General
The main goal of our research is to investigate the therapeutic potential of immunoactivating oligonucleotides for tumor diseases.
Background
The innate immune system has the capability to recognize the presence of invading microorganisms such as virus and bacteria by sensing foreign conserved molecular structures. These molecular structures could be bacterial or viral RNA/DNA oligonucleotides but also bacterial proteins (e.g. flagellin), sugars (e.g. polysaccharides), and lipids. The detection is mediated by the so-called Toll-like receptors (TLRs), that are distributed on plasma membranes and within endosomes. The recognition leads to the activation of the immune system thereby alerting other cells and initiating clearance of the pathogens or infected cells.
The usage of short synthetic DNA or RNA oligonucleotide, that mimic viral or bacterial structures, could therefore serve as a great tool for stimulation of the immune system in cancer patients, which typically display an immunosuppressive status.
To investigate the therapeutic potentials of these oligonucleotides, we utilize different tumor-mouse models. On one hand, we investigate a transgenic mouse line, that spontaneously develops gastric tumors, on the other hand we apply subcutaneous injection of different tumor cell lines of melanoma, colon and gastric adenocarcinoma to induce local tumors. Our group explores different application routes, the frequency of application and various delivery system of oligonucleotides, dendritic cell vaccination, adoptive T cell transfer and modulation of T cells.
Additionally, we are interested to elucidate which factors shape the tumor microenvironment and how oligonucleotides can alter these immune suppressive states.
Picture taken from Hotz C and Bourquin C, Oncoimmunology 2012
Synthetic compounds or viral RNA activate TLR7 on dendritic cells (DC), followed by secretion of proinflammatory cytokines. Those cytokines modulate adaptive immunity fo favor tumor cell destruction by inhibiting regulatory cell types (Treg: regulatory T-cells and MDSC: myeloid derived supressor cells) and promoting effector cells (NK: natural killer cells, Teff: T effector cells).
RESEARCH RESULTS
Systemic cancer immunotherapy with
Toll-like receptor 7 agonists: Timing is everything
Compounds activating innate immunity through pattern-recognition receptors, such as the Toll-like receptor (TLR) 7 agonist imiquimod, have proven very effective in topical application against skin cancer. They have however to date met with limited success when applied systemically, despite the induction of immune responses. The disappointing clinical efficacy may be due to avoidable phenomena such as the development of receptor tolerance.
We have shown that a single injection of R848, a small molecule agonist of TLR7, leads to tolerance towards a second stimulation beginning 24h after injection and lasting for up to five days. In contrast, repeated stimulation within the first 24h results in an enhanced response. Of note, protocols used for clinical trials investigating systemic TLR7 stimulation in cancer have relied on single injections given every two to three days. According to our study, this schedule results in TLR7 tolerance and may explain the limited success of systemic treatment in these studies. Based on our findings, we designed a protocol of fractionated stimulation with R848 in cycles separated by 5-day intervals to circumvent tolerance. This protocol leads to efficient block of tumor growth in a cancer model and.was more effective than the schedule used in clinical studies, although the cumulated dose was lower (Bourquin, Hotz et al., Cancer Research 2011).
Our findings demonstrate that in-depth understanding of the molecular mechanisms of immune activation can foster the design of more effective treatment protocols that circumvent receptor tolerance. We plan to build on these results to include a new effector mechanism, receptor priming, in immunotherapy protocols. Based on a careful study of the signaling events leading to receptor priming, we aim to develop novel treatment protocols that effectively activate antitumor immunity.
The curve shows the kinetics of TLR7 sensitivity after a single injection of a synthetic agonist. Our “fractionated” protocol takes advantage of initial increased sensitivity (see arrows indicating repeated injections) while avoiding stimulation in the tolerant state.
Toll-like receptor signaling blocks tumor-associated immunosuppression
An anticancer immune response can be suppressed by regulatory immune cells such as regulatory T cells or myeloid-derived suppressor cells (MDSC). The presence of these immunosuppressive cells is considered to be one of the main reasons for insufficient immunological tumor control. We have shown that the suppressive function of regulatory T cells and MDSC can be pharmacologically controlled by immunostimulatory oligonucleotides that activate Toll-like receptors (TLR) 7 and 9 (Anz et al., J. Immunol. 2010; Anz et al., Int J Cancer 2010; Zoglmeier et al., Clin Canc Res 2011; Anz et al., Histopath 2011). In particular, we have shown that TLR9 activation promotes maturation and differentiation of MDSC. These cells, which are normally poorly differentiated, lose their immunosuppressive activity through differentiation. We have defined the antitumoral cytokine IFNa, produced by plasmacytoid dendritic cells, as key effector for this phenotypical and functional change. We thus describe a novel mechanism by which TLR9 ligands promote antitumor responses.
We are currently investigating the impact of TLR stimulation on the migration of immune cells into tumors to determine whether, in addition to their regulatory function, the homing pattern of these cells is affected by immunopharmacological intervention. This ongoing project is performed within a research network focusing on cell migration that includes the group of Prof. Rüegg at the University of Fribourg, the Theodor-Kocher Institute in Berne, and the Institute for Biomedical Research in Bellinzona (www.cell-mig.ch).
Toll-like receptor immunostimulation by DNA origami nanostructures
We have shown that nanoparticles represent efficient drug delivery systems for immunoactive DNA and RNA oligonucleotides. Using gelatin-based nanoparticles, we have demonstrated that these carriers protect RNA oligonucleotides from degradation, facilitate their uptake by dendritic cells, and target these nucleic acids to the endosomal compartment where they are recognized by TLR7 (Bourquin, J Immunother 2010). Further, we have shown that immunization with RNA oligonucleotide-loaded nanoparticles leads to the development of an efficient antitumoral response.
The 3D structure of DNA constructs can be controlled by a refolding technique termed DNA origami. We demonstrated that DNA origami nanostructures can function as programmable and nontoxic immunostimulants. In collaboration with Prof. Liedl from the Center of Nanoscience of the Ludwig-Maximilian University of Munich, we used DNA origami tubes decorated with TLR9-activating oligonucleotides to stimulate dendritic cells. The DNA constructs were taken up by dendritic cells and localized in the endosome, a necessary step for the triggering of TLR9. Activated dendritic cells produced cytokine mediators such as interleukin-6 and interleukin-12p70, a process that underlies the initiation of an immune response. In this work we have shown for the first time that DNA origami constructs represent an efficient vehicle for immunoactive oligonucleotides (Schueller et al., ACS Nano, 2011). Collaborations with Prof. Fromm (Dept. of Chemistry, University of Fribourg), and Profs. Fink and Rothen-Rutishauser (Adolphe-Merkle Institute, University of Fribourg) are ongoing to further explore the potential of nanomaterials for the immunopharmacology of cancer.
