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Researchers Submit Patent Application, "Non-Coding Immunomodulatory DNA Construct", for Approval

January 27, 2014

By a News Reporter-Staff News Editor at Clinical Trials Week -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors Schroff, Matthias (Berlin, DE); Kleuss, Christiane (Berlin, DE); Kapp, Kerstin (Berlin, DE), filed on December 23, 2011, was made available online on January 16, 2014 (see also Mologen Ag).

The patent's assignee is Mologen Ag.

News editors obtained the following quote from the background information supplied by the inventors: "An emerging strategy to fight complex diseases, such as cancer, infectious diseases, allergy and asthma, is to utilize the patient's immune system. It is known that the immune system or its activity can be modulated by specific DNA sequences. Most known immunomodifying short DNA sequences contain an unmethylated cytosine guanine motif (CG motif) which has been described by Krieg et al. (Nature 1995 374: 6522 546-549). The occurrence of unmethylated CG motifs is substantially suppressed in the genome of eukaryotes compared to prokaryotes or viruses. Therefore, DNA molecules containing such a motif have evolved as a natural 'danger signal' and trigger the immune system in the fight against prokaryotic or viral pathogens. This can be exploited therapeutically or prophylactically to treat or prevent infectious as well as non-infectious diseases.

"DNA constructs comprising unmethylated CG motifs are able to elicit a considerable physiological effect by strongly stimulating effector cells of the innate immune system including dendritic cells, macrophages, natural killer (NK) and NKT cells. Unmethylated CG motifs are detected by the innate immune pattern recognition receptor Toll-like receptor (TLR) 9. While the exact recognition mechanism is not yet fully understood, significant progress in unraveling the underlying pathways has been made (A. Krieg, Nat. Rev. Drug Disc., 5:471-484, 2006). It is assumed that upon binding of DNA constructs containing unmethylated CGs to the receptor, multiple signal cascades are activated in responding cells. By upregulation of characteristic surface molecules and secretion of cytokines, adaptive immunity with a predominant Th1 pattern is induced. Such constructs can be used in combination with, for example, antibodies, chemotherapy or radiation therapy, vaccines or cytokines. Allergic diseases and asthma are mostly Th2-mediated. By increasing the ratio of Th1/Th2, the Th2-mediated responses are attenuated and thereby these types of diseases can be treated or prevented.

"Surface molecules include, for example, CD40, CD69, CD80 or CD86, depending on the specific cell type analyzed. Secretion of cytokines is also characteristic for distinct cell types; cytokines include, for example, macrophage inflammatory proteins (MIP)-1alpha, MIP-1beta, interleukin (IL)-6, IL-8, interferon (IFN)-alpha, tumor necrosis factor (TNF)-alpha, IFN-gamma, monocyte chemotactic protein (MCP)-1 or IFN-gamma-induced protein of 10 kDa (IP-10).

"In order to prevent or treat diseases, vaccination has been proven as a very effective approach. To ensure a strong and durable immune response, adjuvants capable of stimulating antigen-presenting cells such as dendritic cells, are usually administered together with the antigen, and for that purpose TLR9 agonists have been shown to be potent immunostimulants.

"Independently of any explanations of the underlying mechanisms by which unmethylated CG motifs influence or modulate an immune response, many approaches were developed for modulation of the immune system by using such motifs. The WO 1998/018810 discloses that immunostimulatory sequences containing unmethylated CG motifs are even more effective when they are part of a single strand. However, administering an open-chained single-stranded DNA molecule is not practicable due to the quick degradation of single-stranded nucleic acids. Consequently, different methods for the protection of single- or double-stranded DNA constructs comprising an unmethylated CG motif were developed.

"To achieve resistance against the degradation by DNA nucleases the phosphodiester bonds in the backbone of a nucleic acid polymer are frequently modified to phosphorothioates. Besides a somewhat less stimulatory activity of such phosphorothioate-protected nucleic acids clinical trials within the last years showed that the toxicity of a phosphorothioate-protection exclude or severely limit such nucleic acids from any use in pharmaceutical compositions or medicaments.

"Another approach to protect DNA sequences comprising a CG motif is disclosed for example in EP 1 196 178. This document discloses short deoxyribonucleic acid molecules, comprising a partially single-stranded, dumbbell-shaped, covalently closed sequence of nucleotide residues comprising CG motifs ('dSLIM'). According to the disclosure of the EP 1 196 178 the CG motifs are located within the single-stranded loops at both ends of the double-stranded stem of the disclosed molecule or within the double-stranded stem. The single-stranded hairpin loops protect a double-stranded stem from degradation by DNA nucleases within or outside of the cell.

"Document WO 2010/039137 discloses immune regulatory oligonucleotides as antagonists for TLR mediated diseases having one or more chemical modifications in the sequence flanking an immune stimulatory motif and/or in an oligonucleotide motif that would be immune stimulatory but for the modification. Thus, the intention of the disclosed oligonucleotides of WO 2010/039137 is to suppress an immune response caused by TLRs.

"WO 2005/042018 describes new so-called C-class CpG oligonucleotides, wherein a c-class oligonucleotide is characterised by CpG sequences, generally positioned at or near the 5' end or 3' end of the molecule, and a GC-rich palindrome motif, generally positioned at or near the other end of the molecule. The document discloses variations of the palindromic sequence of a c-class DNA."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "With regard to the state of the art it is an objective of the present disclosure to provide alternative immunomodulating DNA constructs being stable after transfer into eukaryotic cells and avoiding harmful side effects.

"The present disclosure teaches a DNA construct for immunostimulation comprising at least one sequence motif N.sup.1N.sup.2CGN.sup.3N.sup.4, wherein N1N2 .sup.and N.sup.3N.sup.4 is any combination of C, G, A, and T, and C is deoxycytidine, G is deoxyguanosine, A is deoxyadenosine and T is deoxythymidine and wherein the construct is a linear single- or double-chained DNA sequence comprising at least one nucleotide in L-conformation. N.sup.1N.sup.2 might be an element selected from the group comprising GT, GG, GA, AT or AA, N.sup.3N.sup.4 is an element selected from the group comprising CT or TT.

"As a further embodiment of the present disclosure a construct is provided wherein at least one nucleotide in L-conformation is comprised within the terminal five nucleotides located at or near the 5'- and/or the 3'-end of a DNA single strand.

"The invention provides further a DNA construct with at least one G stretch of at least there consecutive deoxyguanosine located near the 5' and/or 3' end, wherein a G stretch can be located between two sequence motifs according to claim 1 or 2.

"The spacing between two sequence motifs according to claim 1 or 2 can be at least five bases, especially when no deoxyguanosine is an element of the sequence.

"It is further intended that the DNA sequence is a linear open-chained DNA construct comprising single or double-stranded DNA or is a linear DNA construct, which comprises at least one end with a single stranded loop.

"The sequence motif N.sup.1N.sup.2CGN.sup.3N.sup.4 as defined above shall be located within a single-stranded and/or a double-stranded region of the DNA sequence.

"As a further embodiment the construct comprises inter- and/or intramolecular base-pairs and at least one unpaired, single-stranded region.

"Furthermore, a multimeric construct is provided, wherein at least two constructs comprising inter- and/or intramolecular base-pairs and at least one unpaired, single-stranded region ligate to one another.

"In addition, the construct may comprise at least one nucleotide in L- or D-conformation which is modified with a functional group selected from the group comprising carboxyl, amine, amide, aldimine, ketal, acetal, ester, ether, disulfide, thiol and aldehyde groups.

"The modified nucleotide may be linked to a compound selected from the group comprising peptides, proteins, carbohydrates, antibodies, synthetic molecules, polymers, micro projectiles, metal particles, nanoparticles, micelles, lipid carriers, or a solid phase.

"The disclosure provides a DNA construct having a first G stretch at the 5' end and three sequence motifs according to claim 1 or 2, wherein at least five bases are located between the first and second motif, excluding deoxyguanosine, and a G stretch, which is located between the second and third sequence motif and wherein two of the three 3' terminal deoxynucleotides are in L-conformation

"The constructs according to the present disclosure can be used for the treatment of cancer or autoimmune diseases or for the modulation of the immune system.

"As a further embodiment of the present disclosure a pharmaceutical composition is provided comprising a DNA construct as described above. The pharmaceutical composition may also comprise a chemotherapeutic.

"Furthermore, a vaccine is provided which comprises a DNA construct as described above. Therein, the DNA construct may be comprised as adjuvant."

For additional information on this patent application, see: Schroff, Matthias; Kleuss, Christiane; Kapp, Kerstin. Non-Coding Immunomodulatory DNA Construct. Filed December 23, 2011 and posted January 16, 2014. Patent URL:

Keywords for this news article include: Antibodies, Antigen-Presenting Cells, Drugs, Cytokines, Immunology, Mologen Ag, Chemotherapy, DNA Research, Blood Proteins, Cancer Vaccines, Dendritic Cells, Immunoglobulins, Radiation Therapy, Hemic and Immune Systems, Clinical Trials and Studies, Mononuclear Phagocyte System.

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Source: Clinical Trials Week

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