Toll-like receptor (TLR) 3 agonists emerged as attractive candidates for vaccination strategies against tumors and pathogens. An important mechanism of action of such agonists is based on the activation of TLR3-expressing dendritic cells (DCs), which display a unique capacity to induce and stimulate T-cell responses. In this context, it has been demonstrated that targeting of TLR3 by double-stranded RNA such as poly(I:C) results in potent activation of DCs. Major disadvantages of poly(I:C) comprise its undefined chemical structure and very poor homogeneity, with subsequent unpredictable pharmacokinetics and high toxicity. In the present study, we evaluated the physicochemical properties and biological activity of the novel TLR3 agonist RGC100. RGC100 has a defined chemical structure, with a defined length (100 bp) and molecular weight (64.9 KDa) and a good solubility. RGC100 is stable in serum and activates myeloid DCs through TLR3 targeting, as evidenced by gene silencing experiments. Activation of mouse and human myeloid CD1c+ DCs by RGC100 leads to secretion of several proinflammatory cytokines. In addition, RGC100 improves the ability of CD1c+ DCs to stimulate T-cell proliferation. Due to its physicochemical properties and its immunostimulatory properties, RGC100 may represent a promising adjuvant for prophylactic and therapeutic vaccination strategies.
In the initial phase of infection, the innate immune system generates a rapid and potent inflammatory response. This response aims at blocking dissemination of the infectious agent, with subsequent activation of T cells and B cells that mount the acquired immune response against the pathogen [
Expression of TLR3 has been evidenced in BDCA1+ myeloid DCs (mDCs), human-monocyte-derived DCs (MoDCs) but not in plasmacytoid DCs [
Polyinosinic-polycytidylic acid poly(I:C) is a potent activator of innate immunity [
In the present study, structure, analytical profile and biological activity of the novel TLR3 agonist RGC100 are presented. RGC100 displays a very well defined chemical structure, length and molecular weight, a good solubility and serum stability, being able to activate DCs in a dose-dependent manner by specifically targeting endosomal TLR3.
Analysis of RGC100 length and integrity was performed on 12% native PAGE. DNA marker (Fermentas, Germany) was used to illustrate molecular size distribution and RNA staining was achieved by using Stains-all (Alfa Aesar, USA). Analysis of poly(I:C) was performed on 1% native agarose gel electrophoresis. Two different poly(I:C) compounds were used: poly(I:C) with a low molecular weight (LMW, 0.2–1 kb, Invivogen, USA), and poly(I:C) with a high molecular weight (HMW, 1.5–8 kb, Invivogen, USA). RNA marker (Promega, Germany) was used to illustrate molecular size distribution.
Physical characterization of RGC100 in solution was performed by size-exclusion chromatography (SEC) with UV, refractive index (RI), and right angle light scattering (RALS) detection on the Viscotek TDAmax (Malvern, UK). A sample volume of 125
Blood samples were obtained with informed consent from healthy donors. The study was approved by the institutional review board of the University Hospital of Dresden (no. EK 27022006). Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque (Biochrom, Germany) density centrifugation. Subsequently, human CD1c+ DC were isolated from freshly prepared PBMCs by immunomagnetic negative depletion and positive selection according to the manufactures instructions (Miltenyi Biotec, Germany). CD3+ T cells were isolated from PBMCs by negative depletion using immunomagnetic separation according to the manufacturer’s instructions (Miltenyi Biotec, Germany). To analyze the purity of the cell preparations, CD1c+ DCs were stained with PE-conjugated anti-CD1c+ and FITC-conjugated anti-CD19 antibodies and CD3+ T cells with PE-conjugated anti-CD2 and FITC-conjugated anti-CD3 antibodies. Purity was determined by FACS analysis, which was performed on a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany).
CD1c+ DCs (1 × 104/well) were cocultured with autologous CD3+ T cells (1 × 105 cells/well) in the presence or absence of 50
JAWS II cell line was obtained from American Type Culture Association (ATCC, USA). JAWS II is an immortalized immature myeloid DC line derived from C57BL/6 mice, which displays a similar phenotypic profile as resting bone-marrow-derived DCs (BMDCs) [
To assess the toxicity of siRNA on JAWs II DCs, a cell proliferation assay was performed as previously described [
Human CD1c+ DCs were plated in round-bottomed 96-well plates at 2.5 × 104/well in RPMI 1640 medium containing 10% human AB serum (CCPRO, Germany), 2 mM L-glutamine, 1% nonessential amino acids, 100 U/mL penicillin, and 100 mg/mL streptomycin (Biochrom, Germany). Then, cells were stimulated with RGC100 or poly(I:C) (Sigma-Aldrich, Deutschland) at different concentrations. After 24 h, supernatants were collected and the concentration of IL-1
Gene silencing was performed using IBONI siRNA (Riboxx, Germany) targeting TLR3 (5′-CTCGGCCTTAATGAAATTGAA-3′) and a nontargeting siRNA (Riboxx, Germany). Therefore, JAWS II cells were plated in round-bottomed 96-well plates at 5 × 104/well and incubated at 37°C (5% CO2) for 16 h. Then, IBONI siRNA (Riboxx. Germany) was mixed to riboxxFECT transfection reagent (Riboxx, Germany) according to manufacturer’s instructions and the mix was added to the wells at a concentration of 20 nM. At 6 h after transfection, RGC100 was added and the cells were incubated for 16 h. Subsequently, cells and supernatants were harvested. RNA was extracted from cells using the RNeasy kit (Qiagen, Germany) and used for subsequent qRT-PCR. Supernatants were used for cytokines measurement with ELISA according to the manufacturer’s instructions (Qiagen, Germany). qRT-PCR was performed on LightCycler using QuantiTect Primer assays (Qiagen, Germany) for mouse TLR3 and mouse
RGC100 (1.6
Analysis of RGC100 (3
Student’s
Design of RGC100 was performed based on the knowledge of structural and biological characteristics of TLR3 agonists. Crystal structure of the ectodomain of TLR3 with its dsRNA ligand [
The choice of sequence composition of RGC100 was based on previous studies on biological activity
Taking these experimental observations on length and sequence composition into consideration, we have designed RGC100 that bears a length of 100 bp, and consists of 100 rC paired to 100 rG. Analysis by native PAGE and SEC with UV, RI and LS detection showed that RGC100 displays a defined physicochemical structure. RGC100 has an observed molecular weight of 64.6 kDa (MWcalc = 64.9 kDa) with low polydispersity (Figures
Determination of physicochemical properties of RGC100. (a) Analysis of RGC100 by 12% native PAGE. RGC100 displays a length of 100 bp as indicated. It consists of 100 rC bases paired to 100 rG bases, perfectly annealed in a double strand. As a reference, a 100 mer consisting of homopolymeric cytidine is shown. (b) Analysis of poly(I:C) by 1% native agarose gel electrophoresis. p(I:C) LMW: poly(I:C) with a low molecular weight. p(I:C) HMW: poly(I:C) with a high molecular weight. (c) Analysis of RGC100 by size-exclusion chromatography (SEC) with UV, RI and RALS detection. Data analysis provides information about molecular size and polydispersity (Mw/Mn = 1.015).
The defined chemical structure and good solubility of RGC100 are of importance to reduce potential toxic effects of TLR3 agonists. As observed for poly(I:C), the homogeneity of the compound plays an essential role in the genesis of toxicity. Poly(I:C) is a polydisperse and heterogeneous compound (Figure
The advantages of using a TLR3 agonist such as RGC100 displaying defined physicochemical properties such as solubility and homogeneity, as well as precise chemical structure, length and molecular weight have been already highlighted by others [
Comparison of the physicochemical and functional properties of RGC100 and poly(I:C).
Length | Molecular weight | Chemical structure | Biostabilitya | Agonist of | |
---|---|---|---|---|---|
RGC100 | 100 bp | 64.6 KDa | dsRNA | 7 days | TLR3 |
Poly(I:C) | ~1500–8000 bp | ~1020–5440 KDa | dsRNA ± ssRNA | <5 minutes | TLR3, RLRsb |
aBiostability measured as resistance to serum nuclease; bRLR: RIG-I-like receptors.
Stability of RGC100 was examined in serum. RGC100 was incubated with FCS, mouse serum and human serum, and its integrity was assessed on native PAGE over time. As shown in Figures
Assessment of the stability of RGC100 in FCS, mouse serum, or human serum. RGC100 (1.6
DCs display an extraordinary capacity to induce and expand CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ T cells [
In the present study, we investigated the impact of RGC100 on TLR3-expressing murine JAWS II cells, representing immature myeloid DCs, which have been used in studies focusing on antitumor and pathogen-specific immunity [
Activation of JAWS II DCs by RGC100 in a dose-dependent manner. (a), cytokine and chemokine profile of JAWS II DCs activated by RGC100. RGC100 was incubated with JAWS II DCs for 16 h at a concentration of 250
To explore the mechanism of action of RGC100, knockdown of TLR3 in JAWS II DCs was performed. In order to prevent cytotoxicity resulting from off-target effects of siRNA, the CC50 of the siRNA was assessed in a cell proliferation assay. The CC50 of siRNA was >200 nM. Consequently, the concentration used to knockdown TLR3 (20 nM) was chosen to be 10-fold lower than the CC50. Additionally, the siRNA used in this assay displays a specific design that prevents off-target effects, as previously reported [
Inhibition of activation of JAWS II DCs by RGC100 using siRNA targeting TLR3. Cells were treated with siRNA targeting TLR3, then incubated with RGC100 at the indicated concentrations. RNA was extracted and supernatant was harvested. (a) Relative expression of TLR3 mRNA in cells treated with siRNA targeting TLR3 or with a nontargeting siRNA (NC siRNA). mRNA levels were normalized to
In a further step, we examined whether endosomal acidification is essential for activation of JAWS II DCs by RGC100. For this purpose, cells were treated by chloroquine followed by incubation with RGC100. As shown in Figure
Inhibition of activation by RGC100 of JAWS II DCs through chloroquine. (a) Cells were first treated with chloroquine (treated, 100
To get novel insights into the impact of RGC100 on the immunostimulatory properties of TLR3-expressing native human DCs, we investigated whether RGC100 promotes the release of proinflammatory cytokines by CD1c+ DCs in comparison to poly(I:C). Therefore, CD1c+ DCs and CD3+ T cells were immunomagnetically isolated from blood of healthy donors and maintained in the presence or absence of RGC100 and poly(I:C). The purity of isolated CD1c+ DC and CD3+ T cells was >90% as assessed by flow cytometric analysis (supplemental Figure S2). As shown in Figure
Activation of human myeloid CD1c+ DCs by RGC100 and poly(I:C). Freshly isolated CD1c+ DCs were cultivated in the presence or absence of RGC100 or poly(I:C). After 24 h, supernatants were harvested and the concentration of IL-1
Impact of RGC100 and poly(I:C) on CD1c+ DC-mediated T-cell proliferation. CD1c+ DCs were cocultured with autologous T cells in the presence or absence of 50
In the present study, experimental data on physicochemical properties and biological activity of the novel TLR3 agonist RGC100 are presented. RGC100 has optimal physicochemical properties, such as defined chemical structure and stability in serum. RGC100 activates murine myeloid DCs through targeting of endosomal TLR3, resulting in secretion of pro-inflammatory cytokines in a dose-dependent manner. In addition, RGC100 efficiently augments the secretion of pro-inflammatory cytokines by native human CD1c+ DCs and improves their capacity to promote T-cell proliferation. Based on these properties, RGC100 may represent a promising candidate for prophylactic and therapeutic vaccination strategies against tumors and pathogens.
Kai Naumann, Christiane Petzold and Jacques Rohayem are employees of Riboxx GmbH, Radebeul, Germany. RGC100 is covered by two PCT patent families (pending).
The authors wish to acknowledge the excellent technical work of Katrin Jäger, Constanze Grunau, Ivonne Böhmer, and Dorothea Kramer, as well as Bärbel Löbel.