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A human in vitro whole blood assay to predict the systemic cytokine response to therapeutic oligonucleotides including siRNA.

Coch C, Lück C, Schwickart A, Putschli B, Renn M, Höller T, Barchet W, Hartmann G, Schlee M - PLoS ONE (2013)

Bottom Line: Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology.Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development.In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, University of Bonn, Bonn, Germany. ccoch@uni-bonn.de

ABSTRACT
Therapeutic oligonucleotides including siRNA and immunostimulatory ligands of Toll-like receptors (TLR) or RIG-I like helicases (RLH) are a promising novel class of drugs. They are in clinical development for a broad spectrum of applications, e.g. as adjuvants in vaccines and for the immunotherapy of cancer. Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology. Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development. To serve this purpose, we here established a human whole blood assay (WBA) that is fast and easy to perform. Its response to synthetic TLR ligands (R848: TLR7/8, LPS: TLR4) was on a comparable threshold to the more time consuming peripheral blood mononuclear cell (PBMC) based assay. By contrast, the type I IFN profile provoked by intravenous CpG-DNA (TLR9 ligand) in humans in vivo was more precisely replicated in the WBA than in stimulated PBMC. Since Heparin and EDTA, but not Hirudin, displaced oligonucleotides from their delivery agent, only Hirudin qualified as the anticoagulant to be used in the WBA. The Hirudin WBA exhibited a similar capacity as the PBMC assay to distinguish between TLR7-activating and modified non-stimulatory siRNA sequences. RNA-based immunoactivating TLR7/8- and RIG-I-ligands induced substantial amounts of IFN-α in the Hirudin-WBA dependent on delivery agent used. In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.

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Comparison of cytokines induced by LPS and R848 in PBMC and in a human WBA.a – PBMC were incubated in 96 well with increasing concentrations of LPS. After 48 h supernatant was analyzed for TNF-α and IL-6 using ELISA. Results of eight donors are shown as mean +/− SD. b–c – Whole blood anticoagulated either with Heparin, EDTA or Hirudin was incubated in 96 wells with increasing concentrations of LPS and after 48 h supernatant was checked for IL-6 (b), TNF-α (c). Mean of six donors is shown +/− SD. d – Done as described in (a) but R848 was used for stimulation and supernatant was analyzed for IFN-α, TNF-α and IL-6. Mean of eight donors (three for IFN-α) is shown +/− SD. e–g – Done as described for (b) but R848 was used for stimulation and IFN-α (e), TNF-α (f) and IL-6 (g) were measured in supernatant. Results of four (IFN-α) and six donors are shown as mean +/− SD.
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pone-0071057-g001: Comparison of cytokines induced by LPS and R848 in PBMC and in a human WBA.a – PBMC were incubated in 96 well with increasing concentrations of LPS. After 48 h supernatant was analyzed for TNF-α and IL-6 using ELISA. Results of eight donors are shown as mean +/− SD. b–c – Whole blood anticoagulated either with Heparin, EDTA or Hirudin was incubated in 96 wells with increasing concentrations of LPS and after 48 h supernatant was checked for IL-6 (b), TNF-α (c). Mean of six donors is shown +/− SD. d – Done as described in (a) but R848 was used for stimulation and supernatant was analyzed for IFN-α, TNF-α and IL-6. Mean of eight donors (three for IFN-α) is shown +/− SD. e–g – Done as described for (b) but R848 was used for stimulation and IFN-α (e), TNF-α (f) and IL-6 (g) were measured in supernatant. Results of four (IFN-α) and six donors are shown as mean +/− SD.

Mentions: As the first step towards a whole blood assay (WBA) that identifies cytokine release induced by therapeutic oligonucleotides, we started with established non-RNA/DNA ligands targeting nucleic acid recognizing- or endotoxin recognizing TLRs. We used the small molecule resiquimod (R848) acting on the RNA-detecting receptors TLR7 and 8 as well as LPS, known to activate TLR4. We compared cytokine induction by R848 and LPS in PBMC with whole blood anticoagulated with Heparin, EDTA or Hirudin (Fig. 1). Induction of typical cytokines in response to LPS (TNF-α, IL-6; Fig. 1a–c) and R848 (IFN-α, TNF-α, IL-6; Fig. 1d–g) were seen in both assays. Although induced cytokine concentrations were in general higher in PBMC, the threshold concentration of agents LPS or R848 leading to detectible induction of cytokines was comparable in both assays, as long as Heparin or Hirudin were used as anticoagulant. By contrast, EDTA anticoagulated blood showed a reduced response to the stimuli. Thus, the WBA assay with Heparin or Hirudin as anticoagulant yields the same qualitative results for cytokine induction in response to non-oligonucleotide TLR4/7/8 ligands as the established but more time consuming PBMC assay.


A human in vitro whole blood assay to predict the systemic cytokine response to therapeutic oligonucleotides including siRNA.

Coch C, Lück C, Schwickart A, Putschli B, Renn M, Höller T, Barchet W, Hartmann G, Schlee M - PLoS ONE (2013)

Comparison of cytokines induced by LPS and R848 in PBMC and in a human WBA.a – PBMC were incubated in 96 well with increasing concentrations of LPS. After 48 h supernatant was analyzed for TNF-α and IL-6 using ELISA. Results of eight donors are shown as mean +/− SD. b–c – Whole blood anticoagulated either with Heparin, EDTA or Hirudin was incubated in 96 wells with increasing concentrations of LPS and after 48 h supernatant was checked for IL-6 (b), TNF-α (c). Mean of six donors is shown +/− SD. d – Done as described in (a) but R848 was used for stimulation and supernatant was analyzed for IFN-α, TNF-α and IL-6. Mean of eight donors (three for IFN-α) is shown +/− SD. e–g – Done as described for (b) but R848 was used for stimulation and IFN-α (e), TNF-α (f) and IL-6 (g) were measured in supernatant. Results of four (IFN-α) and six donors are shown as mean +/− SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3733725&req=5

pone-0071057-g001: Comparison of cytokines induced by LPS and R848 in PBMC and in a human WBA.a – PBMC were incubated in 96 well with increasing concentrations of LPS. After 48 h supernatant was analyzed for TNF-α and IL-6 using ELISA. Results of eight donors are shown as mean +/− SD. b–c – Whole blood anticoagulated either with Heparin, EDTA or Hirudin was incubated in 96 wells with increasing concentrations of LPS and after 48 h supernatant was checked for IL-6 (b), TNF-α (c). Mean of six donors is shown +/− SD. d – Done as described in (a) but R848 was used for stimulation and supernatant was analyzed for IFN-α, TNF-α and IL-6. Mean of eight donors (three for IFN-α) is shown +/− SD. e–g – Done as described for (b) but R848 was used for stimulation and IFN-α (e), TNF-α (f) and IL-6 (g) were measured in supernatant. Results of four (IFN-α) and six donors are shown as mean +/− SD.
Mentions: As the first step towards a whole blood assay (WBA) that identifies cytokine release induced by therapeutic oligonucleotides, we started with established non-RNA/DNA ligands targeting nucleic acid recognizing- or endotoxin recognizing TLRs. We used the small molecule resiquimod (R848) acting on the RNA-detecting receptors TLR7 and 8 as well as LPS, known to activate TLR4. We compared cytokine induction by R848 and LPS in PBMC with whole blood anticoagulated with Heparin, EDTA or Hirudin (Fig. 1). Induction of typical cytokines in response to LPS (TNF-α, IL-6; Fig. 1a–c) and R848 (IFN-α, TNF-α, IL-6; Fig. 1d–g) were seen in both assays. Although induced cytokine concentrations were in general higher in PBMC, the threshold concentration of agents LPS or R848 leading to detectible induction of cytokines was comparable in both assays, as long as Heparin or Hirudin were used as anticoagulant. By contrast, EDTA anticoagulated blood showed a reduced response to the stimuli. Thus, the WBA assay with Heparin or Hirudin as anticoagulant yields the same qualitative results for cytokine induction in response to non-oligonucleotide TLR4/7/8 ligands as the established but more time consuming PBMC assay.

Bottom Line: Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology.Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development.In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, University of Bonn, Bonn, Germany. ccoch@uni-bonn.de

ABSTRACT
Therapeutic oligonucleotides including siRNA and immunostimulatory ligands of Toll-like receptors (TLR) or RIG-I like helicases (RLH) are a promising novel class of drugs. They are in clinical development for a broad spectrum of applications, e.g. as adjuvants in vaccines and for the immunotherapy of cancer. Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology. Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development. To serve this purpose, we here established a human whole blood assay (WBA) that is fast and easy to perform. Its response to synthetic TLR ligands (R848: TLR7/8, LPS: TLR4) was on a comparable threshold to the more time consuming peripheral blood mononuclear cell (PBMC) based assay. By contrast, the type I IFN profile provoked by intravenous CpG-DNA (TLR9 ligand) in humans in vivo was more precisely replicated in the WBA than in stimulated PBMC. Since Heparin and EDTA, but not Hirudin, displaced oligonucleotides from their delivery agent, only Hirudin qualified as the anticoagulant to be used in the WBA. The Hirudin WBA exhibited a similar capacity as the PBMC assay to distinguish between TLR7-activating and modified non-stimulatory siRNA sequences. RNA-based immunoactivating TLR7/8- and RIG-I-ligands induced substantial amounts of IFN-α in the Hirudin-WBA dependent on delivery agent used. In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.

Show MeSH
Related in: MedlinePlus