Abstract 2565: Conformation overrides base sequence - insights from a novel class TLR-9 agonists

Abstract

Abstract Introduction DNA-based TLR-9 agonists are potent activators of immune cell populations and the immune system. Preclinical and ongoing clinical studies support the use of TLR-9 agonists as immunomodulators and/or adjuvants, and proof their anti-tumor effect by enhancing both the humoral and cellular responses. From the 4 classes of known activators with distinct immunomodulation profiles all members except one comprise of linear DNA molecules. The exception is dSLIM®, the active ingredient of MGN1703 in several clinical trials. dSLIM® establishes a new class of TLR-9 agonists, represented by covalently closed dumbbell-like DNA molecules consisting entirely of natural DNA with two single-stranded CG-containing loops separated by a double-stranded stem. Results By cytokine secretion and cellular activation of relevant cells in human PBMC we show that CG-motifs - such as in ODN2006, a class B TLR9 agonist - if incorporated in dSLIM® conformation reveal an improved immunomodulatory potential compared to their corresponding linear CpG oligonucleotides. Increased cytokine secretion encompasses the central anti-cancer cytokine IFN-alpha, the potent angiostatic cytokine IP-10, or IFN-gamma the key activator of NK-, NKT-, and cytotoxic T-cell responses. After 24-48h exposure to dSLIM® TLR-9 agonists, also activation surface markers of relevant cell subpopulations of PBMC are up-regulated including CD80 of pDCs, CD86 of B-cells, CD86 and CD169 of monocytes, CD69 of NK, NKT as well as T cells. Consequently, dSLIM® triggers an explicit specific cytotoxicity against Jurkat target cells while ODN2006 is without significant effect in this readout. All these effects are considered important and relevant cancer-fighting features of dSLIM®. Conclusions Differences in immunomodulation profiles between classes of TLR-9 agonists appear to be more closely related to their conformation than their individual nucleotide sequences. We show individual loop sequences in dSLIM® that differentiate the immunomodulation profile of molecules only to a moderate extent. Inherent to the dumbbell-like structure of dSLIM® TLR-9 agonists is their potential to dimerize loop-binding TLR-9 molecules in the membrane of immune-competent cells. Thereby, dSLIM® TLR-9 agonists most probably not only bind and activate but also cluster TLR-9 molecules thereby changing the TLR-9 triggered immune response in a qualitative way. Stabilizing chemical modifications in linear CpG ODN also impact the immunomodulatory profile of TLR-9 agonists and are discussed. Citation Format: Kerstin Kapp, Christiane Kleuss, Matthias Schroff, Burghardt Wittig. Conformation overrides base sequence - insights from a novel class TLR-9 agonists. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2565. doi:10.1158/1538-7445.AM2014-2565