Design and Structural Requirements of the Potent and Safe TLR-9 Agonistic Immunomodulator MGN1703.
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.
Affiliation: 1Mologen AG, Berlin, Germany.
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.
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Mentions: In keeping with this, MGN1703 also generated the highest stimulation of cytokine production compared with the molecules when IFN-α and IL-6 production from healthy donor PBMC and IL-12p40 from murine spleen cells were analyzed (Fig. 2D–F). To investigate the importance of structure in more detail, we generated further variations of MGN1703 with increased (from 28 bp to 56 bp and 88 bp) or decreased stem size (from 28 bp to 22 bp and 16 bp). Accordingly, we decreased the MGN1703 loop size (from 30 nt to 24 nt and 20 nt) while maintaining the 20-nt immunomodulatory sequence of that encompasses the CG motifs. The resulting molecules Mod-4 to Mod-9 are shown in Figure 3A. Notably, these structure variations of MGN1703 clearly diminished its immunomodulatory potential, as shown by IFN-α, IFN-γ, and IL-6 production from PBMC (Fig. 3B–D). Using even smaller constructs with only one CG motif per loop or two CG motifs in the stem confirmed the necessity of CG motifs in both loops but not in the stem for a high potency (Supplementary Fig. S2). These data clearly demonstrate the importance of a certain structure, size, CG motif content, and positioning of CG motifs on the efficacy of MGN1703 in both human and murine cells.
No MeSH data available.