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Design and Structural Requirements of the Potent and Safe TLR-9 Agonistic Immunomodulator MGN1703.

Schmidt M, Hagner N, Marco A, König-Merediz SA, Schroff M, Wittig B - Nucleic Acid Ther (2015)

Bottom Line: Efficacy of immunomodulation strictly depends on the descriptive dumbbell shape and size of the molecule.Variations in stem length and loop size lead to reduced potency of the respective members of the dSLIM(®) class.The CpG ODN-PT group showed severe organ damage, whereas no such or other pathologies were found in the MGN1703 group.

View Article: PubMed Central - PubMed

Affiliation: 1Mologen AG, Berlin, Germany.

ABSTRACT
Single-stranded oligodeoxynucleotides (ODN), containing nonmethylated cytosine-guanine motifs (CpG ODN), are recognized by the innate immune system as "danger signals." CpG ODN are efficacious immunomodulators but require phosphorothioate (PT) or other backbone modifications for metabolic stability, which cause toxicities in mice and primates. We therefore designed a covalently closed DNA molecule (dSLIM(®)) where two single-stranded loops containing CG motifs are connected through a double-stranded stem in the absence of any nonnatural DNA component. The most promising immunomodulator, MGN1703, comprises two loops of 30 nucleotides containing three CG motifs each, and a connecting stem stem of 28 base pairs. MGN1703 stimulates cytokine secretion [interferon (IFN)-α, IFN-γ, interleukin (IL)-12, IL-6, and IL-2] and activates immune cells by increased expression of CD80, CD40, human leukocyte antigen (HLA)-DR and ICAM-1. Efficacy of immunomodulation strictly depends on the descriptive dumbbell shape and size of the molecule. Variations in stem length and loop size lead to reduced potency of the respective members of the dSLIM(®) class. In a representative mouse model, toxicities from injections of high amounts of a CpG ODN-PT and of MGN1703 were evaluated. The CpG ODN-PT group showed severe organ damage, whereas no such or other pathologies were found in the MGN1703 group. Oncological clinical trials of MGN1703 already confirmed our design.

No MeSH data available.


Related in: MedlinePlus

Histopathology of the liver after seven daily injections of MGN1703 or PT-based ODN1826 into mice. (A) Injection of PBS (control); (B) 7×2.5 μg MGN1703; (C) 7×2.5 μg ODN1826; (D) 7×60 μg MGN1703; (E, F) 7×60 μg ODN1826. (A, B, D) Minor inflammatory reaction (triangles) predominantly associated to the presence of macrophages and neutrophils. (C) Multiple inflammatory areas with necrotic hepatocytes (black arrows) and incipient necrosis (red arrows): intense presence of macrophages and some neutrophils. (E, F) Multiple inflammatory areas with incipient necrosis of hepatocytes (black arrows): intense eosinophilic response in the cytoplasm, nuclear pyknosis, and karyolysis. Representative pictures are shown.
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f5: Histopathology of the liver after seven daily injections of MGN1703 or PT-based ODN1826 into mice. (A) Injection of PBS (control); (B) 7×2.5 μg MGN1703; (C) 7×2.5 μg ODN1826; (D) 7×60 μg MGN1703; (E, F) 7×60 μg ODN1826. (A, B, D) Minor inflammatory reaction (triangles) predominantly associated to the presence of macrophages and neutrophils. (C) Multiple inflammatory areas with necrotic hepatocytes (black arrows) and incipient necrosis (red arrows): intense presence of macrophages and some neutrophils. (E, F) Multiple inflammatory areas with incipient necrosis of hepatocytes (black arrows): intense eosinophilic response in the cytoplasm, nuclear pyknosis, and karyolysis. Representative pictures are shown.

Mentions: In contrast, ODN1826 led to significant weight increase of these organs—with the 2.5 μg dose and even more with 60 μg of the CpG ODN-PT (Fig. 4A, B). This repeated the experiments with and confirmed the high toxicity of the CpG ODN described in the paper by Heikenwalder et al. [9]. The absence of toxicity after MGN1703 injections and the drastic toxicity from ODN1826 injections was not due to the difference in molecular weight (MGN1703>ODN1826) and the resulting higher number of ODN1826 molecules compared with MGN1703 at equal masses of injected molecules. As shown in Figure 4C, the highly significant differences were also observed if equimolar amounts of the respective molecules were injected, though both MGN1703 and ODN1826 were able to raise IL-12p40 levels in mice sera (Fig. 4C). Histopathological analyses of liver and spleen tissues confirmed the differences as well (Figs. 5, 6). While MGN1703 had only a slight effect on histopathology, ODN1826 resulted in vastly increased liver damage from inflammatory reaction and hepatocyte necrosis (regarding number and size of lesions). Damage of spleen produced similar impressive evidence, such as red pulp hyperplasia with intense extramedullary hematopoiesis and alteration of the white pulp (Table 2). The organ alterations caused by ODN1826 administration are not the equivalent of a normal immune response but do indicate toxic effects on the tissues analyzed.


Design and Structural Requirements of the Potent and Safe TLR-9 Agonistic Immunomodulator MGN1703.

Schmidt M, Hagner N, Marco A, König-Merediz SA, Schroff M, Wittig B - Nucleic Acid Ther (2015)

Histopathology of the liver after seven daily injections of MGN1703 or PT-based ODN1826 into mice. (A) Injection of PBS (control); (B) 7×2.5 μg MGN1703; (C) 7×2.5 μg ODN1826; (D) 7×60 μg MGN1703; (E, F) 7×60 μg ODN1826. (A, B, D) Minor inflammatory reaction (triangles) predominantly associated to the presence of macrophages and neutrophils. (C) Multiple inflammatory areas with necrotic hepatocytes (black arrows) and incipient necrosis (red arrows): intense presence of macrophages and some neutrophils. (E, F) Multiple inflammatory areas with incipient necrosis of hepatocytes (black arrows): intense eosinophilic response in the cytoplasm, nuclear pyknosis, and karyolysis. Representative pictures are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4440985&req=5

f5: Histopathology of the liver after seven daily injections of MGN1703 or PT-based ODN1826 into mice. (A) Injection of PBS (control); (B) 7×2.5 μg MGN1703; (C) 7×2.5 μg ODN1826; (D) 7×60 μg MGN1703; (E, F) 7×60 μg ODN1826. (A, B, D) Minor inflammatory reaction (triangles) predominantly associated to the presence of macrophages and neutrophils. (C) Multiple inflammatory areas with necrotic hepatocytes (black arrows) and incipient necrosis (red arrows): intense presence of macrophages and some neutrophils. (E, F) Multiple inflammatory areas with incipient necrosis of hepatocytes (black arrows): intense eosinophilic response in the cytoplasm, nuclear pyknosis, and karyolysis. Representative pictures are shown.
Mentions: In contrast, ODN1826 led to significant weight increase of these organs—with the 2.5 μg dose and even more with 60 μg of the CpG ODN-PT (Fig. 4A, B). This repeated the experiments with and confirmed the high toxicity of the CpG ODN described in the paper by Heikenwalder et al. [9]. The absence of toxicity after MGN1703 injections and the drastic toxicity from ODN1826 injections was not due to the difference in molecular weight (MGN1703>ODN1826) and the resulting higher number of ODN1826 molecules compared with MGN1703 at equal masses of injected molecules. As shown in Figure 4C, the highly significant differences were also observed if equimolar amounts of the respective molecules were injected, though both MGN1703 and ODN1826 were able to raise IL-12p40 levels in mice sera (Fig. 4C). Histopathological analyses of liver and spleen tissues confirmed the differences as well (Figs. 5, 6). While MGN1703 had only a slight effect on histopathology, ODN1826 resulted in vastly increased liver damage from inflammatory reaction and hepatocyte necrosis (regarding number and size of lesions). Damage of spleen produced similar impressive evidence, such as red pulp hyperplasia with intense extramedullary hematopoiesis and alteration of the white pulp (Table 2). The organ alterations caused by ODN1826 administration are not the equivalent of a normal immune response but do indicate toxic effects on the tissues analyzed.

Bottom Line: Efficacy of immunomodulation strictly depends on the descriptive dumbbell shape and size of the molecule.Variations in stem length and loop size lead to reduced potency of the respective members of the dSLIM(®) class.The CpG ODN-PT group showed severe organ damage, whereas no such or other pathologies were found in the MGN1703 group.

View Article: PubMed Central - PubMed

Affiliation: 1Mologen AG, Berlin, Germany.

ABSTRACT
Single-stranded oligodeoxynucleotides (ODN), containing nonmethylated cytosine-guanine motifs (CpG ODN), are recognized by the innate immune system as "danger signals." CpG ODN are efficacious immunomodulators but require phosphorothioate (PT) or other backbone modifications for metabolic stability, which cause toxicities in mice and primates. We therefore designed a covalently closed DNA molecule (dSLIM(®)) where two single-stranded loops containing CG motifs are connected through a double-stranded stem in the absence of any nonnatural DNA component. The most promising immunomodulator, MGN1703, comprises two loops of 30 nucleotides containing three CG motifs each, and a connecting stem stem of 28 base pairs. MGN1703 stimulates cytokine secretion [interferon (IFN)-α, IFN-γ, interleukin (IL)-12, IL-6, and IL-2] and activates immune cells by increased expression of CD80, CD40, human leukocyte antigen (HLA)-DR and ICAM-1. Efficacy of immunomodulation strictly depends on the descriptive dumbbell shape and size of the molecule. Variations in stem length and loop size lead to reduced potency of the respective members of the dSLIM(®) class. In a representative mouse model, toxicities from injections of high amounts of a CpG ODN-PT and of MGN1703 were evaluated. The CpG ODN-PT group showed severe organ damage, whereas no such or other pathologies were found in the MGN1703 group. Oncological clinical trials of MGN1703 already confirmed our design.

No MeSH data available.


Related in: MedlinePlus