracil (5-FU) hydroxamide flufamide flufamide -2a, alpha-2b, leuprolide acetate (analogue of LHRH releasing factor), lomustine (CCNU), mechlorethamine-HCl (nitrogen analogue of mustard gas), mercaptopurine, mesna, mitotane (o.r.- DDD), mitoxantrone- HCl, octreotide, plikamycin, procarbazine-HCl, streptozocin, tamoxifen citrate, thiogua nin, thiotepa, vinblastine sulfate, amsacrine (m-AMSA), azacitidine, erythropoietin, hexamethylmelamine (HMM), interleukin-2, mitoguazone (methyl-GAG, bis-guanylhydrazone methylglyoxal, MGBG), 2-penta-pentostin (methyl-CCNU), teniposide (VM-26) and vindesine sulfate, but not limited to.

The immunotherapeutic agent can be selected from the group consisting of ributaxin, herceptin, quadramet, panorex, IDEC-Y2B8, BEC2, C225, oncolim, SMART M195, ATRAGEN, ovarex, bexar, LDP-03, ior t6, MDX-210, MDX- 11, MDX-22, OV103, 3622W94, antibodies against VEGF, zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMab-G2, TNT, Gliomab-H, GHI-250, EMD-72000, LymphoCide, СМА-676, Monopharma-C, 4B5, ior egf.r3, ior c5, BABS, antibodies against FLK-2, MDX-260, antibodies against ANA, antibodies against SMART 1D10, antibodies against SMART ABL364 and ImmuRAIT-CEA, but not limited to.

The anti-cancer vaccine can be selected from the group consisting of EGF, anti-idiotypic anti-cancer vaccines, Gp75 antigen, GMK melanoma vaccines, MGV-ganglioside conjugate vaccines, Her2 / neu, ovarex, M-Vax, O-Vax, L- Vax, STn-KHL teratopes, BLP25 (MUC-1), idiotypic liposome vaccines, melacin, peptide antigen vaccines, toxin / antigen vaccines, MVA vaccines, PACIS, BCG vaccines, TA-HPV, TA-CIN, DISC virus, and ImmuCyst / TheraCys, but not limited to.

The use of immunostimulatory CpG oligonucleotides in combination with immunotherapeutic agents, such as monoclonal antibodies, can increase longevity by a number of mechanisms, including a significant increase in ADCC (antibody-dependent cellular cytotoxicity) (as discussed above), activation of natural killer (NK) and increased levels of IFN-α. Nucleic acids when used in combination with monoclonal antibodies can reduce the dose of the antibody necessary to achieve a biological effect.

As used herein, the terms “cancer antigen” and “tumor antigen” are used interchangeably to mean antigens that are differentially expressed by cancer cells, and therefore can be used to target cancer cells. Cancer antigens are antigens that can potentially stimulate possible tumor-specific immune responses. Some of these antigens are encoded but not necessarily expressed by normal cells. These antigens can be characterized as antigens that are usually silent (i.e. not expressed) in normal cells; or as antigens that are expressed only at specific stages of differentiation; or as antigens that are temporarily expressed, for example, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cell genes, such as oncogenes (e.g., activated ras oncogen), suppressor genes (e.g., p53 mutant), and fusion proteins resulting from internal deletions or chromosomal translocations. Other cancer antigens can be encoded by viral genes, for example, such as antigens present on tumor RNA and DNA viruses.

Immunostimulatory CpG oligonucleotides can also be used to treat and prevent autoimmune disease. An autoimmune disease is a class of diseases in which the subject's own antibodies react with the host tissue or in which immune effector T cells are auto-reactive with their own endogenous peptides and cause tissue destruction. In this case, the immune response is directed against the subject's own antigens, called autoantigens. Autoimmune diseases include, but are not limited to, rheumatoid arthritis, Crohn’s disease, multiple sclerosis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, severe myasthenia gravis (TM), Hashimoto thyroiditis, Goodpasture syndrome, pemphigus vulgaris (eg, vulvar bladder disease) autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma associated with anti-collagen antibodies, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Single Addissona disease, infertility, associated with an autoimmune disease, glomerulonephritis (e.g., crescentic glomerulonephritis, proliferative glomerulonephritis), bullous pemphigoid, Sjogren's syndrome, insulin resistance, and autoimmune diabetes.

As used herein, the term “autoantigen” means an antigen of normal host tissue. Normal host tissue does not contain cancer cells. Thus, in the case of an autoimmune disease, an immune response generated against an autoantigen is an undesirable immune response and leads to destruction and damage to normal tissue, while an immune response generated against a cancer antigen is a desirable immune response and leads to destruction of a tumor or cancer. Thus, in certain aspects of the present invention related to the treatment of autoimmune diseases, it is not recommended to administer immunostimulatory CpG oligonucleotides together with autoantigens, and in particular antigens that are targets in an autoimmune disorder.

In other cases, immunostimulatory CpG-containing nucleic acids can be delivered with low doses of autoantigens. Studies in a number of animals have demonstrated that the administration of small doses of antigen to the mucosa can lead to sustained immune hyporeactivity or “tolerance”. Obviously, in this case, the active mechanism is a cytokine-mediated deviation from the Th1 immune response predominantly towards the Th2 and Th3 response (i.e., with a predominance of the TGF-β response). Active suppression with low-dose antigen delivery may also suppress the irrelevant immune response (“suppression”), which is of particular interest in the treatment of autoimmune diseases such as rheumatoid arthritis and SLE. "Witness suppression" involves the secretion of Th1 -regulatory suppressor cytokines in a local environment where pro-inflammatory and Th1-cytokines are released by an antigen-specific or antigen-nonspecific mechanism. This phenomenon is defined by the term “tolerance” as used herein. Indeed, oral tolerance is effective in treating a number of autoimmune diseases in animals, including experimental autoimmune encephalomyelitis (EAE), experimental autoimmune severe myasthenia gravis, collagen-induced arthritis (ICA), and insulin-dependent diabetes mellitus. In these models, the prevention and suppression of autoimmune diseases is associated with a shift in the antigen-specific humoral and cellular responses from the Th1 to the Th2 / Th3 response.

The present invention also relates to a method for inducing antigen-nonspecific activation of the natural immune system and a wide spectrum of resistance to infectious stimulation using immunostimulatory CpG oligonucleotides. As used herein, the term “antigen-nonspecific activation of the natural immune system” means activation of non-B cell immune cells, and it may, for example, comprise activation of NK cells, T cells or other immune cells that may respond in an antigen-dependent manner , or a combination of these cells. The induction of resistance to broad-spectrum infections is due to the fact that immune cells are present in an active form and are primed to respond to any invasive compound or invasive microorganism. These cells should not be specifically primed against a particular antigen. This is especially important in the case of bacteriological warfare and in other cases described above, for example, when traveling.

The present invention also relates to oligonucleotides having chiral internucleotide bonds. As described above, the soft and semi-soft oligonucleotides of the present invention may have phosphodiester-like bonds between C and G. One example of a phosphodiester-like bond is the phosphorothioate bond in the Rp conformation.

In at least one study, the effect of p-chirality on the immunostimulatory effects of CpG oligonucleotides was analyzed. Yu et al. compared stereo-enriched (not stereo-pure) phosphorothioate (FT) oligonucleotides to their ability to induce proliferation of spleen cells (Yu et al., 2000). In this study, it was found that a 19-dimensional sequence containing one CpG motif induces high levels of proliferation of mouse spleen cells if the indicated oligonucleotide was synthesized with randomized p-chirality or enriched with internucleotide Sp-bonds, but this proliferation was markedly reduced in that case when said oligonucleotide was enriched in internucleotide Rp bonds (Yu et al., 2000). However, this study did not evaluate the specific role of the p-chirality of CpG dinucleotide, nor did it determine whether Rp-CpG oligonucleotides are active in short-term stimulation assays.

In accordance with the present invention, it was found that the p-chirality of the oligonucleotide can obviously have the opposite effect on the immune activity of the CpG oligonucleotide depending on the time of measurement of activity. At an early time, after 40 minutes, phosphorylation of JNK in mouse spleen cells induced the Rp stereoisomer rather than the Sp stereoisomer of the phosphorthioate CpG oligonucleotide (as discussed in the examples). In contrast, when analyzed at a later time, after 44 hours, the active stimulator of spleen cell proliferation was the Sp stereoisomer rather than the Rp stereoisomer. The authors of the present invention have demonstrated that such a difference in the kinetics and biological activity of the Rp and Sp stereoisomers was not due to any difference in their uptake by cells, and in all likelihood this was due to two opposite biological mechanisms of p-chirality. First, the increased activity of the Rp stereoisomer compared to the Sp stereoisomer in stimulating immune cells at an early stage of time indicates that Rp may be more effective when interacting with the CpG receptor, TLR9, or when inducing subsequent signal transmission pathways. On the other hand, faster degradation of Rp-PS oligonucleotides compared to Sp oligonucleotides leads to a significant reduction in signal transmission time, and therefore, it is likely that Sp-PS oligonucleotides have greater biological activity when tested at a later time.

In some of its aspects, the present invention is based on the finding that the previously reported relative lack of immune stimulation by Rp-PS oligonucleotides is due only to their sensitivity to nuclease and not to their inability to stimulate the CpG receptor and subsequent signal transmission pathways. When testing for their ability to stimulate JNK phosphorylation, which indicates the activity of this mitogen-activated protein kinase pathway, the Rp oligonucleotide was the most active, followed by the stereo-randomized oligonucleotide, while the Sp oligonucleotide did not have any detectable activity. However, when comparing these oligonucleotides for the ability to activate the NF-kB pathway, which was evaluated by degradation of the inhibitory protein IkB-α, it was found that all CpG oligonucleotides were active, although the control oligonucleotide not containing CpG did not induce degradation of IkB-α . Thus, the Sp-oligonucleotide still remained biologically active. This lack of JNK pathway induction may be due to differences in the kinetics of activation of the JNK and NF-kB pathways, but due to the limited amounts of stereospecific oligonucleotide required for testing, the authors of the present invention could not confirm this hypothesis.

In the experiments described in the examples, a strong p-chirality effect of the CpG dinucleotide itself was unexpectedly discovered. Compared to a stereo randomized CpG oligonucleotide, another oligonucleotide of the same type in which one CpG dinucleotide has an Rp link was slightly more active, whereas an oligonucleotide of the same type but containing an Sp link was almost inactive in inducing cell proliferation the spleen. The loss of activity by the Sp analog confirms the hypothesis of the authors of the present invention that the TLR9 receptor may not be inert with respect to the chirality of the CpG dinucleotide in the DNA with which it interacts, but in fact it can be better stimulated by the Rp stereoisomer. Thus, the stimulating effect of the stereo-randomized oligonucleotide is probably due not only to the presence of 50% Sp bonds that slow down degradation, but also due to the fact that half of the oligonucleotide molecules will have Rp chirality in the CpG dinucleotide, which will obviously be lead to increased immunostimulating effects.

The sensitivity of Rp-PS bonds to nuclease cleavage is important for the interpretation of pharmacokinetic (PK) studies and studies of the metabolism of PS oligonucleotides in humans or animals. It is known that the predominant activity of serum nuclease is the activity of 3'-exonuclease. In a typical stereo-randomized PS oligonucleotide solution, the last internucleotide 3′-bond is believed to represent Rp chirality in one half of these molecules. Therefore, in these 50% PS-oligonucleotide molecules, the terminal 3'-base will cleave off rather quickly after i.v. infusion. The second internucleotide bond from the 3'-end should represent Rp-chirality in one half of these molecules, and therefore it can be assumed that in 25% of the original PS-oligonucleotide molecules, the 3'-end can be relatively quickly shortened to two bases. It can be assumed that this in vivo cleavage of the base, including internucleotide 3'-Rp bonds, will continue until the internucleotide 3'-bond acquires the Sp configuration. Therefore, if PS oligonucleotides were synthesized so that they contained a 3'-terminal Sp bond, then they should be subject to much slower degradation and have a different PK profile than stereo-randomized PS oligonucleotides. This would allow the use of slightly shorter oligonucleotides for in vivo use. When constructing optimized oligonucleotides to obtain antisense structures, enhanced binding of the Rp stereoisomer to RNA will indicate the need to use only the inner part of the oligonucleotide in the Rp configuration, if possible. Alternatively, the optimized CpG oligonucleotide used for immunostimulation may be one of the oligonucleotides in which all internucleotide bonds except for CpG must represent Sp-chirality.

Immunostimulatory CpG oligonucleotides can be directly delivered to the subject, or they can be administered in combination with a complex for the delivery of nucleic acids. A nucleic acid delivery complex is a nucleic acid molecule associated with a targeting delivery vehicle (for example, bound to this agent by an ionic or covalent bond, or encapsulated in this vehicle), for example, with a molecule that provides a higher binding affinity for the cell- the target. Examples of nucleic acid delivery complexes are nucleic acids associated with a sterol (e.g., cholesterol), a lipid (e.g., a cationic lipid, a virosome or a liposome), or agents that specifically bind to a target cell (e.g., a ligand recognized by cell surface receptor). Preferred complexes can be complexes that are sufficiently stable in vivo, which avoids the production of significant levels of unbound nucleic acid before it is internalized by the target cell. However, this complex can be cleaved under appropriate conditions within the cell, as a result of which the nucleic acid will be released in functional form.

Carriers for delivery or devices for the delivery of antigens and oligonucleotides on the surface have been described in the literature. An immunostimulatory CpG oligonucleotide and / or antigen and / or other therapeutic agents can be administered separately (for example, in saline or in buffer) or using any delivery vehicles known to those skilled in the art. For example, the following delivery vehicles have been described: cochleates (Gould-Fogerite et al., 1994, 1996), emulsions (Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993, Carlsson et al., 1999, Hu et al., 1998, Morein et al., 1999); liposomes (Childers et al., 1999, Michalek et al., 1989, 1992, de Haan 1995a, 1995b), live bacterial vectors (e.g., Salmonella, Escherichia coli, Bacillus calmatte-guerin, Shigella, Lactobacillus) (Hone et al. , 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991, Nugent et al., 1998); live viral vectors (e.g., vaccinia virus, adenovirus, herpes simplex virus) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugent et al., 1998, Morrow et al., 1999); microspheres (Gupta et al., 1998, Jones et al., 1996, Maloy et al., 1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989); nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada et al., 1997, Ishii et al., 1997); polymers (e.g. carboxymethyl cellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill et al., 1998); polymer rings (Wyatt et al., 1998); proteosomes (Vancott et al., 1998, Lowell et al., 1988, 1996, 1997); sodium fluoride (Hashi et al., 1998); transgenic plants (Tacket et al., Mason et al., 1998, Haq et al., 1995); virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al., 1998); virus-like particles (Jiang et al., 1999, Leibl et al., 1998). Other delivery vehicles are known to those skilled in the art, and some additional examples are described below in the discussion of vectors.

As used herein, the term “effective amount of an immunostimulatory CpG oligonucleotide” means an amount necessary or sufficient to achieve the desired biological effect. So, for example, an effective amount of an immunostimulatory CpG oligonucleotide, administered together with an antigen to induce an immune response produced in the mucosa, means the amount needed to produce IgA in response to the antigen, whereas the amount needed to induce a systemic immune response necessary for the production of IgG in response to exposure to the specified antigen. By combining with the methods described herein and by selecting a particular compound from various active compounds, taking into account such important factors as their effectiveness, relative bioavailability, patient body weight, severity of negative side effects, and a preferred administration schedule, an effective prophylactic or therapeutic regimen can be developed. treatment that will not cause significant toxic effects and will also be absolutely effective for the treatment of a particular subject. The effective amount of a compound for any particular application may vary depending on factors such as the particular disease or condition being treated, the particular immunostimulatory CpG oligonucleotide administered, the weight of the subject or the severity of the disease or condition. Each specialist can empirically determine the effective amount of a specific immunostimulating CpG oligonucleotide and / or antigen and / or other therapeutic agent without undue experimentation.

Dosages of the compounds described herein for mucosal or local delivery to a subject are typically in the range of 0.1 μg to 10 mg per administration, depending on the frequency of administration, which can be carried out daily, once a week, or once a month or at other time intervals. In most cases, doses for mucosal administration or for local administration are in the range of about 10 μg to 5 mg per administration, and generally about 100 μg to 1 mg, and such administration can be carried out 2-4 times at intervals in a few days or weeks. In general, doses of an immunostimulating agent range from 1 μg to 10 mg per administration, and in most cases, from 10 μg to 1 mg daily or once a week. Doses of the compounds described herein for parenteral delivery to a subject in order to induce an angiogenic specific immune response, wherein said compound is administered with an antigen, but not with another therapeutic agent, usually 5-10000 times higher, mostly 10-1000 times higher, and in most cases, 20-100 times higher than the effective dose of the vaccine adjuvant or immunostimulants delivered through the mucosa. Dosages of the compounds described herein for parenteral delivery in order to induce a natural immune response in a subject, or to enhance ADCC, or to induce an antigen-specific immune response when immunostimulatory CpG oligonucleotides are administered in combination with other therapeutic agents or in special delivery vehicles, are usually about 0.1 μg to 10 mg per administration, depending on the route of administration, which can be carried out daily, once a week or once a month or through a friend other time intervals. In most cases, parenteral doses for such purposes are from about 10 μg to 5 mg per administration, and generally from about 100 μg to 1 mg, where such administration can be carried out 2-4 times at intervals of several days or weeks. However, in some embodiments, parenteral doses administered for these purposes may be 5 to 10,000 times the typical doses described above.

For any compound described herein, a therapeutically effective amount may first be determined using animal models. A therapeutically effective dose can also be determined from data obtained for human CpG oligonucleotides that have been tested in humans (clinical trials involving humans) and for compounds known to have similar pharmacological activities, for example, like other adjuvants, for example, LT and other antigens used for vaccination. For parenteral administration, higher doses are required. The administered dose can be adjusted based on the relative bioavailability and effectiveness of the administered compound. Dose adjustment to achieve maximum effectiveness by the methods described above and other known methods well known in the art can be carried out by any specialist.

The compositions of the present invention are administered in pharmaceutically acceptable solutions, which typically contain pharmaceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, adjuvants and optionally other therapeutic ingredients.

For therapeutic use, an effective amount of an immunostimulatory CpG oligonucleotide can be administered to a subject by any means capable of delivering said oligonucleotide to a desired surface, such as a mucosa, or systemic delivery. Administration of the pharmaceutical composition of the present invention can be carried out by any method known to those skilled in the art. Preferred routes of administration include, but are not limited to, oral administration, intramuscular administration, intranasal administration, sublingual administration, intratracheal administration, administration by inhalation, intraocular administration, vaginal and rectal administration.

For oral administration, the compounds (i.e., immunostimulatory CpG oligonucleotides, antigens, and other therapeutic agents) can be easily prepared by combining the active (s) compound (s) with pharmaceutically acceptable carriers well known in the art. Using such carriers, the compounds of the present invention can be prepared in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, suspensions, and the like. for oral administration to a subject being treated. Pharmaceutical preparations for oral administration can be obtained in solid excipient by optionally grinding the resulting mixture and processing the mixture of granules after adding, if necessary, suitable additives to obtain the core tablets or dragees. Suitable excipients are, in particular, excipients such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methyl cellulose, hydroxypropyl methyl cellulose, sodium substituted carboxymethyl cellulose and / or polyvinylpyrrolidone (PVP). If necessary, disintegrating agents, such as structured polyvinylpyrrolidone, agar or alginic acid, or a salt thereof, such as sodium alginate, can be added. Compositions for oral administration may also be prepared, but not necessarily, in saline or in buffers to neutralize internal acidity, or they may be administered without any carriers.

Oral medicines containing the above (e) component or components are also specifically contemplated. Such a component or components may be chemically modified to achieve effective oral delivery of said derivative. In general terms, such a chemical modification is that at least one constituent moiety is attached to the component molecule itself, which (a) inhibits proteolysis and (b) facilitates the entry of the molecule into the bloodstream from the stomach or intestines. It is also desirable to increase the overall stability of this component or components and increase the time of their circulation in the body. Examples of such a component are polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline. Abuchowski & Davis, 1981, "Soluble Polymer-Enzyme Adducts", Enzymes as Drugs, Hocenberg & Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383; Newmark et al., 1982, J. Appl. Biochem. 4: 185-189. Other polymers that can be used for these purposes are poly-1,3-dioxolane and poly-1,3,6-trioxolane. As indicated above, polypropylene glycol compounds are preferred for pharmaceutical use.

The release site of the component (or derivative) may be the stomach, small intestine (duodenum, jejunum or ileum) or large intestine. Those skilled in the art will recognize compositions that do not dissolve in the stomach, but release the active material into the duodenum or some other part of the intestine. It is preferable that this release does not cause a negative effect on the gastric environment, which can be achieved either by protecting the oligonucleotide (or derivative), or by releasing the biologically active substance outside the gastric environment, for example, in the intestine.

To ensure complete resistance to the gastric environment, the coating should be substantially impermeable, at least at pH 5.0. Examples of the most common inert ingredients that are commonly used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMC), HPMC 50, HPMC 55, polyvinyl acetate phthalate (PVAF), eudragit L30D, hydrated; cellulose acetate phthalate (ATS), eudragit L, eudragit S and shellac. These coatings can be used as mixed films.

Coatings or a coating mixture may also be applied to tablets which are not intended to protect against the action of the gastric environment. Such coatings may be sugar coatings or coatings that facilitate swallowing of the tablet. Capsules may consist of a hard shell (such as gelatin) to deliver a dry therapeutic substance, i.e. powder; and for liquid forms, a soft gelatin shell may be used. For cachets, the shell material may be thick starch or other edible paper. For the manufacture of pills, tablets in the form of lozenges, molded tablets or triturate for tablets, a wet grinding technique may be used.

The therapeutic agent may be included in the composition in the form of a plurality of particulates in the form of granules or spheres of particles of 1 mm in size. A composition consisting of capsule administration material can be prepared in the form of a powder, a slightly compressed briquette, or even tablets. A therapeutic agent can be obtained by compression.

Dyes or flavoring agents may be included. So, for example, an oligonucleotide (or derivative) can be obtained (for example, by encapsulation in a liposome or microsphere), and then it is introduced into a food product, such as a chilled drink containing dyes or flavoring agents.

The therapeutic agent can be diluted, or its volume can be increased by adding inert material. Such diluents can be carbohydrates, and in particular mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts, including calcium triphosphate, magnesium carbonate and sodium chloride, can also be used as fillers. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress, and Avicell.

Disintegrants may be included in a therapeutic composition in solid dosage form. Materials used as disintegrants include, but are not limited to, starch, including the commercially available starch based disintegrator, Explotab. Starch sodium glycolate, amberlite, sodium carboxymethyl cellulose, ultramilopectin, sodium alginate, gelatin, orange peel, acidic carboxymethyl cellulose, natural sponges and bentonite can be used. Another form of disintegrating agents is insoluble cation exchange resins. Powdered resins can be used as disintegrating agents and binders, and such resins can be agar, karaya gum and tragacanth gum. Alginic acid and its sodium salt may also be used as disintegrating agents.

Binders can be used to bond the therapeutic agent together with other components to form a solid tablet, and such materials are materials from natural products such as gum arabic, tragacanth gum, starch and gelatin. Other binding agents are methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). To granulate the therapeutic substance, polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) in alcohol solutions can be used.

To prevent sticking during the preparation of the therapeutic composition, an antifriction agent may be included in the composition. Lubricants may be used as a layer between the therapeutic material and the impermeable wall, and such lubricants are, but are not limited to, stearic acid, including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol with various molecular weights, Carbovax 4000 and 6000 can also be used.

To facilitate mold changes during the pressing process, substances can be added that enhance the sliding and improve the fluidity of the drug during the preparation of the composition. Such glidants may contain starch, talc, pyrogenic silica and hydrated silicoaluminate.

To facilitate the dissolution of the therapeutic agent in an aqueous medium, a surfactant may be added as a wetting agent. Such surfactants are anionic detergents such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate. Cationic detergents that contain benzalkonium chloride or benzethonium chloride can be used. Possible nonionic detergents that can be included as surfactants in the composition are lauromacrogol 400, polyoxyl stearate 40, polyoxyethylated hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, fatty acid ester and sucrose, methyl cellulose and carboxymethyl cellulose. These surfactants may be present in the composition of the oligonucleotide or its derivative either separately or in a mixture in various ratios.

Pharmaceutical preparations that can be used for oral administration are push-fit capsules made from gelatin, as well as soft, hermetically sealed capsules made from gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain the active ingredients in admixture with an excipient, such as lactose, binders, such as starches, and / or lubricants, such as talc or magnesium stearate, and, optionally, with stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. Microspheres for oral administration may also be used. Such microspheres are well known in the art. All compositions for oral administration should be prepared in doses suitable for such administration.

For buccal administration, the compositions may be prepared in the form of tablets or lozenges formulated in a standard manner.

For administration by inhalation, the compounds of the present invention can be easily delivered as an aerosol spray delivered from an aerosol package or inhaler using a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosol packaging, the unit dosage can be determined using a metering valve used to deliver a specific amount of drug. Capsules and cartridges can be prepared, for example, from gelatin, used in an inhaler or in an insufflator, and containing a powder mixture of the compound and a suitable powder base, such as lactose or starch.

The present invention also contemplates pulmonary delivery of oligonucleotides (or their derivatives). An oligonucleotide (or derivative) is delivered to the lungs of a mammal by inhalation, and through the epithelial lining of the lungs it enters the bloodstream. Other descriptions of molecules that can be administered by inhalation are provided by Adjei et al., 1990, Pharmaceutical Research, 7: 565-569; Adjei et al., 1990, International Journal of Pharmaceuticals, 63: 135-144 (leuprolide acetate); Braquet et al., 1989, Journal of Cardiovascular Pharmacology, 13 (suppl. 5); 143-146 (endothelin-1); Hubbard et al., 1989, Annals of Internal Medicine, Vol. III, pp.206-212 (a1-antitrypsin); Smith et al., 1989, J. Clin. Invest. 84: 1145-1146 (a-1 proteinase); Oswein et al., 1990, "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March (Recombinant Human Growth Hormone); Debs et al., 1988, J. Immunol. 140: 3482-3488 (interferon-γ and tumor necrosis factor-alpha) and Platz et al., US patent No. 5284656 (granulocyte colony stimulating factor). A method and composition for pulmonary drug delivery in order to produce a systemic effect are described in US Pat. No. 5,451,569, issued September 19, 1995, to Wong et al.

For the implementation of the present invention provides the use of a wide range of mechanical devices for the pulmonary delivery of therapeutic products, including, but not limited to, nebulizers, metered-dose inhalers and insufflators, all of which are known to those skilled in the art.

Some specific examples of commercially available devices suitable for carrying out the present invention are an Ultravent atomizer manufactured by Mallinckrodt, Inc., St. Louis, Missouri; Acorn II nebulizer manufactured by Marquest Medical Products, Engelwood, Colorado; Ventolin dosing valve inhaler manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and Spinhaler insufflator manufactured by Fisons Corp., Bedford, Massachusetts.

All of these devices require the use of compositions suitable for dosing an oligonucleotide (or derivative). Typically, a specific composition is used for each particular type of device, which, in addition to the usual diluents, adjuvants or carriers used in therapy, may contain an appropriate propellant. In addition, the use of liposomes, microcapsules or microspheres, including their complexes, or other types of carriers is also contemplated. Chemically modified oligonucleotide can also be obtained in the form of various compositions depending on the type of chemical modification or on the type of device used.

Compositions suitable for use with nebulizers, either jet or ultrasonic, typically contain an oligonucleotide (or derivative) dissolved in water at a concentration of about 0.1-25 mg of biologically active oligonucleotide per ml of solution. The composition may also contain a buffer and simple sugar (for example, to stabilize the oligonucleotide and regulate the osmotic pressure). To reduce surface-induced aggregation of the oligonucleotide caused by spraying the solution during aerosol formation, the nebulizer composition may also contain a surfactant.

Compositions intended for use in a metered dose inhaler inhaler typically comprise a finely divided powder containing an oligonucleotide (or derivative) suspended in a propellant with a surfactant. Such a propellant can be any standard substance used for this purpose, for example, such as chlorofluorocarbon, chlorofluorocarbon, fluorocarbon or hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,2,2-tetrafluoroethane, or combinations thereof. Suitable surfactants are sorbitan trioleate and soya lecithin. Oleic acid may also be used as a surfactant.

Insufflator dosed delivery compositions may contain finely divided powder containing an oligonucleotide (or derivative) and may also contain a bulking agent such as lactose, sorbitol, sucrose or mannitol in amounts that facilitate atomization of the powder from this device, for example, in an amount 50-90% by weight of the composition. For the most efficient delivery to the distal regions of the lung, the oligonucleotide (or derivative) should preferably be prepared in the form of particles with an average size of less than 10 mm (or microns), and more preferably 0.5-5 mm.

In addition, intranasal delivery of the pharmaceutical composition of the present invention is contemplated. Intranasal delivery allows the pharmaceutical composition of the present invention to enter the bloodstream immediately after administration of the therapeutic product into the nose without sedimentation of the product in the lungs. Compositions for intranasal delivery contain dextran or cyclodextran.

A suitable device for intranasal administration is a small rigid vessel to which a nebulizer with a metering valve is connected. In one embodiment, a specific dose is delivered by feeding a solution of the pharmaceutical composition of the present invention into a chamber of a certain volume, which has an aperture of a size that provides spraying of the aerosol and aerosol composition by forming a spray when the liquid is compressed in this chamber. In order to administer the pharmaceutical composition of the present invention, the chamber is compressed. In a specific embodiment, this chamber is a piston device. The devices described are commercially available.

Alternatively, a plastic pressure vessel with an aperture or orifice provides spraying of the aerosol composition by forming a pressure spray. This opening is usually located in the upper part of the vessel, and this upper part of the vessel usually tapers in a cone shape, which allows it to partially pass into the nasal passages for effective administration of the aerosol composition. Preferably, the inhaler for intranasal administration provides a metered amount of the aerosol composition to administer the desired dose of drug.

Compounds intended for systemic delivery may be formulated for parenteral administration by injection, for example, by injection of a loading dose or by continuous infusion. Compositions for injection can be obtained in the form of a standard dosage form, for example in ampoules or in vials for repeated administration with the addition of preservatives. These compositions can also be prepared in the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and they may contain processing agents, such as suspending, stabilizing and / or dispersing agents.

Pharmaceutical compositions for parenteral administration contain aqueous solutions of the active compounds obtained in a water-soluble form. In addition, if necessary, suspensions of the active compounds can be prepared as oily injectable suspensions. Suitable lipophilic solutions or carriers are fatty oils, such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium-substituted carboxymethyl cellulose, sorbitol, or dextran. This suspension may also contain, but not necessarily, suitable stabilizers or agents that increase the solubility of the compounds, which allows to obtain highly concentrated solutions.

Alternatively, the active compounds may be prepared in powder form, which may be reconstituted with a suitable vehicle, for example sterile pyrogen-free water, before use.

These compounds can also be formulated for rectal or vaginal administration, such as suppositories or retention enemas, containing, for example, standard suppository bases, such as cocoa butter or other glycerides.

In addition to the compositions described previously, these compounds can also be prepared as a depot preparation. Such sustained release formulations may be prepared using suitable polymeric or hydrophobic materials (for example, as an emulsion in a suitable oil), or ion exchange resins, or as sparingly soluble derivatives, for example, sparingly soluble salts.

The pharmaceutical compositions may also contain suitable solid or gel carriers or excipients. Examples of such carriers or excipients are, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.

Suitable forms of liquid or solid pharmaceutical preparations are, for example, aqueous or physiological solutions for inhalation, microencapsulated preparations, cochleate preparations; preparations applied to microscopic gold particles; preparations contained in liposomes, in inhalers and aerosols; pills for implantation in the skin, or drugs that dry on the damaged area with penetration into the skin. The pharmaceutical compositions can also be granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops or sustained-release preparations of active compounds using standard excipients and additives and / or auxiliary substances described above, such as disintegrants, binders, coating agents, swelling agents, lubricants, perfumes, sweeteners and solubilizing agents. These pharmaceutical compositions are suitable for use in a number of drug delivery systems. A brief overview of drug delivery methods is provided by Langer, Science 249: 1527-1533, 1990, which is incorporated herein by reference.

Immunostimulatory CpG oligonucleotides and optionally other therapeutic agents and / or antigens can be administered per se (in pure form) or in the form of a pharmaceutically acceptable salt. If these salts are used in medicine, they must be pharmaceutically acceptable, but salts that are not pharmaceutically acceptable are usually used to prepare such pharmaceutically acceptable salts. Such salts include, but are not limited to, salts derived from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, malonic, succinic, naphthalene 2-sulfonic and benzenesulfonic acids. In addition, such salts can be obtained in the form of salts of alkali or alkaline earth metals, such as sodium, potassium or calcium salts of carboxylic acids.

Suitable buffering agents are acetic acid and salt (1-2% w / v); citric acid and salt (1-3% w / v); boric acid and salt (0.5-2.5% w / v) and phosphoric acid and salt (0.8-2% w / v). Suitable preservatives are benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01-0.25% w / v) and thimerosal (0.004-0.02% w / v).

The pharmaceutical compositions of the present invention contain an effective amount of an immunostimulatory CpG oligonucleotide and optionally antigens and / or other therapeutic agents, optionally included in a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” means one or more compatible solid or liquid excipients, diluents, or encapsulation promoters that are suitable for administration to a human or other vertebrate animal. The term “carrier” means an organic or inorganic ingredient, natural or synthetic, with which the active ingredient can be combined to facilitate administration. The components of the pharmaceutical compositions should also be such that when mixed with the compounds of the present invention or with each other, they do not interact with each other and do not have a significant negative effect on the desired pharmaceutical efficacy.

The present invention is further illustrated in the following examples, which should not be construed as limiting the invention. The full contents of all these works (including literature, granted patents, published patent applications and simultaneously pending applications) cited in this application are hereby incorporated by reference.

EXAMPLES

Materials and methods

Oligodeoxynucleotides (ODN)

All ODNs were purchased from Biospring (Frankfurt, Germany) or Sigma-Ark (Darmstadt, Germany) and were verified for identity and purity by Coley Pharmaceuticals GmbH (Langenfeld, Germany). ODN was diluted in phosphate buffered saline (Sigma, Germany) and stored at -20 ° C. All dilutions were performed using pyrogen-free reagents.

Cell cleaning

The peripheral blood leukocyte preparations taken from healthy donors, men and women, were obtained from the blood bank of the University of Dusseldorf (Germany), and MCPC was isolated from these preparations by centrifugation at Fikoll-Gipak (Sigma). Purified PBMCs were either used fresh (for most analyzes) or suspended in a freezing medium and stored at -70 ° C. If necessary, aliquots of these cells were thawed, washed and resuspended in RPMI 1640 culture medium (BioWhittaker, Belgium), to which 5% (v / v) thermally inactivated human AB serum (BioWhittaker, Belgium) or 10% (vol. (v / v) thermally inactivated FCS, 2 mM L-glutamine (BioWhittaker), 100 units / ml penicillin and 100 μg / ml streptomycin (Invitrogen (Karlsruhe, Germany)).

Cytokine detection

Thawed or fresh PBMCs were plated on 48-well flat-bottom plates or on 96-well round-bottom plates, and incubated with ODN at the indicated concentrations in an incubator with high humidity at 37 ° C. Culture supernatants were harvested and either used directly or stored frozen at -20 ° C until use. The number of cytokines in supernatants was evaluated using commercially available ELISA kits (Diaclone, USA) or laboratory ELISA kits developed using commercially available antibodies (from Becton Dickinson / Pharmingen or PBL).

Culture for flow cytometric analysis for the activation of NK cells

Fluorochrome-conjugated monoclonal antibodies against CD3 (T cell marker), CD56 (NK cell marker), and CD69 (early activation marker on NK cells and T cells) were purchased from Becton Dickinson. MCPCs were incubated for 24 hours with or without the addition of various concentrations of ODN in 96-well round-bottom plates. NK cells were identified as CD56-positive and CD3-negative by flow cytometric analysis. Flow cytometric analysis data was obtained on a FACSCalibur device (Becton Dickinson). These data were analyzed using the CellQuest computer program (Becton Dickinson).

Flow cytometric analysis of cell surface activation markers

To measure the expression of a co-stimulating CD86 molecule as an activation marker on B cells, PBMCs were incubated for 48 hours with ODNs at indicated concentrations and cells were stained with anti-CD19 and CD86 mAbs (Pharmingen, Germany). The expression of CD86 on CD19-positive B cells was evaluated by flow cytometry.

To measure the expression level of a costimulating CD80 molecule as an activation marker on monocytes, PBMCs were incubated for 48 hours with ODNs at the indicated concentrations and cells were stained with anti-CD14, CD19 and CD80 mAbs (Pharmingen, Germany). The expression of CD80 on CD14-positive and CD19-negative monocytes was evaluated using flow cytometry. The results of these measurements are given as mean fluorescence intensity (MFI).

Example 1

. Тестируемыми олигонуклеотидами, показанными на фиг. 1А, 2А, 3А, 4А и 5А, являются SEQ ID NO:322, SEQ ID NO:323 и SEQ ID NO:324. Тестируемыми олигонуклеотидами, показанными на фиг. 1В, 2В, 3В, 4В и 5В, являются SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327 и SEQ ID NO:328. Концентрация олигонуклеотида, используемого для получения исходных точек на графике, отложена по оси Х (мкМ). Ниже графиков указаны уровни цитокина, секретируемого клетками, обработанными негативным контролем (средой), а в некоторых случаях ЛПС, как указано для каждого эксперимента.The levels of interferon alpha (IFN-α), IFN-γ, IL-10, IL-6, and TNF-α secreted from human PBMCs after treatment of these cells with the CpG oligonucleotides described herein are indicated in the attached FIG. 1-5. Test oligonucleotides are indicated on these graphs ▲. An oligonucleotide that serves as a positive control is indicated

. The test oligonucleotides shown in FIG. 1A, 2A, 3A, 4A and 5A are SEQ ID NO: 322, SEQ ID NO: 323, and SEQ ID NO: 324. The test oligonucleotides shown in FIG. 1B, 2B, 3B, 4B and 5B are SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327 and SEQ ID NO: 328. The concentration of the oligonucleotide used to obtain the starting points on the graph is plotted along the X axis (μM). The graphs below show the levels of the cytokine secreted by cells treated with the negative control (medium), and in some cases LPS, as indicated for each experiment.

As shown in FIG. 1-5, the oligonucleotides tested in these assays are capable of inducing cytokine secretion at approximately equivalent or higher levels than those induced by positive control oligonucleotides having a fully phosphorothioate backbone. Negative control stimulated the production of significantly lower levels of cytokines.

Example 2

. Тестируемыми олигонуклеотидами, показанными на фиг.6А, являются SEQ ID NO:322, SEQ ID NO:323 и SEQ ID NO:324. Тестируемыми олигонуклеотидами, показанными на фиг.6В, являются SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327 и SEQ ID NO:328. Олигонуклеотидом позитивного контроля, используемым в этих исследованиях, является SEQ ID NO:329. Ниже графиков указаны уровни экспрессии CD69 на Т- и NK-клетках, обработанных негативным контролем (средой) и ЛПС, для каждого эксперимента.CD69 (MFI) expression levels on NK cells were evaluated in response to the treatment of these cells with test oligonucleotides compared to control oligonucleotides. CD69 expression is an indicator of the activation of T cells and NK cells. These cells were treated with test oligonucleotides, designated ▲ in FIG. 6, or a positive control oligonucleotide, designated

. The test oligonucleotides shown in FIG. 6A are SEQ ID NO: 322, SEQ ID NO: 323, and SEQ ID NO: 324. The test oligonucleotides shown in FIG. 6B are SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, and SEQ ID NO: 328. The positive control oligonucleotide used in these studies is SEQ ID NO: 329. The graphs below show the expression levels of CD69 on T and NK cells treated with negative control (medium) and LPS for each experiment.

As shown in FIG. 6, oligonucleotides tested in these assays are capable of inducing expression of CD69 at approximately equivalent or higher levels than those induced by positive control oligonucleotides having a fully phosphorothioate backbone. Negative control stimulated the production of significantly lower levels of CD69.

Example 3

. Олигонуклеотид, который служит в качестве позитивного контроля, SEQ ID NO:242, обозначен ●. Олигонуклеотид, который служит в качестве негативного контроля, SEQ ID NO:330, обозначен

. Тестируемым олигонуклеотидом, показанным на фиг. 7А и 7В, является SEQ ID NO:313. Тестируемым олигонуклеотидом, показанным на фиг. 8А и 8В, является SEQ ID NO:314. Тестируемым олигонуклеотидом, показанным на фиг. 9А и 9В, является SEQ ID NO:319. Тестируемым олигонуклеотидом, показанным на фиг. 10А и 10В, является SEQ ID NO:316. Тестируемым олигонуклеотидом, показанным на фиг. 11А и 11В, является SEQ ID NO:317. Тестируемым олигонуклеотидом, показанным на фиг. 12А и 12В, является SEQ ID NO:320. Тестируемым олигонуклеотидом, показанным на фиг. 17А и 17В, является SEQ ID NO:321. Концентрация олигонуклеотида, используемая для получения исходных точек на графике, отложена по оси Х (мкМ).The levels of interferon alpha (IFN-α) and IL-10 secreted by human PBMCs after treatment of these cells with the CpG oligonucleotides described herein are shown in the attached FIG. 7-12 and 17. In these graphs, the test oligonucleotides are indicated

. The oligonucleotide, which serves as a positive control, SEQ ID NO: 242, is indicated by ●. An oligonucleotide that serves as a negative control, SEQ ID NO: 330, is indicated

. The test oligonucleotide shown in FIG. 7A and 7B, is SEQ ID NO: 313. The test oligonucleotide shown in FIG. 8A and 8B, is SEQ ID NO: 314. The test oligonucleotide shown in FIG. 9A and 9B, is SEQ ID NO: 319. The test oligonucleotide shown in FIG. 10A and 10B, is SEQ ID NO: 316. The test oligonucleotide shown in FIG. 11A and 11B, is SEQ ID NO: 317. The test oligonucleotide shown in FIG. 12A and 12B, is SEQ ID NO: 320. The test oligonucleotide shown in FIG. 17A and 17B, is SEQ ID NO: 321. The concentration of the oligonucleotide used to obtain the starting points on the graph is plotted along the X axis (μM).

As shown in FIG. 7-12 and 17, each of the oligonucleotides tested in these assays is capable of inducing different levels and patterns of cytokine secretion. For example, most of the tested ODNs taken at approximately equivalent or lower concentrations showed better induction of one or more cytokines than the positive control oligonucleotide having a fully phosphorothioate backbone. Negative control induced the production of significantly lower levels of cytokines.

After incubation with SEQ ID NO: 313, PBMC secreted the same levels of interferon-alpha (IFN-α) and interleukin-10 (IL-10) as after incubation with SEQ ID NO: 242. SEQ ID NO: 314 induced the same levels of IL-10 secretion from human PBMCs as SEQ ID NO: 242, but much higher levels of IFNα secretion. Unlike SEQ ID NO: 242, SEQ ID NO: 319 induced only small levels of IFN-α secretion from human PBMCs, but IL-10 secretion levels induced by these two oligonucleotides were similar. SEQ ID NO: 316 is able to induce several times higher levels of IFNα from human PBMCs than SEQ ID NO: 242. An increase in the total amount of secreted IL-10 was also observed. SEQ ID NO: 317 showed similar properties to SEQ ID NO: 316, however, it induced significantly higher levels of IFN-α secretion from human PBMCs than SEQ ID NO: 242. IL-10 secretion levels were slightly elevated. Although SEQ ID NO: 320 induced the secretion of IFNα and IL-10 from human PBMCs, this level of induction was lower than for SEQ ID NO: 242. SEQ ID NO: 321 is capable of inducing IFN-α levels from human MCPCs that are more than ten times the levels of IFNα from human MCPC induced by the oligonucleotide SEQ ID NO: 242 (Fig. 17A). Compared to SEQ ID NO: 242, the secretion of IL-10 from human PBMCs induced by SEQ ID NO: 321 was slightly higher at higher concentrations of this oligonucleotide (FIG. 17B).

Example 4

. Олигонуклеотид, который служит в качестве позитивного контроля, SEQ ID NO:242, обозначен ●. Олигонуклеотид, который служит в качестве негативного контроля, SEQ ID NO:330, обозначен

. Тестируемым олигонуклеотидом, показанным на фиг. 13А и 13В, является SEQ ID NO:313. Тестируемым олигонуклеотидом, показанным на фиг. 14А и 14В, является SEQ ID NO:314. Тестируемым олигонуклеотидом, показанным на фиг. 15А и 15В, является SEQ ID NO:319. Тестируемым олигонуклеотидом, показанным на фиг. 16А и 16В, является SEQ ID NO:316. Тестируемым олигонуклеотидом, показанным на фиг. 18А и 18В, является SEQ ID NO:321. Тестируемым олигонуклеотидом, показанным на фиг. 19А и 19В, является SEQ ID NO:317. Тестируемым олигонуклеотидом, показанным на фиг. 20А и 20В, является SEQ ID NO:320. Концентрация олигонуклеотида, используемая для получения исходных точек на графиках, отложена по оси Х (мкМ).The levels of activation of B cells and monocytes after treatment of these cells with the CpG oligonucleotides described herein are shown in the attached FIG. 13-15, 16 and 18-20. In these graphs, the tested oligonucleotides are indicated

. The oligonucleotide, which serves as a positive control, SEQ ID NO: 242, is indicated by ●. An oligonucleotide that serves as a negative control, SEQ ID NO: 330, is indicated

. The test oligonucleotide shown in FIG. 13A and 13B, is SEQ ID NO: 313. The test oligonucleotide shown in FIG. 14A and 14B, is SEQ ID NO: 314. The test oligonucleotide shown in FIG. 15A and 15B, is SEQ ID NO: 319. The test oligonucleotide shown in FIG. 16A and 16B, is SEQ ID NO: 316. The test oligonucleotide shown in FIG. 18A and 18B, is SEQ ID NO: 321. The test oligonucleotide shown in FIG. 19A and 19B, is SEQ ID NO: 317. The test oligonucleotide shown in FIG. 20A and 20B, is SEQ ID NO: 320. The concentration of the oligonucleotide used to obtain the starting points on the graphs is plotted along the X axis (μM).

As shown in FIG. 13-15, 16 and 18-20, each of the oligonucleotides tested in these assays is able to induce different levels and patterns of expression of the cell surface marker. For example, most of the tested ODNs taken at approximately equivalent or lower concentrations showed better induction of cell surface markers than a positive control oligonucleotide having a fully phosphorothioate backbone.

The expression level of CD86 on B cells and the expression of CD80 on monocytes induced by SEQ ID NO: 313 was similar to the level induced by SEQ ID NO: 242. In contrast to SEQ ID NO: 242, SEQ ID NO: 313 stimulated cells at lower concentrations than SEQ ID NO: 242, which suggests its higher activity. The levels of CD86 expression on B cells and CD80 expression on monocytes induced by SEQ ID NO: 314 were similar. The effect of SEQ ID NO: 314 was observed when applied at lower concentrations than SEQ ID NO: 242, indicating an increased activity of SEQ ID NO: 314. The expression of CD86 on the surface of B cells was strongly activated by SEQ ID NO: 319, while the signal level was comparable to the signal level induced by SEQ ID NO: 242. As for the levels of expression of CD80 on monocytes, in this case only a slight increase induced by SEQ ID NO: 319 could be detected. The effectiveness of SEQ ID NO: 319 in inducing activation of CD86 on B cells was slightly lower compared with the effectiveness of SEQ ID NO: 242. Compared to SEQ ID NO: 242, SEQ ID NO: 316 induced higher levels of the CD86 activation marker on B cells (FIG. 16A) and the CD80 activation marker on monocytes (FIG. 16B). B cells were strongly activated after incubation of human PBMCs with SEQ ID NO: 321, as indicated by expression of CD86 (Fig. 18A). The level of CD86 exceeded the level induced by SEQ ID NO: 242. Monocyte activation induced by SEQ ID NO: 321, as determined by expression of CD80, was also stronger than activation induced by SEQ ID NO: 242 (Fig. 18B). SEQ ID NO: 317 induced the same expression levels of CD86 on B cells as SEQ ID NO: 242 (FIG. 19A), while expression levels of the CD80 activation marker on monocytes were higher than the activation levels induced by SEQ ID NO: 242 (FIG. 19B). SEQ ID NO: 320 induced the same expression levels of CD86 on B cells as SEQ ID NO: 242 (FIG. 20A).

Example 5

Semi-soft oligonucleotides have an immunostimulating effect on human PBMCs in vitro.

In this example, semi-soft oligonucleotides were analyzed for their ability to induce cytokines and chemokines in vitro. Peripheral blood mononuclear cells (PBMCs) were obtained from three healthy human donors and cultured in the presence of various concentrations (0.05; 0.1; 0.2; 0.5; 1.0 and 5.0 μM) of fully stabilized CpG SEQ ID NO: 242 or semi-soft SEQ ID NO: 241. After 6, 16 or 48 hours, culture supernatants were collected and the levels of various cytokines (IFN-α, TNF-α, IL-10) and IP-10 chemokine in supernatants were measured by ELISA. At low oligonucleotide concentrations, semi-soft and fully stabilized oligonucleotides induced the same levels of IFN-α in culture after 16 or 48 hours. However, the maximum induction of IFN-α by the oligonucleotide ODN 5476 was achieved at about half the concentration of this oligonucleotide required for SEQ ID NO: 242. At intermediate concentrations, SEQ ID NO: 242 induced higher levels of IFN-α than SEQ ID NO: 241, and at higher concentrations, none of SEQ ID NO: 242 and SEQ ID NO: 241 induced significant levels of IFN-α. The chemokine IP-10 was stimulated by semi-soft and fully stabilized oligonucleotides at a similar level and with a similar dependence on their concentrations. In both cases, at lower oligonucleotide concentrations, approximately 700 pg / ml IP-10 was observed, and lower IP-10 was induced at higher oligonucleotide concentrations. The nature of the induction of IP-10 was similar to that of the cytokine IL-10, except that the semi-soft oligonucleotide at 0.05 μM induced a significant amount of IL-10, while the fully stabilized oligonucleotide at 0.05 μM induced a small amount of IL-10, or did not induce IL-10 at all. Semi-soft and fully stabilized oligonucleotides had a similar ability to induce TNF-α, that is, oligonucleotides of both types strongly induced TNF-α, especially at high concentrations.

Table 1

Cytokines and chemokine (pg / ml) ^one induced by oligonucleotides (μM)

¹ Values are given as mean (standard deviation)

Example 6

In vitro stimulation of murine macrophages with semi-soft SEQ ID NO: 241 compared to fully stabilized ODN.

The murine macrophage cell line (RAW264) was incubated for 16 hours with the semi-soft oligonucleotide SEQ ID NO: 241, with the fully stabilized oligonucleotide SEQ ID NO: 242, with the fully stabilized ODN 1826, with lipopolysaccharide (LPS) or with PBS. Semi-soft ODN and fully stabilized ODN were evaluated at concentrations of 0.02; 0.05 and 0.1 μM. Supernatants were collected and the p40 subunit of IL-12 (IL-12p40, pg / ml) was measured by ELISA. The results are shown in Table 2. The semi-soft oligonucleotide SEQ ID NO: 241 was significantly more active in inducing secretion of IL-12p40 macrophages than any of the fully stabilized ODNs.

table 2

The secretion of IL-12p40 mouse macrophages stimulated with a semi-soft oligonucleotide SEQ ID NO: 241

Example 7

Semi-soft B-class oligonucleotides with a sequence optimized for stimulating human immune cells are potent immunostimulants of murine immune cells.

It has been reported that human and mouse immune cells respond to the effects of various CpG-ODNs. Fully stabilized CpG SEQ ID NO: 242 was considered “optimal” for stimulating human immune cells, but was not considered “optimal” for stimulating murine immune cells. Conversely, the fully stabilized CpG-ODN 5890 (5 'T * C * A * A * C * G * T * T3') was considered "optimal" for stimulating murine immune cells, but was not considered as "optimal" for stimulating human immune cells. Both human and mouse B cells have been reported to express TLR9. TLK9-expressing mouse HEK293 splenocytes were cultured in the presence of various concentrations of fully stabilized CpG SEQ ID NO: 242, fully stabilized CpG-ODN 5890 or semi-soft SEQ ID NO: 241, and TLR9 activation was measured as described below. The cells used for this assay expressed murine TLR9 and contained a reporter gene construct. Cells were incubated with ODN for 16 hours at 37 ° C in a humidified incubator. Each experimental point was obtained with three repetitions. Cells were lysed and assayed for reporter gene activity. Stimulation indices were calculated with respect to the activity of the reporter gene in the medium without the addition of ODN. The semi-soft oligonucleotide SEQ ID NO: 241 and the fully stabilized oligonucleotide SEQ ID NO: 242 had the same nucleotide sequence. The results are shown in table 3. At the lowest concentrations of SEQ ID NO: 241 and SEQ ID NO: 242 had a minimal immunostimulating effect. However, as the concentration increased to 14 nM and higher, SEQ ID NO: 241 had a clearly higher immunostimulating effect than SEQ ID NO: 242. At the highest concentrations studied in this experiment, SEQ ID NO: 241 had at least the same stimulatory activity as the fully stabilized oligonucleotide ODN 5890 optimized for mice.

Table 3

Stimulation index of murine HEK293 TLR-expressing cells with a semi-soft ODN with a sequence optimized for human cells

Examples 8-9

Semi-soft oligonucleotides induce activation of NK cells.

Semi-soft and fully stabilized oligonucleotides were also compared in terms of their ability to stimulate NK cell activation. Using a standard chromium release assay, 10 × 10 ⁶ spleen cells of BALB / c mice were cultured for 48 hours in 2 ml of RPMI medium containing 10% FBS (thermally inactivated to 65 ° C for 30 minutes), 50 μM 2-mercaptoethanol , 100 units / ml penicillin, 100 μg / ml streptomycin and 2 mM L-glutamate with the addition of either semi-soft SEQ ID NO: 241 or fully stabilized SEQ ID NO: 242, with each ODN being added to a final concentration of 1, 3 or 10 μg / ml, or without the addition of these oligonucleotides. Cells were washed and then used as effector cells in a short-term ⁵¹ Cr release assay using YAC-1 and 2C11, two NK-sensitive target cell lines (Ballas ZK et al. (1993) J. Immunol., 150: 17 -thirty). Effector cells at various concentrations were added to 10 ^{4 51} Cr-labeled target cells in 0.2 ml, in V-bottom microtiter plates and incubated in 5% CO ₂ for 4 hours at 37 ° C. The tested effector cell: target cell (E: T) ratios were 6.25: 1, 25: 1, 50: 1, and 100: 1. The plates were then centrifuged and aliquots of the supernatant were counted for radioactivity. The percentage of specific lysis was determined by calculating the ratio ⁵¹ Cr released in the presence of effector cells, minus ⁵¹ Cr released during cultivation with only target cells, to the total released after cell lysis in 2% acetic acid (100% lysis) minus ⁵¹ Cr (im / min), cultured only The results are presented below in Table 5. On the whole, the semi-soft oligonucleotide SEQ ID NO: 241 and fully stabilized SEQ ID NO: 242 induced basically comparable levels of NK cell activation at all ODN concentrations and for all analyzed E: T ratios.

Table 5

NK cell specific lysis

Example 10

Semi-soft oligonucleotides have mainly greater immunostimulating activity than fully phosphorothioate oligonucleotides with the same or similar sequence.

All tested semi-soft variants are more active in the analysis for human TLR9 than the corresponding molecule with a homogeneous phosphorothioate backbone (table 6). The average stimulation index was calculated from experimental points obtained for four concentrations (0.1 μM, 0.5 μM, 2 μM and 8 μM). In this table, U is 2'-deoxyuracil.

Table 6

Relative average stimulation indices of semi-soft oligonucleotides compared to fully phosphorothioate oligonucleotides having the same or similar sequence

Example 11

Increased in vitro activity of semi-soft variants of fully stabilized oligonucleotides with weak immunostimulating ability.

(T * G * T * C * G * T * T * G * T * C * G * T * T * G * T * C * G * T * T * G * T * C * G * T * T , SEQ ID NO: 244) is a fully stabilized, fully phosphorothioate CpG oligonucleotide having low immunostimulatory activity compared to SEQ ID NO: 242. Related semi-soft oligonucleotides (T * G * T * C_G * T * T * G * T * C_G * T * T * G * T * C_G * T * T * G * T * C_G * T * T, SEQ ID NO: 258) and (T * G * T * C_G * T * T * G * T * C_G * T * T_ * G * T * C_G * T * T_G * T * C_G * T * T, SEQ ID NO: 243) were many times more active than SEQ ID NO: 244 and even more active than SEQ ID NO: 242.

Table 7

Increased immunostimulating activity of semi-soft variants of a fully stabilized oligonucleotide with weak immunostimulating ability

Example 12

Semi-soft oligonucleotides with shorter lengths have immunostimulating activity in vitro.

Semi-soft 16-mer, SEQ ID NO: 283, 16-mer, SEQ ID NO: 245, 17-mer, SEQ ID NO: 284 and 24-mer, SEQ ID NO: 241 compared with fully stabilized ODN, 24-mer, SEQ ID NO: 242, and 18-mer, SEQ ID NO: 285 in terms of their ability to stimulate TLR9 signaling. Each of the oligonucleotides and reporter gene constructs at concentrations of 1, 6, 12, or 24 μg / ml were added to HEK293 cells transfected with human TLR9, after which the level of TLR9 activation was determined as described above.

The 18-dimensional fully stabilized oligonucleotide ODN of SEQ ID NO: 285 had lower immunostimulating activity than the 24-dimensional fully stabilized oligonucleotide of SEQ ID NO: 242 at all estimated concentrations, and the 16-dimensional and 17-dimensional semi-soft oligonucleotides had at least the same stimulating activity as the 24-mer SEQ ID NO: 242 at concentrations of 6 μg / ml and above. In addition, the 16-dimensional and 17-dimensional semi-soft oligonucleotides had almost the same immunostimulating activity as the 24-dimensional semi-soft oligonucleotide SEQ ID NO: 241.

Table 8

Immunostimulatory activity of short semi-soft oligonucleotides compared with short and long fully stabilized and semi-soft oligonucleotides

Example 13

Semi-soft oligonucleotides have immunostimulatory activity in vivo.

BALB / c mice were divided into groups and 400 μg of semi-soft oligonucleotide SEQ ID NO: 241, a fully stabilized immunostimulatory oligonucleotide SEQ ID NO: 242, a fully stabilized negative control oligonucleotide (TGCTGCTTTTTTGTGCTTTTTGTFGTFGTFGTTTGTGTFGTTFGTTGTFGTTFGTFG 28C), or: solution (PBS). 3 hours after injection, animals were bled and serum IP-10, IFN-γ and TNF-α were determined using an appropriate cytokine-specific ELISA assay. Serum IP-10 levels of animals treated with the semi-soft SEQ ID NO: 241 (8000-12000 pg / ml) were approximately two times higher than the IP-10 levels of animals treated with the fully stabilized immunostimulating oligonucleotide SEQ ID NO: 242 (3500-8000 pg / ml). Serum IP-10 levels of animals that were administered with control SEQ ID NO: 286 were as low as those of animals that were injected with PBS. The semi-soft oligonucleotide SEQ ID NO: 241 and the fully stabilized immunostimulatory oligonucleotide SEQ ID NO: 242 induced similar amounts of IFN-γ of approximately 150 pg / ml. The semi-soft oligonucleotide SEQ ID NO: 241 induced TNF-α 30-45% more than the fully stabilized immunostimulatory oligonucleotide SEQ ID NO: 242 (approximately 1550 pg / ml compared to about 1175 pg / ml in one experiment and about 710 pg / ml compared to 490 pg / ml in another experiment).

In another series of in vivo experiments, semi-soft and fully stabilized oligonucleotides were evaluated for their ability to kill tumors in BALB / c mice. Three groups of BALB / c mice were injected with ip spontaneous mouse renal adenocarcinoma (Renca) cells using a developing tumor model. Salup RR et al. (1985) J. Immunopharmacol 7: 417-36. Mice of each group were also injected with either 100 mg of the semi-soft oligonucleotide SEQ ID NO: 241, or 100 mg of the fully stabilized immunostimulating oligonucleotide SEQ ID NO: 242, or an equivalent volume of PBS. Then the survival rate of the mice was monitored and after their death, the tumor size was determined. In mice treated with PBS as a control, the survival time averaged 44 days, and in 20% of mice it was 50 days. In contrast, in mice injected with the semi-soft oligonucleotide SEQ ID NO: 241, survival after 50 days was 80%, and in mice injected with the fully stabilized immunostimulatory oligonucleotide SEQ ID NO: 242, survival after 50 days was 70%. With regard to tumor size (in cubic millimeters), then after 52 days in mice treated with PBS, tumor volumes were nearly 1200 mm ^3, and mice treated with a semi-soft oligonucleotide SEQ ID NO: 241 or fully stabilized immunostimulatory oligonucleotide SEQ ID NO : 242, tumor volumes were approximately 250 mm ³ and 180 mm ^3, respectively. Thus, both the semi-soft oligonucleotide and the fully stabilized oligonucleotide were highly effective in reducing tumor mass and increasing longevity in these experimental models.

Example 14

Soft or semi-soft oligonucleotides have reduced nephrotoxicity.

It has been noted that administration of fully stabilized immunostimulatory oligonucleotides to monkeys may be associated with the development of glomerulonephritis, i.e., kidney inflammation. Diagnosis and observation of the development of glomerulonephritis can be carried out by the presence of red blood cells and protein in the urine, which is often accompanied by a decrease in glomerular filtration rate (azotemia), water and salt retention, hypertension and edema. Typically, urine is mostly free of blood cells and plasma proteins. Diagnosis can also be made by histological analysis of renal tissue. Renal tissue is known to be rich in nucleases, and therefore, soft oligonucleotides are expected to be more active than fully stabilized immunostimulatory oligonucleotides.

Monkeys were divided into two groups, while one group was administered soft oligonucleotides, and the other group was administered fully stabilized immunostimulatory oligonucleotides. Soft oligonucleotides and fully stabilized immunostimulatory oligonucleotides have identical sequences and differ only in internucleotide bonds. The monkeys of both groups were administered the same dose of an immunostimulating oligonucleotide. Preliminary (basic) processing and periodic measurements during processing allowed us to obtain at least one parameter used for analyzes for the presence of glomerulonephritis, including, for example, a detailed analysis of urine for proteinuria and / or hematuria, a microscopic analysis of urine for the presence of red blood cells and / or cylindrical red blood cells, analysis of protein concentration in urine, blood urea nitrogen (BUN) and serum creatinine, blood pressure measurement, determination of body weight, and kidney biopsy on optical and / or electronic micro osprey for tissue analysis. Clinical data correlated with the type of immunostimulatory oligonucleotide administered to each monkey, and the results obtained for each group were compared in order to assess statistical significance.

In addition, but not necessarily, to evaluate the results, depending on the dose of oligonucleotides, additional pairs of groups of monkeys were introduced into the studies, which were administered either soft or semi-soft oligonucleotides or fully stabilized immunostimulatory oligonucleotides, as described above, but at higher or lower doses.

Monkeys injected with mild oligonucleotides were significantly less likely to develop glomerulonephritis than monkeys injected with fully stabilized immunostimulatory oligonucleotides.

Example 15

Soft oligonucleotides have increased immunostimulating activity at high concentrations.

Soft oligonucleotides were compared with SEQ ID NO: 242 in terms of their ability to induce TLR9 activity. The mild ODN and control SEQ ID NO: 242 were compared at each of four concentrations: 1, 6, 12, and 24 μg / ml. The ratios of the level of activation of each soft oligonucleotide compared to the activation of the oligonucleotide SEQ ID NO: 242, for each concentration, are presented below in table 9. These results indicate that, at higher test concentrations, soft oligonucleotides have greater immunostimulatory activity than SEQ ID NO: 242.

Table 9

The relative activity of soft oligonucleotides compared with the oligonucleotide SEQ ID NO: 242 at each concentration

Example 16

Stability of serum and tissue oligonucleotides

Mice were injected subcutaneously with 25 mg / kg of the semi-soft oligonucleotide SEQ ID NO: 241, soft oligonucleotide (T * C * G * T * C * G * T * T * T * T_G_T_C_G_T * T * T * T * G * T * C * G * T * T; SEQ ID NO: 294) or the fully stabilized oligonucleotide SEQ ID NO: 242. Tissue and serum samples were taken after a certain amount of time (hours) and analyzed for the presence of an intact oligonucleotide and its fragments.

A known amount of internal standard ODN (1.25 μg polyT) was injected into the tissue or serum samples with a syringe, and ODN was isolated from tissue and plasma samples by the solid-phase extraction (SPE) methods described below. The resulting solutions containing analyte, metabolites and internal standard were analyzed by capillary gel electrophoresis (CGE), as well as by the MALDI-TOF methods described below. Then, the total amounts of isolated ODN (i.e., analyte plus metabolites) from the kidney, liver, spleen, and serum samples analyzed by CGE were determined. The standard deviation was calculated. For each metabolite, a relative amount was determined, expressed as a percentage of the total peak area.

SPE To isolate ODN from serum, 100 μg of the sample was injected with 1.25 μg of the internal ODN standard, mixed and dissolved in 5 ml of SAX buffer. This solution was applied to an anion exchange column (SAX, Agilent), then the column was washed and the ODN was eluted with increased ionic strength buffer. The resulting eluate was desalted on a reverse phase (RP) column (Glen Research) or on a similar column (HLB, Waters). The eluates from the RP column containing only water and acetonitrile were dried and solubilized in the same tube in 60 μl of deionized water. For further desalination of the samples, membrane dialysis was performed. Samples were analyzed directly by capillary gel electrophoresis. For MALDI-TOF MS, the samples were used either undiluted or in concentrated form, i.e. 50 μl of the ODN sample was dried in vacuo and dissolved in deionized water, and then analyzed as described below.

ODN was isolated from tissues in accordance with the same SPE protocol. 100 mg of tissue was homogenized using a FastPrep device. Then, proteinase K was added and the proteins were hydrolyzed for 2 hours. In the SPE method described above, phenol extraction was performed before passing the water-soluble fraction.

CGE. Desalted samples containing the analyte, its metabolites and a certain amount of the internal ODN standard were electrokinetically injected into the gel-filled capillaries (neutral, 30 cm, eCAP DNA capillary, Beckman # 477477) on the side of the sample with a preliminary injection of water. In this case, a voltage of 300 V / cm was applied, and detection was carried out continuously at 260 nm. Separation was carried out at 25 ° C in Tris / boric acid / EDTA buffer containing 7M urea. The analyte was identified by the relative time of its migration (MT _oligo / MT _internal ), that is, in relation to the time of migration of the standard, obtained in a similar way and simultaneously analyzed. The relative migration time and the relative area percentage of any electrophoretic peak that was> 3 × signal-to-noise ratio (S: N) were recorded. Quantification of peak heights between 3 × and 10 × signal: noise ratios was not performed.

% Oligo = (peak area / total peak area> 3 × S: N) × 100%

MALDI-TOF. Desalted samples containing the analyte and its metabolites were analyzed on an Apllied Biosystems MALDI-TOF mass spectrometer equipped with a delayed extraction source, a nitrogen laser operating at a wavelength of 337 nm, and a cathode ray tube with a 1.2-meter span. The following instrumental parameters were used: voltage 25 kV; grid voltage, 95.4%; on the waveguide, 0.1%; lag time 1200 nsec. As the matrix, 3-hydroxypicolinic acid containing diammonium citrate was used. The spectra of the ODN samples were subjected to external calibration on the same plate under identical conditions using a series of standard ODNs with known molecular weights.

The results obtained in 48 hours are presented in Fig.20. Figure 20 shows that in the kidneys, the levels of semi-soft SEQ ID NO: 241 and soft ODN SEQ ID NO: 294 were much lower (93% and 87%, respectively) than the levels of the fully phosphorothioate oligonucleotide SEQ ID NO: 242.

Example 17

Oligonucleotides C have immunostimulatory activity in vitro.

Semi-soft C-class oligonucleotides with phosphodiester bonds were obtained in the 5'-non-palindromic region (ODN SEQ ID NO: 225), in the 3'-palindromic region (ODN SEQ ID NO: 251), and in both 5'-non-palindromic and 3'- palindromic regions (ODN SEQ ID NO: 295). In addition, ODN SEQ ID NO: 252 was obtained with the same bonds as in ODN SEQ ID NO: 295, but with 2'-O-Meribose sugars in the nucleotides constituting the 3'-palindromic part (in the following sequence, underlined). These oligonucleotides were then evaluated using the TLR9 assay described above.

Fully stabilized backbone C-class oligonucleotides typically have relatively low TLR9 activity compared to B-class oligonucleotides. As shown in Table 10 below, the inclusion of a semi-soft sequence in the 5'-non-palindromic region (ODN SEQ ID NO: 255) led to a significant increase in TLR9 activity compared to the inclusion of a semi-soft sequence in the 3'-palindromic region (ODN SEQ ID NO : 251). The inclusion of a semi-soft sequence in both the 5'-non-palindromic region and the 3'-palindromic region (ODN SEQ ID NO: 295) led to an increase in TLR9 activity compared to the inclusion of a semi-soft sequence only in the 3'-palindromic region (ODN SEQ ID NO : 251).

Table 10

TLR9 Stimulation with Semi-Soft C-Class Oligonucleotides

Semi-soft C-class oligonucleotides not only retain their ability to induce IFN-α by human PBMCs, but they also have much greater activity at low concentrations. Increased activity was most pronounced in those C-class oligonucleotides that included a semi-soft sequence in the 5'-non-palindromic region (ODN SEQ ID NO: 255 and ODN SEQ ID NO: 295). ODN SEQ ID NO: 255, ODN SEQ ID NO: 251 and ODN SEQ ID NO: 295 were analyzed by ELISA and compared with SEQ ID NO: 242, i.e. the fully stabilized form of these three semi-soft oligonucleotides, as well as the C-class oligonucleotide being a strong inducer of IFN-α. The results are presented in table 11.

Table 11

Induction of IFN-α (pg / ml) semi-soft variants of the C-class oligonucleotide

Example 18

Physico-chemical characteristics of SEQ ID NO: 313

Methods

The X-ray powder diffraction pattern obtained for SEQ ID NO: 313 showed a halo characteristic of the amorphous phase. Analysis of the absorption of water vapor showed that SEQ ID NO: 313 is highly hygroscopic. The tendency of the drug to change in humidity can lead to fluctuations in the amount of moisture depending on the humidity of the environment. The specified compound has a high water solubility (> 100 mg / ml), and therefore has adequate solubility at all used pH ranges. Analysis of aqueous solutions of the drug, carried out at elevated temperatures, showed that they quickly decompose in weakly acidic and acidic environments, while solutions buffered to a pH value exceeding 6 had adequate stability in the solution.

Results :

It has been found that SEQ ID NO: 313 is inherently amorphous and highly hygroscopic. The specified compound has a high water solubility (> 100 mg / ml), and therefore has adequate solubility at all used pH ranges. This ODN quickly decomposes in a weakly acidic and acidic environment. Solutions buffered to a pH greater than 6 had adequate stability in solution.

Example 19

In vitro stimulation of TLR9-transfected cells

Methods

HEK 293 cells transfected with human TLR9 were incubated with SEQ ID NO: 313 or SEQ ID NO: 329 for 16 hours. The signal level was determined by reading data for lyciferase.

Results :

Compared to SEQ ID NO: 329, SEQ ID NO: 313 was a stronger TLR9 target receptor stimulator.

Example 20

In vitro stimulation of human immune cells

Methods

Human peripheral blood mononuclear cells taken from 6 donors were incubated with SEQ ID NO: 313 or with SEQ ID NO: 329 for 24 or 48 hours. Then measured the secretion of cytokines.

Results :

The results are presented in Fig.23. Compared to SEQ ID NO: 329, SEQ ID NO: 313 showed increased or at least similar efficacy and / or activity as an inducer of TLR9-associated cytokines IL-6, IL-10, IFNα and IP-10.

Example 21

In vitro stimulation of murine splenocytes

Methods

Mouse (BALB / c) splenocytes were incubated with SEQ ID NO: 313 or with SEQ ID NO: 329 for 48 hours. Then measured the secretion of cytokines and IP-10.

Results :

Compared to SEQ ID NO: 329, SEQ ID NO: 313 showed increased or at least similar efficacy and / or activity as an inducer of the cytokines IL-6, IL-10, IL-12p40, IFNα, TNFα and IP-10 . The data are presented in Fig.24. These data demonstrated that the activity of SEQ ID NO: 13 with respect to murine immune cells is comparable to its activity with respect to human cells (see above) and also correlates with activation by TLR9.

Example 22

In vivo cytokine gene induction in mice

Methods

This study evaluated the expression of cytokines in the lungs of mice after administration of a dose of SEQ ID NO: 313 into the respiratory tract. To study their effects on the kidneys, the induction of the same cytokines (as described in examples 10 and 21) in this organ was also evaluated. Mice (male BALB / c) were dosed with SEQ ID NO: 313 or SEQ ID NO: 329 (1 mg / kg each) either by intranasal instillation or by the administration of an intravenous loading dose. 8 or 15 hours after the dose, the lungs and kidneys were removed. Then, RNA was extracted, which was reverse transcribed into cDNA. The desired cDNA fragments were amplified and detected by real-time PCR (Roche LightCycler thermal cell, detection method using SYBR Green (in the green spectrum)). Primers for GAPDH, IFN-gamma, IL-6, IP-10 and TNF-alpha were designed using the Roche LC PROBE DESIGN program (Version 1.0 Roche catalog No. 3139174). Primers for IFN-alpha were designed using the PRIMER 3 program. Product yields were normalized as a ratio to control gene expression (GAPDH).

Results :

After dosing the oligonucleotide SEQ ID NO: 313 into the respiratory tract, he induced the expression of TLR9-associated genes (IL-6, TNF-α, IFN-α, IFN-γ and IP-10) in the lungs. The results are shown in Fig.25. However, with the exception of IP-10, these genes were not expressed in the kidneys of mice that were dosed in this way. Since IP-10 is usually induced by interferons, expression of this chemokine can occur indirectly as a result of the secretion of interferons into the bloodstream systemically circulating from the lungs. If SEQ ID NO: 313 was administered intravenously, then each of these genes, with the exception of the IFN-γ gene, was induced in the kidneys. Therefore, the absence of the effect of SEQ ID NO: 313 on the kidneys after its introduction into the respiratory tract is probably due to its low systemic effect.

CpG-ODNs can affect the kidney through a number of mechanisms. Acute renal granulomatous inflammation caused by the TLR9-dependent mechanism was observed after systemic exposure to some CpG-ODNs. The results obtained by the authors suggest that the systemic effect of SEQ ID NO: 313 introduced into the respiratory tract is insufficient to directly induce TLR9-associated genes in the kidneys.

Example 23

Effect on antigen-induced lymph node development in mice in vivo

Methods

This study evaluated the ability of SEQ ID NO: 313 to induce a deviation of the immune response from a Th2-type response in draining lymph nodes in mice. Mice (BALB / c males) were sensitized by injection of antigen (ovalbumin, 100 μg) in Freund's complete adjuvant into the right hind paw pad. Mice in the same paw pad were simultaneously injected with SEQ ID NO: 313 or SEQ ID NO: 329 (1.5 mg / kg) or vehicle (saline). Six days after injection into the paw pad, the draining popliteal lymph node was removed. T cells (CD3 ⁺ ) and B cells (B220 ⁺ ) were counted by flow cytometry. An ex vivo analysis of the secondary immune response to the antigen was carried out as follows: 1 × 10 ⁶ cells (from the draining popliteal lymph node) were incubated in 220 μl of RPMI 1640 medium + 10% fetal bovine serum containing either ovalbumin (100 μg / ml) or diluent. After 36 hours, the culture medium was removed, and the concentrations of IL-beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, GM-CSF, IFN-gamma and TNF-alpha were measured with using a kit from LINCO Research, Inc. 14 Research Park Drive, St Charles, Missouri 63304, and analyzed on a Luminex multiplex system (Luminex Corporation, 12212 Technology Boulevard, Austin, Texas 78727-6115).

Results :

The number of cells in the popliteal lymph nodes: Sensitization led to the accumulation of T cells and B cells in the draining popliteal lymph nodes. In mice that were injected with CpG-ODN, basically no further increase in antigen-induced accumulation was observed. However, each CpG-ODN that was individually injected with a non-sensitized mouse did not cause accumulation of both T cells and B cells. The data are shown in Fig.26.

Analysis of the secondary immune response. Cells of the draining lymph node taken from antigen-sensitized mice secreted IL-4, IL-5, IL-10, and IFN-γ upon re-stimulation with ex vivo antigen. In sensitized mice that were also injected with CpG-ODN, levels of secretion of Th2-type cytokines, namely IL-4, IL-5 and IL-10, decreased, and levels of secretion of Th1-type cytokine, IFN-γ increased. The data obtained by the authors, presented in Fig. 27, confirmed the hypothesis that SEQ ID NO: 313, like SEQ ID NO: 329, suppresses the Th2 response to sensitization by antigen. The results are presented as mean ± Sq. (n = 9-10). * P <0.05 compared with the group of sensitized and vehicle-treated mice (Kruskal-Wallis multiple comparison criterion).

Example 24

Effect on antigen-induced IgE production in mice in vivo

Methods

Mice (BALB / c males) on days 0-7 of the study were sensitized with an antigen (ovalbumin, 10 μg, i.p.) with adjuvant (aluminum hydroxide). Two days before each sensitization and on the day of each sensitization, mice were administered SEQ ID NO: 313 (0.15 or 1.5 mg / kg, i.p.) or SEQ ID NO: 17 (1.5 mg / kg, i.p.). On the 18th day of the study, serum was taken. Antigen (ovalbumin) -specific IgE and IgG2a titers were measured by ELISA. A brief description of the protocol is given in table 12.

Table 12
Research Protocol Summary Sensitization Sensitization

ODN ODN ODN ODN

Day: -2 0 5 7 eighteen

end point

Results :

In mice treated with SEQ ID NO: 313 or SEQ ID NO: 329, the production of antigen-specific IgE was completely prevented. In contrast, IgG2a production increased. Since the production of IgE and IgG2a represents the characteristic Th2-type and Th1-type responses, respectively, this effect is additional evidence that SEQ ID NO: 313 can inhibit Th2-type responses to sensitization by antigen. Alternatively, CpG-ODNs can directly induce T-beta expression and the switch of the IgE class in B cells to another class. The data are presented in FIG. The results are presented as mean ± Sq. (n = 10-12 except that for the group of SEQ ID NO: 329, n = 5). * P <0.05 compared with the group of sensitized vehicle-treated mice (Kruskal-Wallis multiple comparison test).

Example 25

The effect on antigen-induced airway inflammation in mice in vivo

Methods

Mice (BALB / c males) on days 0-7 of the study were sensitized with an antigen (ovalbumin, 10 μg, i.p.) with adjuvant (aluminum hydroxide). Twice a week for the next two weeks, the mice were subjected to antigenic stimulation by treatment with an ovalbumin-containing aerosol. The first stimulation was performed on the 21st day of the study. SEQ ID NO: 313 (0.1-1000 μg / kg), SEQ ID NO: 329 (1-1000 μg / kg) or vehicle (saline, 20 μl) was administered by intranasal instillation into the respiratory tract once a week for two days before the first weekly antigenic stimulation. Endpoints were determined 48 hours after the last antigenic stimulation. Airway cells were isolated using bronchoalveolar lavage and differential cell counts were performed. The number of eosinophils (bulk density of eosinophils) and mucus secretion (PAS staining) in lung tissue was determined using histopathological analysis. The protocol is presented in table 13.

Table 13
Research Protocol Summary Sensitization Stimulation Stimulation

ODN ODN

Day: 0 7 19 21 24 26 28 31 33

end points

Results :

Antigenic stimulation led to an increase in the total number of leukocytes, and mainly eosinophils, in the airway lumen. The data are presented in FIG. 29. Eosinophilia was significantly suppressed depending on the administered dose of SEQ ID NO: 313 or SEQ ID NO: 329. The ED ₅₀ values needed to suppress eosinophilia were: SEQ ID NO: 313: 23 μg / kg; SEQ ID NO: 329: 47 mcg / kg. Stimulation also led to the accumulation of CD4 ⁺ T cells (CD3 ⁺ , CD4 ⁺ cells), which was significantly suppressed by the oligonucleotide SEQ ID NO: 313. The oligonucleotide SEQ ID NO: 313 also significantly inhibited antigen-induced accumulation of eosinophils in lung tissue and secretion of epithelial mucus. The results in Fig. 29 are presented as mean ± Sq. (n = 15). * P <0.05 compared with the group of antigen-stimulated and vehicle-treated mice (Kruskal-Wallis multiple comparison test). The results in FIG. 30 are presented as mean ± Sq. (n = 6). * P <0.05, ** P <0.001 compared with the group of antigen-stimulated and vehicle-treated mice (ANOVA, Dunnet’s multiple comparison criterion).

Example 26

Effect on antigen-induced respiratory hyperactivity in mice in vivo

Methods

Mice (BALB / c males) on days 0-7 of the study were sensitized with an antigen (ovalbumin, 10 μg, i.p.) with adjuvant (aluminum hydroxide). Twice a week for the next two weeks, the mice were subjected to antigenic stimulation by treatment with an ovalbumin-containing aerosol. The first stimulation was carried out on the 19th day of the study. SEQ ID NO: 313 (10-1000 μg / kg) or vehicle (saline, 20 μl) was intranasally administered once a week two days before the first weekly antigenic stimulation. Airway hyperactivity was analyzed 24 hours after the last antigenic stimulation by evaluating bronchostenosis (an increase in airway resistance) in response to the intravenous administration of methacholine. For each animal, a dose-response curve for methacholine was constructed and airway reactivity was quantified as the area under the curve. The protocol is presented in table 14.

Table 14
Research Protocol Summary Sensitization Stimulation Stimulation

ODN ODN

Day: 0 7 17 19 22 24 26 29th thirty

end points

Results :

Antigenic stimulation led to respiratory hyperactivity. SEQ ID NO: 313 inhibited the development of antigen-induced airway hyperactivity depending on the dose administered. The data are presented in FIG. 31 and 32 in the form of dose-response curves of a sample for the administration of methacholine, which illustrate the effect of SEQ ID NO: 313 (1000 μg / kg). Dose-dependent response curves for methacholine administration were quantified as the area under the curve. The results are presented as mean ± Sq. (n = 6-8). * P <0.05 compared with the group of antigen-stimulated and vehicle-treated mice (Kruskal-Wallis multiple comparison test).

The analysis of the curves of the maximum dose-response (RL), carried out by comparing mice stimulated with antigen and treated with a carrier, and each of the corresponding mice stimulated with antigen and treated with SEQ ID NO: 313, was carried out using multiple measurements in the MANOVA analysis. Between the dose-response curves for the groups treated with 100 and 1000 μg / kg of SEQ ID NO: 313, a significant difference was observed (P <0.05), while in the groups of mice stimulated with antigen and treated with a vehicle, and animals similarly treated with a dose 10 μg / kg of SEQ ID NO: 313, no significant difference was observed.

Example 27

Pharmacokinetic studies (FC) in rats in vivo

Pharmacokinetic (FC) studies were performed on rats to determine whether SEQ ID NO: 313 (semi-soft ODN) is excreted from plasma and tissues, and in particular from the kidneys, faster than SEQ ID NO: 329 (fully phosphorothioate ODN ) having a nucleotide sequence identical to the sequence of SEQ ID NO: 313.

Methods

56 rats were intravenously (i.v.) and intrathecal (i.t.) administered 5 μg / kg (both i.v. and i.t.) SEQ ID NO: 313 and SEQ ID NO: 329. Then, plasma, lung, and kidney samples were taken. The study lasted 5 days, with 14 readings taken at specific points in time for each group to which the dose was administered. For this, 3 rats / time point for the i.v. group (total - 42 rats) and 4 rats / time point for the i.t. group were used.

Results :

On Fig shows the concentration of ODN in plasma of rats after i.v.- and i.t.-administration at a dose of 5 mg / kg Data obtained for plasma showed that SEQ ID NO: 313 is excreted from plasma faster than SEQ ID NO: 329 after i.v. and i.t. administration.

Fig. 34 shows ODN concentrations in rat lungs after i.v. and i.t. administration at a dose of 5 mg / kg. After i.v. administration at the same dose, the concentrations of SEQ ID NO: 313 in the lungs were lower than the concentrations of SEQ ID NO: 329. After i.t.-administration, the difference was less noticeable. Data obtained after the introduction of SEQ ID NO: 329 into the lungs could only be obtained within 48 hours after the dose was administered.

On Fig shows the concentration of ODN in the kidneys of rats after i.v.- and i.t.-administration of a dose of 5 mg / kg Data for the kidneys showed that the absolute levels of SEQ ID NO: 313 in the kidneys were lower than the corresponding concentrations of SEQ ID NO: 329 after i.v. and i.t. administration. The effect of SEQ ID NO: 313 on the kidneys after i.t.-administration, in particular, was significantly lower than the effect of SEQ ID NO: 329 in the same dose. This is more clearly seen in FIG. 36 and 37.

On Fig shows the concentration of ODN in the kidneys of rats after i.v.-administration of a dose of 5 mg / kg On Fig shows the concentration of ODN in the kidneys of rats after i.t.-administration of a dose of 5 mg / kg After i.t. administration, the levels of SEQ ID NO: 313 and SEQ ID NO: 329 were below the lower limit of the quantitative range (0.4-0.6 μg / g) in the kidneys within 1 hour after the dose. After 1 hour, SEQ ID NO: 329 could be detected in all kidney samples taken during the study (48 hours). On the other hand, SEQ ID NO: 313 was present only at detectable levels for 7 hours after dosing.

Table 15
Systematized data of mean values of PK parameters for SEQ ID NO: 313 and SEQ ID NO: 329 after i.v.- and i.t.-administration of 5 mg / kg dose to rats Dose group the cloth ODN C _max (μg / ml) T _max (hour) T _1/2 (hour) AUC _0-INF
(h μg / ml) AUC _{0-48h *}
(h μg / ml) iv
(5 mg / kg) Plasma 10 na na 020 9.5 9.3 17 na na 0.62 62.8 62,2 Lungs 10 1.4 0.25 0.17 0.47 0.35 17 14,4 0,083 2,5 23.7 20.8 Kidney 10 6.6 0,083 24.9 184 123 17 11,4 0,083 nc nc 346 it
(5 mg / kg) Plasma 10 1.9 0.75 1.20 2.68 2,35 17 2.1 2 2,3 9.01 7.46 Lungs 10 632 0.25 28.1 5540 5350 17 692 one (31) ** (7908) ** 6505 Kidney 10 0.49 2 7.8 5.81 2,34 17 3.8 7 nc nc 134 na - could not be determined
nc - not calculated. They could not be estimated precisely because of the lack of experimental data in the final phase or because the final elimination phase was not reached during this period of the study.
* - AUC _0-48h or AUC _0-LAST at the last measured concentration for a period of up to 48 hours
** - Very rough estimate (obtained from only 2 experimental points in the final phase)
10 - SEQ ID NO: 313
17 - SEQ ID NO: 329.

Tables 16 (a) - (c)
Systemic and tissue effects of SEQ ID NO: 313 and SEQ ID NO: 329 after i.t. and i.v.-administration to rats at a dose of 5 mg / kg (a) Data for plasma ODN Dosing Method AUC _0-48h
(h μg / ml) The ratio of SEQ ID NO: 313: SEQ ID NO: 329 SEQ ID NO: 313 i.t. 2,35 0.32 (i.t.) i.v. 9.30 0.15 (i.v.) Attitude i.t.:i.v. 0.25 SEQ ID NO: 329 i.t. 7.46 i.v. 62,2 Attitude i.t.:i.v. 0.12 (b) Data for the lungs ODN Dosing Method AUC _0-48h
(h μg / ml) The ratio of SEQ ID NO: 313: SEQ ID NO: 329 SEQ ID NO: 313 i.t. 5350 0.82 (i.t.) i.v. 0.35 0.017 (i.v.) Attitude i.t.:i.v. 15286 SEQ ID NO: 329 i.t. 6505 i.v. 21 Attitude i.t.:i.v. 313 (c) Data for the kidneys ODN Dosing Method AUC _0-48h
(h μg / ml) The ratio of SEQ ID NO: 313: SEQ ID NO: 329 SEQ ID NO: 313 i.t. 2,34 0.017 (i.t.) i.v. 123 0.36 (i.v.) Attitude i.t.:i.v. 0.019 SEQ ID NO: 329 i.t. 134 i.v. 346 Attitude i.t.:i.v. 0.39

It was found that the systemic and renal effects of SEQ ID NO: 313 after 2 ODN administration, either intravenously (i.v.) or intratrecheally (i.t.), were significantly lower than the effect of SEQ ID NO: 329.

The AUC for SEQ ID NO: 313 in plasma after i.t.-administration at a dose of 5 mg / kg was 2.7 h · μg / ml. The corresponding value for SEQ ID NO: 329 was 9.0 h · μg / ml. Thus, the systemic effect of SEQ ID NO: 313 was one third of the action of SEQ ID NO: 329.

The AUC for SEQ ID NO: 313 in the kidneys after i.t.-administration at a dose of 5 mg / kg was 2.35 h · μg / ml. The corresponding value for SEQ ID NO: 329 was 134 h · μg / ml. Thus, at the same doses, the systemic effect of SEQ ID NO: 313 was only about 2% of the effect of SEQ ID NO: 329.

In contrast to plasma and kidney, the effect of SEQ ID NO: 313 on the lungs after i.t. administration was not significantly reduced compared to the effect of SEQ ID NO: 329. The AUC for SEQ ID NO: 313 in the lungs was approximately 70-80% compared to the AUC for SEQ ID NO: 329 in the lungs at the same dose. Since the lungs are the target tissue, it is preferable that the level of excretion of ODN from the lungs does not exceed the level of excretion of ODN from plasma and kidneys.

On Fig shows the concentration of SEQ ID NO: 313 and its 8-dimensional metabolite (s) in the kidneys of rats after i.v. administration of SEQ ID NO: 313 at a dose of 5 mg / kg.

Figure 39 shows the concentrations of SEQ ID NO: 313 and its 8-dimensional metabolite (s) in rat kidneys after i.t.-administration of SEQ ID NO: 313 at a dose of 5 mg / kg. Due to methodological problems, the data for the 8-dimensional metabolite of SEQ ID NO: 313 in plasma and tissues are incomplete. However, data for the 8-measure were obtained for some i.v. samples and all i.t. samples of the kidneys. These data showed that in most kidney samples, where 8-mer concentrations were successfully measured, the levels of this metabolite exceeded the levels of SEQ ID NO: 313, indicating that endonuclease activity is an important metabolic pathway for SEQ ID NO: 313.

The introduction of a number of phosphodiester bonds (SEQ ID NO: 313) into the fully phosphorothioate backbone (SEQ ID NO: 329), obviously, leads to an increase in the rate of ODN degradation and thereby to its more rapid elimination, especially from the kidneys.

Example 28

Activation of TLR9 using a semi-soft ODN compared to fully phosphorothioate ODN

Methods

Stably transfected HEK293 cells expressing TLR9 have been described previously [Bauer et al., PNAS; 2001]. In short, HEK293 cells were transfected by electroporation with vectors expressing human TLR9 and a reporter plasmid containing 6 × NF-kB luciferase. Stable transfectants (3 × 10 ⁴ cells / well) were incubated with ODN for 16 hours at 37 ° C in an incubator with high humidity. Each experimental point was determined with three repetitions. Cells were lysed and analyzed for luciferase gene activity (using the Brightlite kit, Perkin-Elmer, Ueberlingen, Germany). Stimulation indices were calculated relative to the activity of the reporter gene in the medium without the addition of ODN.

Results :

TLR9 is readily activated by ODNs containing optimal immunostimulatory CpG sequences. The authors incubated a cell line stably expressing human TLR9 with various semi-soft ODNs and with various fully phosphorothioate ODNs having the same ODN sequence as semi-soft ODNs. The results are shown in FIG.

These results demonstrated that each of the semi-soft ODNs listed in the following table, SEQ ID NO: 376, 378, 380, 382, 384, 241 activated higher TLR9 levels than the same ODN sequence having a fully phosphoric acid backbone, SEQ ID NO: 377, 379, 381, 383, 385, and 242, respectively.

Example 29

Internucleotide Rp bonds as phosphodiester-like bonds in semi-soft oligonucleotides

Methods

Cell Culture Conditions and Reagents

For B-cell proliferation assays, spleen cells taken from BALB / c mice (4-18 weeks old) were cultured at a concentration of 2-5 × 10 ⁵ -10 ⁶ cells / ml in RPMI for 44 h in 96-well microtiter plates, and then treated with 1 μCi ³ H-thymidine for 4-6 hours, after which the cells were collected and the number of pulses per minute was determined on a scintillation counter, as described previously (Krieg et al., 1995). To obtain Western blots, WEHI-231 cells (American Type Culture Collection, Rockville, MD) were cultured at 37 ° C. in a 5% CO ₂ humidity incubator and maintained in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) to which 10% thermally inactivated FCS (Life Technologies, Gaithersburg, MD), 1.5 mM L-glutamine, 50 μM 2-ME, 100 units / ml penicillin and 100 μg / ml streptomycin were added.

Oligonucleotides

Oligodeoxynucleotides (PO-Oligos) and steroidally randomized oligo (deoxynucleoside) phosphorthioates, [Mix-PS] -Oligos, were purchased from Operon Technologies (Alameda, CA) or obtained by the standard phosphoamidite method (Caruthers, 1985) (Stec et al., 1984) ) The oligonucleotide [Mix-PS] -d (TCCATGACGTTCCTGACGTT) ([Mix-PS] -SEQ ID NO: 386) was used as a positive control since it was previously found to have a strong immunostimulating effect on mouse cells (Yi et al. , 1996). For a CpG-PS-Oligo with a minimal stimulating motif, the PS-d sequence (TCAACGTT) -2066 was chosen for studies as a typical CpG motif with broad-spectrum immunostimulatory effects characteristic of a large family of CpG DNA. This sequence was named [Mix-PS] -2006 if it had a stereo randomized backbone. If this octameric sequence had a fully or partially stereo-main chain, then PS-Oligo was designated either [All-Rp-PS] -2066 or [All-Sp-PS] -2066 if the entire main chain was a stereo the main chain, or [CG-Rp-PS] -2066, or [CG-Sp-PS] -2066 if the stereo main chain had only CpG dinucleotide. Other used PS-Oligos are CpG-PS-d (TCAACGTTGA) ([Mix-PS] -SEQ ID NO: 387 and its A11-Rp and All-Sp stereo analogs, and the control non-CpG PS-d ( TCAAGCTTGA) (Mix-PS] -SEQ ID NO: 388.

The phosphorothioate stereo oligodeoxynucleotides were obtained by the oxathiophospholane method described previously (Stec et al., 1995) (Stec et al., 1998). The synthesis was carried out manually. The first nucleoside units from the 3'-end were immobilized on a solid support by means of a DBU-resistant sarcosinyl linker (Brown et al., 1989). Appropriately protected deoxynucleoside monomers having a 3'-O-2-thio-spiro-4,4-pentamethylene-1,3,2-oxathiophospholane group were synthesized and chromatographed to pure P-diastereomers. For the synthesis of [CG-Rp-PS] -2066 and [CG-Sp-PS] -2066, undivided mixtures of both P-diastereomers (with an Rp: Sp ratio of approximately 1: 1) (Stec et al., 1998) were used to assemble internucleotide bonds in a randomized configuration of P. atoms. All synthesized oligomers were purified by two-stage RP-HPLC: DMT-on (retention time within 23-24 minutes) and DMT-off (retention time 14-16 minutes); chromatographic system: ODS Hypersil column, 5 μm, 240 × 4.6 mm, 0-40% CH ₃ CN in 0.1 M triethylammonium bicarbonate, pH 7.5, gradient 1% / min. The purity of these oligomers was evaluated by polyacrylamide gel electrophoresis.

To study the absorption level of PS-Oligo, fluorescin-conjugated stereoregular PS-Oligos were prepared by solid-phase elongation of manually synthesized stereo-PS oligomers. After the detritylation step, fluorescein-phosphoramidite (ChemGenes Corporation, Ashland, MA; working concentration 125 mg / ml) and 1H-tetrazole (binding time-120 s) were added in the conventional manner, and then sulfonation with S-Tetra reagent (Stec et al. , 1993). Cleavage from the carrier and deprotection was carried out using concentrated ammonium hydroxide for 1 hour at room temperature and for 4 hours at 55 ° C, respectively. The resulting oligomers were purified by one-step RP-HPLC (see above). Due to the increased hydrophobicity of the fluorescein part, the Rp and Sp oligomers eluted with retention times of 14.5, 14.8, 14.7 and 15.0 min, respectively, i.e. at the end of attached sequences. In both cases, the two P-diastereomers eluted due to the non-stereospecificity of the phosphoramidite method / method of elongation of the fluorescein monomer by sulfonation.

Western blot analysis

Cells were harvested and resuspended in fresh medium at a concentration of 2 × 10 ⁶ cells / ml. Then the cells were left for four hours before the 40-minute stimulation. Cells were harvested and washed three times with cold PBS. Cells were lysed in 0.05 M Tris (pH 7.4), 0.14 M NaCl, 1% NP-40, 0.001 M Na ₃ NO ₄ , 0.01 M NaF, 4.3 mg / ml β-glycerophosphate, 0.002 M DTT , 50 μg / ml PMSF, 12.5 μg / ml antipain, 12.5 μg / ml aprotinin, 12.5 μg / ml leipeptin, 1.25 μg / ml pepstatin, 19 μg / ml bestatin, 10 μg / ml phosphoramidone 12.5 μg / ml trypsin inhibitor by freezing and thawing, followed by 30 minutes incubation on ice. Then the samples were centrifuged at 10,000 × g for 10 minutes at 4 ° C. The supernatant was stored as whole cell lysates for further analysis. Equal amounts of whole cell lysates (20 μg) were boiled in SDS sample buffer for 5 minutes and then electrophoresed in denaturing 11% polyacrylamide gel. After electrophoresis, proteins were transferred onto nitrocellulose membranes by semi-dry blotting (Bio-Rad Laboratories, Hercules, CA). Blots were blocked with 5% skim milk, and then hybridized with phospho-SAPK / JNK (Cell Signaling Technology, Beverly, MA), IkB-α and JNK1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Blots were visualized using enhanced chemiluminescence reagents (ECL, Amersham International) according to the manufacturer's protocol.

Results :

Induction of the CpG PS-Oligos Sp stereoisomer by incorporation of ³ H-thymidine into spleen cells. To determine the stereo-specificity of the immunostimulating effects of CpG DNA, BALB / c spleen cells were cultured with PS-d stereo-octanucleotides (TCAACGTT) -2066, in which all internucleotide bonds had either the Rp or Sp configuration at the concentrations indicated in Table 17 Cells were cultured for 48 hours, and this time was sufficient to induce proliferation of B cells with CpG motifs (Krieg et al., 1995). Stero-randomized [Mix-PS] -2066, having a CpG motif, induced strong proliferation of spleen cells depending on the dose (table 17). The Sp isomer also induced cell proliferation, and it had slightly stronger activity than [Mix-PS] -2066. In contrast, the Rp stereoisomer did not induce any detectable proliferation, which is consistent with the data obtained by Yu et al. (Yu et al., 2000).

Table 17
Induction of spleen cell proliferation by the Sp stereoisomer of CpG octamers Oligo Concentration pulse / min SI None (Wednesday) 2170 one 2066 (stereo randomized CpG) 0.4 μM 3154 1,5 " 2.4 μM 16525 7.6 " 4.8 μM 30811 14.2 Rp (2066) 0.4 μM 1207 0.6 " 2.4 μM 985 0.5 " 4.8 μM 640 0.3 Sp (2066) 0.4 μM 9567 4.4 " 2.4 μM 35372 16.3 " 4.8 μM 45591 20.1 Rp (2066) +1066 ¹ 0.4 μM 1597 0.7 " 2.4 μM 10255 4.7 " 4.8 μM 15841 7.3 SI = stimulation index compared to control (medium)
¹ each of two PS-Oligos was added at the indicated concentration at the beginning of cultivation

Preliminary studies of the authors of the present invention have demonstrated that the decamer CpG-PS-Oligos had an improved immunostimulating effect compared to the octamers used in the first experiment. Therefore, these experiments were repeated using the PS-SEQ ID NO: 387 construct, which was synthesized either as a stereo randomized [Mix-PS] -SEQ ID NO: 387, either in the form of (All-Rp) or in the form of (All -Sp). Again, both [Mix-PS] -SEQ ID NO: 387 and [All-Sp-PS] -SEQ ID NO: 387 significantly induced ³ H-thymidine incorporation depending on the dose. However, in this case, [All-Rp-PS] -SEQ ID NO: 387 also induced a significant increase in cell proliferation at the highest concentrations, indicating that it had at least partial stimulating activity.

Preferred Rp-chirality of CpG dinucleotide in octameric PS-Oligos .

It remains unclear whether the preference for the Sp stereoisomer in preliminary experiments is the result of an effect produced in the CG dinucleotide itself, or whether this effect can occur outside of the CG. In order to determine this, two PS-2066 octamers were synthesized that have a stereo-randomized backbone except for the link between the central CG, which were identified as Sp or Rp. In this experiment, unexpected results were obtained opposite to those obtained using PS-Oligos, in which the entire main chain was stereoregular, since [CG-Rp-PS] -2066 induced the same strong increase in the level of incorporation of ³ H-thymidine into cells spleen, as well as control stereo-randomized PS-Oligo. In contrast, PS-Oligo [CG-Rp-PS] -2066 was mostly inactive.

Inhibition of the incorporation of ³ H-thymidine into spleen cells by the CpG PS-Oligos R-stereoisomer .

The level of ³ H-thymidine incorporation in the wells treated with the Rp stereoisomer was lower than in the control wells, which suggests its possible inhibitory activity, although microscopic evaluation of these cells did not reveal any cytotoxicity. Indeed, if the cells were cultured with an equimolar mixture of [Mix-PS] -2066 and the All-Rp stereoisomer, there was an approximately 50% decrease in the level of ³ H-thymidine incorporation compared to cells cultured only with [Mix-PS] -2066 (table 17).

Preferred immune stimulation of [Rp-PS] oligonucleotides in the early stages .

Assays for ³ H-thymidine incorporation performed in previous experiments are vulnerable to artifact resulting from PS-Oligo degradation, and cold thymidine was released that competed with labeled material, which artificially suppressed its incorporation (Matson et al ., 1992). Previous studies have shown that [Rp-PS] -Oligos are much more sensitive to nuclease digestion than Sp analogues. Thus, it is possible that the apparent absence of the stimulating effect of [Rp-PS] -Oligo in the authors' analyzes on the incorporation of ³ H-thymidine could be an artifact leading to an incorrect conclusion that does not reflect the true effects of [Rp-PS] -Oligo. To detect the stimulatory effects of [Rp-PS] -Oligo at an early stage, that is, before PS-Oligo could undergo degradation, the authors analyzed the ability of these PS-oligonucleotides to induce rapid phosphorylation as an independent biological analysis for CpG-induced stimulation regulatory mitogen-activated protein kinase, JNK. The authors unexpectedly found that after processing the CpG sequences of PS-SEQ ID NO: 386 and PS-SEQ ID NO: 387 for 40 minutes, JNK phosphorylation was strongly induced not by [Sp-PS] isomers, but by stereo randomized [Mix- PS] - and [Rp-PS] -isomers. The control non-CpG [Mix-PS] -SEQ ID NO: 388 did not induce detectable JNK phosphorylation. All samples contained comparable amounts of total JNK protein.

Although the effect of CpG [Sp-PS] -Oligo was not observed in the JNK phosphorylation assay, this oligonucleotide was biologically active in this experiment because the level of inhibitory protein and IkB-α decreased under the action of all CpG-PS-Oligos regardless stereoisomer, but not under the influence of the control non-CpG-PS SEQ ID NO: 388.

Stereo-independent binding of PS-Oligo to the cell surface and its absorption .

One possible explanation for the observed differences in the bioactivity of PS-Oligo stereoisomers may be that the binding of PS-Oligos to cells or their uptake by cells is stereo-dependent. To evaluate this possibility, P-stereo-PS-Oligos with fluorescent labels were synthesized, and then they were incubated with cells. PS-Oligos showed a concentration and temperature dependent pattern of absorption by cells, which was consistent with previous studies. Namely, no detectable difference in binding or uptake of Rp or Sp-PS-Oligo was observed.

Example 30

The semi-soft oligonucleotide ODN 316 C-class and the semi-soft oligonucleotide ODN 313 B-class helped to reduce antigen-induced airway inflammation in vivo.

This study evaluated the in vivo effect of ODN 316 in a mouse model of antigen-induced airway inflammation. In this study, ODN 313 B-grade was used for comparison.

Methods

Mice (BALB / c males) on days 0-7 of the study were sensitized with an antigen (ovalbumin, 10 μg, i.p.) with adjuvant (aluminum hydroxide) (Pierce Alum).

Twice a week for the next two weeks, the mice were subjected to antigenic stimulation by treatment with an ovalbumin-containing aerosol. The first stimulation was performed on day 21 of the study. The aerosol was sprayed for 1 hour from a 1% solution of ovalbumin in PBS using a DeVilbiss Ultraneb nebulizer. Individual mice were used as a non-sensitized control.

Two days before the first antigenic stimulation, ODN 316 or ODN 313 (1-100 μg / kg) or vehicle (saline, 20 μl) was intranasally administered once a week every week.

Endpoints were determined on the 33rd day of the study (i.e., 48 hours after the last antigenic stimulation). Airway cells were isolated using bronchoalveolar lavage. Then, differential cell counting was performed using an automatic Advia cell counter using randomized samples verified by visual counting of cells on Wright-Giemsa stained cytocentrifuge preparations. The number of CD4 ⁺ T cells (CD3 ⁺ , CD4 ⁺ cells) was counted by flow cytometry. The results were expressed as mean ± Sq. for each group. Significance was determined using the Kruskal-Wallis multiple comparison criterion.

Results :

Antigenic stimulation led to an increase in the total number of leukocytes in the airway lumen. This increase was mainly due to the accumulation of eosinophils (for example, 3 × 10 ⁵ eosinophils / ml in antigen-stimulated, vehicle-treated mice, as opposed to unstimulated mice in which <1 × 10 ⁴ eosinophils / ml were observed). Eosinophilia was significantly suppressed by ODN 316 or ODN 313 (for example, approximately 5 × 10 ⁴ eosinophils / ml (P <0.05) in antigen-stimulated mice treated with 100 μg / ml of any ODN).

Antigen stimulation also resulted in accumulation of CD4 ⁺ T cells, which was significantly suppressed by any ODN (e.g., approximately 2 × 10 ⁴ CD4 ⁺ T cells / ml in antigen stimulated mice treated with 100 μg / ml of any ODN, and approximately 1 , 3 × 10 ⁵ CD4 ⁺ T cells / ml (P <0.05), in antigen-stimulated and vehicle-treated mice.

Conclusions :

Each of the semi-soft oligonucleotides of ODN 316 C-class and the semi-soft oligonucleotides of ODN 313 B-class suppressed antigen-induced airway eosinophilia and accumulation of CD4 ⁺ T cells in vivo.

Example 31

Comparison of the semi-soft ODNs of class B, C, and T: induction of cytokine secretion from murine splenocytes in vitro

These studies evaluated the ability of semi-soft ODNs of class B, C, and T to induce cytokine secretion from murine splenocytes in vitro.

Methods

BALB / c mouse splenocytes were collected and pooled. These splenocytes were incubated in 48-well culture plates with 1 × 10 ⁷ cells per 1 ml in RPMI 1640 + 10% fetal serum containing a separate ODN (1; 0.001; 0.01; 0.1; 1 or 10 μg / ml ) The tested ODNs were semi-soft ODN 20674 class B, semi-soft ODN 316 and ODN 317 class C, semi-soft ODN 319 and ODN 320 class T.

After 48-hour incubation (37 ° C, 5% CO ₂ ), the culture medium was removed and the concentrations of IL-β, IL-2, IL-4, IL-5, IL-6, IL-10, GM-CSF, IFN- gamma and TNF-alpha were measured using the Luminex multiplex cytokine system. Concentrations of IL-12p70, TNF-α, and IP-10 were measured by ELISA. The lower limits of accurate detectability were 3.2-10 pg / ml. Cell activation status was assessed by measuring the expression of CD40, CD69 and CD86 on CD3 ⁺ and B220 ⁺ cells using flow cytometry.

Results :

Each of the ODNs induced activation of B cells (B220 ⁺ cells) as determined by increased expression of CD40, CD69 and CD86, and activation of T cells (CD3 ⁺ cells) as determined by increased expression of CD69.

ODN induced secretion of IL-6, IL-10, IL-12p70, IFN-α, TNF-α and IP-10. The titers of the other measured cytokines were not increased. So, for example, it was found that at an ODN concentration of 1 μg / ml, the levels of cytokine secretion were as described below (all levels are expressed in PG / ml).

Table 18

In vitro cytokine secretion in response to the administration of a semi-soft ODN of class B, C and T

nd - not detected

Compared to the semi-soft ODN 313 B-class, two semi-soft ODN C-class induced higher titers of IL-10, IL-12p40, IFN-α, TNF-α and IP-10, but did not induce increased levels of B-cell activation. Two semi-soft T-class ODNs were obviously less effective as cytokine inducers than semi-soft B- and C-class ODNs.

Conclusions :

Each of the B-Class and C-Class ODNs gave a cytokine induction profile that corresponded to TLR9 activation, and each of them induced B-cell activation. T-class ODNs were less effective inducers of cytokines.

Compared to the semi-soft ODN 313 of B-class, each of the semi-soft ODN 316 and 317 of C-class induced higher concentrations of immunomodifying cytokines, but did not induce B-cell activation. These data allow us to conclude about the therapeutic value of the C-class ODN.

Example 32

Induction of cytokines, antibodies and CTLs in vivo in response to the administration of CpG-ODN

Measurement of cytokine levels: BALB / c mice were injected by subcutaneous (s.c.) injection of 400 mg ODN (SEQ ID NO: 294 (mild), 241 (semi-soft), 242 and 286). 3 hours after injection, animals were bled and plasma levels of IP-10, IFN-gamma and TNF-alpha were measured by ELISA. The results are shown in FIG. 41A and B (IP-10), C (IFN), D and E (TNF).

Humoral response: BALB / c mice were immunized with 1 mg of HBsAg administered alone or in combination with CpG-ODN by intramuscular (i.m.) injection. 4 weeks after the primary immunization, animals were given booster injections. Antibody titers were measured at the endpoint using an ELISA. 2 weeks after administration of the booster injection, IgG titers were measured at the endpoint using an ELISA. The results are shown in FIG. 42A and B.

Cytotoxic T lymphocyte response: BALB / c mice were immunized with 1 mg of HBsAg, administered alone or in combination with CpG-ODN by intramuscular (im) injection. 4 weeks after the primary immunization, animals were given booster injections. 4 weeks after administration of the booster injection, CTL activity was measured using a ⁵¹ Cr release assay. The results are presented in figs.

Thus, mild and semi-soft ODNs have a similar or stronger activating effect on the mouse immune system, as has been shown in vitro and in vivo studies, and can enhance antigen-specific immune responses.

Example 33

The use of CpG-ODN in in vivo anticancer therapy

The ODNs of the present invention were tested for efficacy in monotherapy on three cancer models. First, ODN was administered to mice with renal cell carcinoma (renca). These tests were performed using the following methods: tumors were induced by subcutaneous (sc) injection of 2 × 10 ⁵ renca cells in the left flank of the mouse on day 0. Treatment was performed by sc injection of PBS, CpG-ODN 241 or 242 weekly for 5 weeks starting at 10 day after the injection of tumor cells. The results are presented in FIG. 43A and B.

As the second test model, murine non-small cell lung cancer (Lewis lung carcinoma) was used. Tumors were induced by sc injection of 2 × 10 ⁶ Lewis carcinoma cells in the left flank of the mouse on day 0. Treatment was carried out by sc injection of PBS, 100 mg CpG-ODN 241 or 242 on the 1st, 3rd, 7th day and weekly within 2 months. The results are presented in FIG. 43E and F.

As a third model, mouse neuroblastoma was used. 1 × 10 ⁶ Neuro2a sc cells were injected into the left flank on day 0. Subcutaneous injections of PBS, 100 mg of CpG-ODN 241 or 242 were administered daily from day 10 to day 15. The results are shown in FIG. 43C and D.

Thus, a semi-soft ODN can inhibit the growth of a malignant tumor (mouse renca, LLC, neuroblastoma) and increase the survival of mice with these malignant tumors.

Example 34

Perinephral inflammation resulting from the administration of soft, semi-soft, and hard ODNs in TLR9-deficient BALB / c mice

TLR9-deficient BALB / c mice were examined for perinephral inflammation. The results are presented in tables 19 and 20, respectively. The semi-soft ODN (241) induced a lesser degree of inflammation in the injection area, but did not induce (at a dose of 100 mg) or induce a slight (at a dose of 250 mg) perinephral inflammation and was better tolerated by mice after its repeated administration.

Table 19 Groups Kidney Parenchyma Inflammation Granulomatous inflammation of the renal capsule Granulomatous inflammation of adipose tissue Pbs Norm 5/5 Norm 5/5 Norm 5/5 242, 100 mg Weak, 2/5 Mild or moderate 5/5 Mild or moderate 5/5 242, 250 mg Weak, 1/4 Mild or moderate 4/4 Noticeable 4/4 241, 100 mg Norm 5/5 Norm 5/5 Norm 5/5 241, 250 mg Weak, 2/5 Weak, 2/5 Weak or moderate, 3/5

Table 20 Groups Kidney Parenchyma Inflammation Granulomatous inflammation of the renal capsule Granulomatous inflammation of adipose tissue Pbs Norm 5/5 Norm 5/5 Norm 5/5 242, 100 mg Norm 5/5 Norm 5/5 Norm 5/5 242, 250 mg Norm 5/5 Norm 5/5 Norm 5/5 241, 100 mg Norm 5/5 Norm 5/5 Norm 5/5 241, 250 mg Norm 5/5 Norm 5/5 Norm 5/5

The above description is sufficient for the practical implementation of the present invention by any specialist. The scope of the present invention is not limited to the examples described above, since these examples are provided only to illustrate one aspect of the present invention, and other functionally equivalent variants may be included in the scope of the present invention. Based on the above description, in addition to the above and the above options, the specialist can make various modifications without departing from the scope of the attached claims. The advantages and objectives of the present invention are not necessarily covered by each of the presented variants of the invention.

Claims (52)

1. An immunostimulatory oligonucleotide having at least one internal cytosine-guanosine (CG) dinucleotide, where at least one internal CG dinucleotide has a phosphodiester or phosphodiester-like internucleotide bond, where optionally each additional internal dinucleotide CG or phosphodiester has, a stabilized phosphorothioate internucleotide bond and where all other internucleotide bonds are stabilized by a phosphorothioate bond. 2. The oligonucleotide according to claim 1, where the immunostimulatory oligonucleotide contains many internal CG dinucleotides having a phosphodiester internucleotide bond. 3. The oligonucleotide according to claim 2, where each internal CG dinucleotide has a phosphodiester internucleotide bond. 4. The oligonucleotide according to claim 3, wherein the immunostimulatory oligonucleotide molecule is a B-class immunostimulatory nucleic acid molecule. 5. The oligonucleotide according to claim 3, wherein the immunostimulatory oligonucleotide molecule is a C-class immunostimulatory nucleic acid molecule. 6. The oligonucleotide according to claim 3, where the molecule of the immunostimulatory oligonucleotide has a length of 4-100 nucleotides. 7. The oligonucleotide according to claim 3, not containing antisense sequences. 8. An immunostimulatory oligonucleotide containing at least one sequence N ₁ CGN ₂ , where in each sequence N ₁ CGN ₂ CG independently represents an internal dinucleotide cytosine-guanosine, each of N ₁ and N ₂ independently represents any nucleotide, and where, at least in one sequence N ₁ CGN ₂ and optionally in each additional sequence N ₁ CGN ₂ : the CG dinucleotide has a phosphodiester or phosphodiester-like internucleotide bond, and (a) N ₁ and C are linked by a phosphodiester or phosphodiester-like internucleotide bond, if N ₁ is an internal nucleotide, (b) G and N ₂ are connected by a phosphodiester or phosphodiester-like internucleotide bond, if N ₂ is an internal nucleotide, or (c) N ₁ and C are linked by a phosphodiester or phosphodiester-like internucleotide bond if N ₁ is an internal nucleotide and G and N ₂ are connected by a phosphodiester or phosphodiester-like internucleotide bond if N ₂ is an internal nucleotide, where all other internucleotide bonds are stabilized by phosphorus . 9. An immunostimulatory oligonucleotide containing:

where each of N ₁ and N ₃ independently represents a nucleic acid sequence with a length of 1-20 nucleotides, and where _ means an internal phosphodiester or phosphodiester-like internucleotide bond, N ₂ independently represents a nucleic acid sequence with a length of 0-20 nucleotides, and where GN ₂ - C contains 1 or 2 bonds stabilized by a phosphorioate bond. 10. Immunostimulatory oligonucleotide containing:

where each of N ₁ and N ₃ independently represents a nucleic acid sequence with a length of 1-20 nucleotides, and where _ means an internal phosphodiester or phosphodiester-like internucleotide bond, N ₂ independently represents a nucleic acid sequence with a length of 4-20 nucleotides, and where GN ₂ - C contains at least 5 phosphorothioate-stabilized bonds. 11. An immunostimulatory oligonucleotide containing:

where each of N ₁ , N ₂ and N ₃ independently represents a nucleic acid sequence with a length of 0-20 nucleotides, where _ means an internal phosphodiester or phosphodiester-like internucleotide bond, and where the specified oligonucleotide does not contain antisense sequences. 12. The compound containing the oligonucleotide according to claim 3, conjugated to an adjuvant, cytokine or antigen. 13. An immunostimulatory oligonucleotide containing: 5'T * C * G * (T * / A *) TN ₃ CGTTTTN ₄ CGN ₅ * T * T3 '(SEQ ID NO: 301), where N ₃ consists of 0-4 nucleotides, N ₄ consists of 1- 5 nucleotides and optionally 1-2 nucleotides, N ₅ consists of 0-7 nucleotides, where the symbol * means the presence of a stabilized phosphorothioate internucleotide bond, and where the oligonucleotide contains at least 3 phosphodiester internucleotide bonds and optionally 16 -24 nucleotide. 14. The oligonucleotide according to item 13, where the specified oligonucleotide has one of the following structures: 5'T * C * G * (T * / A *) TN ₃ CGTTTTN ₄ C * G * N ₅ * T * T 3 '(SEQ ID NO: 301), 5 'T * C * G * A * T * N ₃ C * G * TTTTN ₄ C_G_ * N ₅ * T * T 3' (SEQ ID NO: 302), 5'T * C * G * A * T * C * G * T * T * T * T_T_C_G * T * G * C * G * T * T * T * T * T3 '(SEQ ID NO: 304), 5'T * C * G * T * T * T * T * G * A_C_G_T * T * T * T * G * T * C * G * T * T3 '(SEQ ID NO: 305), or 5'T * C * G * T * T * N ₃ C_G_TTTTN ₄ CGN ₅ * T * T 3 '(SEQ ID NO: 303). 15. An immunostimulatory oligonucleotide containing: 5'T * CGCGN ₈ CGCGC * GN ₉ 3 '(SEQ ID NO: 315), where N ₈ has a length of 4-10 nucleotides and contains at least 1 C_G motif and optionally at least 2 or 3 CG motifs, N ₉ has a 0-3 nucleotide length, the symbol * means the presence of a stabilized phosphorothioate internucleotide linkage, and where _ means the presence of a phosphodiester internucleotide linkage, and where said oligonucleotide has a length of 15-40 nucleotides. 16. The oligonucleotide according to clause 15, where N ₈ represents PuCGPyPyCG, PuCGPyPyCGCG or ACGTTCG. 17. The oligonucleotide according to clause 15, where N ₉ contains at least one CG motif. 18. The oligonucleotide according to clause 15, where N ₉ represents CCG. 19. The oligonucleotide according to clause 15, where the specified oligonucleotide has the following structure: 5'T * C_G * C_G * A * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G3 '(SEQ ID NO: 316) or 5'T * C * G * C * G * A * C_G * T * T * C * G * C * G * C_G * C * G * C * G3 '(SEQ ID NO: 317). 20. An immunostimulatory oligonucleotide containing: 5'T * C_G (N ₆ C_GN ₇ ) _2-3 T * C_G * T * T3 '(SEQ ID NO: 311-312), where N ₆ and N ₇ independently have a length of 1-5 nucleotides and N ₆ , optional represents one nucleotide, preferably T or A, and N ₇ optionally consists of five nucleotides, preferably five pyrimidines or TTTTG, where * means the presence of a stabilized phosphorothioate internucleotide bond, and where _ means the presence of a phosphodiester internucleotide bond, and where indicated the oligonucleotide has a length of 16-40 nucleotides. 21. The oligonucleotide according to claim 20, where the specified oligonucleotide has one of the following structures: 5'T * C_G * T * C_G * T * T * T * T * G * A * C_G * T * T * T * T * G * T * C_G * T * T 3 '(SEQ ID NO: 313) or 5'T * C_G * A * C_G * T * T * T * T * G * T * C_G * T * T * 'T * T * G * T * C_G * T * T 3' (SEQ ID NO: 314). 22. An immunostimulatory oligonucleotide containing: 5'T * T * GX ₁ X ₂ TGX ₃ X ₄ T * T * T * T * N ₁₀ T * T * T * T * T * T * T3 '(SEQ ID NO: 318), where N ₁₀ has 4-8 nucleotides in length and contains at least 1 C_G motif and optionally at least 2 or 3 CG motifs, where X ₁ , X ₂ , X ₃ and X _{4 are} independently C or G, * means presence stabilized phosphorothioate internucleotide linkage, and where _ means the presence of a phosphodiester internucleotide linkage, and where the specified oligonucleotide has a length of 24-40 nucleotides. 23. The oligonucleotide according to item 22, where the specified oligonucleotide has one of the following structures: 5'T * T * G * C_G * T * G * C_G * T * T * T * T * G * A * C_G * T * T * T * T * T * T * T3 '(SEQ ID NO: 319 ) or 5'T * T * G * G_C * T * G * G_C * T * T * T * T * G * A * C_G * T * T * T * T * T * T * T3 '(SEQ ID NO : 320). 24. An immunostimulatory oligonucleotide containing: 5'T * C * G * C_G * A * C * G * T * T * C_G * G * C * G * C_G * C * G * C * C * G3 '(SEQ ID NO: 321), where * means the presence of a stabilized phosphorothioate internucleotide bond, where _ means the presence of a phosphodiester internucleotide bond, and optionally wherein said oligonucleotide is 21-40 nucleotides in length. 25. An immunostimulatory oligonucleotide containing: an octameric sequence containing at least one CG dinucleotide having a phosphodiester or phosphodiester-like internucleotide linkage and at least 4 nucleotides T, where C is cytosine or modified cytosine, and G is guanosine or modified guanosine, and where indicated the oligonucleotide contains at least one stabilized phosphorothioate internucleotide linkage. 26. The oligonucleotide of claim 25, wherein said octameric sequence comprises a TTTT motif. 27. The oligonucleotide of claim 25, wherein said octameric sequence comprises two CG dinucleotides. 28. The oligonucleotide according to item 27, where both CG dinucleotides have a phosphodiester or phosphodiester-like internucleotide bond. 29. The oligonucleotide of claim 25, wherein said octameric sequence is selected from the group consisting of: T * C-G * T * C-G * T * T, C-G * T * C-G * T * T * T, G * T * C-G * T * T * T * T, T * C-G * T * T * T * T * G, C-G * T * T * T * T * G * A, T * T * T * T * G * A * C-G, T * T * T * G * A * CG * T, T * T * G * A * CG * T * T, T * G * A * CG * T * T * T, G * A * CG * T * T * T * T A * C-G * T * T * T * T * G, C-G * T * T * T * T * G * T, T * T * T * T * G * T * C-G, T * T * T * G * T * C-G * T, and T * T * G * T * C-G * T * T, where * means the presence of a stabilized phosphorothioate internucleotide bond, and where _ means the presence of a phosphodiester internucleotide bond. 30. The oligonucleotide according A.25, where the specified oligonucleotide has a length of 8-40 nucleotides. 31. The oligonucleotide of claim 25, wherein C is cytosine or modified cytosine bases selected from the group consisting of 5-methylcytosine, 5-methylisocytosine, 5-hydroxycytosine, 5-halogenocytosine, uracil, N4-ethylcytosine, 5-fluorouracil, and hydrogen. 32. The oligonucleotide of claim 25, wherein G is guanine or a modified guanine base selected from the group consisting of 7-deazaguanine, 7-dease-7-substituted guanine (such as 7-dease-7- (C2-C6) alkynylguanine), 7-dease-8-substituted guanine, hypoxanthine, 2,6-diaminopurine, 2-aminopurine, purine, 8-substituted guanine such as 8-hydroxyguanine, 6-thioguanine, 2-aminopurine. 33. The oligonucleotide according A.25, where the specified oligonucleotide has a sequence selected from the group consisting of CGTCGTTTTGACGTTTTGTCGTT (SEQ ID NO: 333), GTCGTTTTGACGTTTTGTCGTT (SEQ ID NO: 334), TCGTTTTGACGTTTTGTCGTT (SEQ ID NO: 335), CGTTTTGACGTTTTGTCGTT (SEQ ID NO: 336), GTTTTGACGTTTTGTCGTT (SEQ ID NO; 337), TTTTGACGTTTTGTCGTT (SEQ ID NO: 338), TTTGACGTTTTTGTCGTT (SEQ ID NO: 339), TTGACGTTTTTTCTGTT (SEQ ID NO: 340), TGACGTTTTTGTCGTT (SEQ ID NO: 341), GTGTTTTTTTTTTTTTTTTTTTTTTTGTGTGTGTGTGTGTGTGTGTGTGTG ; 343), GTTTTGTCGTT (SEQ ID NO: 344), GTTTTGTCGTT (SEQ ID NO: 345). 34. The oligonucleotide according A.25, where the specified oligonucleotide has a sequence selected from the group consisting of TCGTCGTTTTGACGTTTTGTCGT (SEQ ID NO: 347), TCGTCGTTTTGACGTTTTGTCG (SEQ ID NO: 348), TCGTCGTTTTGACGTTTTGTC (SEQ ID NO: 349), TCGTCGTTTTGACGTTTTGT (SEQ ID NO: 350), TCGTCGTTTTGACGTTTTG (SEQ ID NO: 351), TCGTCGTTTTGACGTTTT (SEQ ID NO: 352), TCGTCGTTTTTGACGTTT (SEQ ID NO: 353), TCGTCGTTTTGACGTT (SEQ ID NO: 354), TCGTCGTTTTGACGT (SEQ ID NO: 355), TCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGG : 357), TCGTCGTTTTGA (SEQ ID NO: 358), TCGTCGTTTTG (SEQ ID NO: 359), TCGTCGTTTT (SEQ ID NO: 360). 35. The oligonucleotide of claim 25, wherein said oligonucleotide has a sequence selected from the group consisting of CGTCGTTTTGACGTTTTGTCGT (SEQ ID N0: 361), GTCGTTTTGACGTTTTGTCG (SEQ ID NO: 362), TCGTTTTGACGTTTTGTC (SEQ ID NO 363), CGTTTTGACGTTTTTGT (SEQ ID NO: 364), GTTTTGACGTTTTG (SEQ ID NOTGT (SEQ ID NOTGTTTTTTG) 367). 36. An immunostimulatory oligonucleotide containing: 5'TCGTCGTTTTGACGTTTTGTCGTT3 '(SEQ ID NO: 368), wherein at least one CG dinucleotide has a phosphodiester internucleotide bond and wherein said oligonucleotide contains at least one stabilized phosphorothioate internucleotide bond. 37. An immunostimulatory oligonucleotide containing: 5'GNC3 ', where N is a 4-10 nucleotide nucleic acid sequence that is at least 50% T and does not contain a CG dinucleotide, and where the oligonucleotide contains at least one stabilized phosphorothioate internucleotide communication. 38. The oligonucleotide according to clause 37, where N contains the TTTT motif. 39. The oligonucleotide of claim 38, wherein said oligonucleotide is selected from the group consisting of G * T * T * T * T * G * T * C and G * T * T * T * T * G * A * C, where * means the presence of a stabilized phosphorothioate internucleotide bond. 40. An immunostimulatory oligonucleotide containing T * C-G * T * C-G * A * C-G * T * T * C-G * G * C * G * C-G * C * G * C * C * G (SEQ ID NO: 369). 41. An immunostimulatory oligonucleotide containing T * C-G * G * A * C-G * T * T * C-G * G * C * G * C * G * C * C * G (SEQ ID NO: 371). 42. A method of modulating an immune response, comprising administering to a subject an oligonucleotide according to any one of claims 1 to 41 in an amount effective to modulate the immune response. 43. The method according to § 42, where the specified oligonucleotide is administered to the specified subject for the treatment of asthma or allergies. 44. The method according to § 42, where the specified subject suffers from a malignant tumor, infectious disease or autoimmune disease. 45. The method according to § 42, which further provides for the treatment of the specified subject in accordance with such a technique as surgical intervention, radiation therapy or drug therapy. 46. The method according to § 42, where the specified oligonucleotide is part of the drug. 47. The method according to § 42, where the specified oligonucleotide is administered by a method selected from the group consisting of oral, intranasal, sublingual, intravenous, subcutaneous, transmucosal route of administration, administration through the respiratory tract, direct injection and intradermal administration. 48. The method according to § 42, where the specified oligonucleotide is administered to the specified subject in an amount effective to induce the expression of cytokines, where the cytokines optionally include IL-6, TNF-α, IFN-α, IFN-γ IP-10. 49. The method according to § 42, wherein said oligonucleotide is administered to said subject in an amount effective to switch the immune response from a Th2 response to a Th1 response. Priority on points and signs: 08/19/2002 according to claims 1-14, claims 42-49 regarding the use of oligonucleotides according to claims 1-14; November 27, 2002 according to claims 15-23, claims 42-49 regarding the use of oligonucleotides according to claims 15-23; 02/14/2003 according to paragraph 24, paragraphs 42-49 regarding the use of oligonucleotides according to paragraph 24.

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