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Distinct immunological activation profiles of dSLIM ® and ProMune ® depend on their different structural context

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: DNA‐based TLR9 agonists are potent activators of the immune system. ProMune® and dSLIM® belong to different families of TLR9 agonists and both have been established as cancer immunotherapeutics in clinical proof‐of‐concept studies. Unfortunately, ProMune® failed in pivotal oncological trials. dSLIM®, the active ingredient of Lefitolimod (MGN1703), successfully finished a double‐blinded, placebo‐controlled phase II study in patients with advanced colorectal cancer, exhibiting improved progression‐free survival and durable disease control.

Methods: To explain the different systemic efficacies of dSLIM® and ProMune®, both TLR9 agonists and chimeric molecules thereof are analyzed side‐by‐side in a panel of in vitro assays for immune activation.

Results and conclusions: Indeed, dSLIM® exposure results in an IFN‐α dependent broad activation of immune cells whereas ProMune® strongly stimulates B cells. Moreover, all functional effects of dSLIM® strictly depend on the presence of CG‐motifs within its dumbbell‐shaped, covalently closed structural context. Conversely, several immunological effects of ProMune® like IL‐8 secretion are independent of CG‐motifs and could be ascribed to the phosphorothioate‐modifications of its DNA backbone, which may have caused the side effects of ProMune® in clinical trials. Finally, we showed that the implementation of ProMune® (ODN2006) base sequence into the characteristic dSLIM® dumbbell form resulted in dSLIM2006 with all beneficial effects for immunostimulation combined from both TLR9 classes without any CG‐independent effects.

No MeSH data available.


Related in: MedlinePlus

Dependency of dSLIM® and CpG‐ODN effects on type I interferon. PBMC were treated with the TLR9 agonists dSLIM®, ProMune®, or class A CpG‐ODN (ODN2216) at final concentrations of 3 μM or medium alone for 48 h. To block type I interferons, cell cultures were co‐incubated with B18R, a vaccinia virus‐encoded receptor with specificity to type I interferons, at 0.5 μg/mL final concentration (open bars). Cytokine levels in the supernatants were determined by a bead‐based multiplex immunoassay or ELISA. Cells were stained with antibodies against lineage‐ and activation markers and analyzed by flow cytometry as described in Materials and Methods section. Frequencies or MFI of activation markers within the cell population are shown. Means and SEM resulting from four individual experiments are displayed.
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iid3126-fig-0003: Dependency of dSLIM® and CpG‐ODN effects on type I interferon. PBMC were treated with the TLR9 agonists dSLIM®, ProMune®, or class A CpG‐ODN (ODN2216) at final concentrations of 3 μM or medium alone for 48 h. To block type I interferons, cell cultures were co‐incubated with B18R, a vaccinia virus‐encoded receptor with specificity to type I interferons, at 0.5 μg/mL final concentration (open bars). Cytokine levels in the supernatants were determined by a bead‐based multiplex immunoassay or ELISA. Cells were stained with antibodies against lineage‐ and activation markers and analyzed by flow cytometry as described in Materials and Methods section. Frequencies or MFI of activation markers within the cell population are shown. Means and SEM resulting from four individual experiments are displayed.

Mentions: The CG‐dependent activation of TLR9 negative cells within PBMC by dSLIM® is probably mediated by IFN‐α. Co‐incubation of dSLIM‐activated PBMC with the vaccinia virus protein B18R, that is known to complex type I interferon 36, abolishes the secretion of IP‐10 as well as the activation of monocytes, NK cells, and NKT cells (Fig. 3). Blocking of type I interferon also impairs the stimulation of PBMC by ODN2216 (see Fig. 3 and others 19), a class A CpG‐ODN with partial PTO‐protection at the termini, known for strong induction of IFN‐α, whereas the ProMune® effects depicted in Figure 3 are hardly affected.


Distinct immunological activation profiles of dSLIM ® and ProMune ® depend on their different structural context
Dependency of dSLIM® and CpG‐ODN effects on type I interferon. PBMC were treated with the TLR9 agonists dSLIM®, ProMune®, or class A CpG‐ODN (ODN2216) at final concentrations of 3 μM or medium alone for 48 h. To block type I interferons, cell cultures were co‐incubated with B18R, a vaccinia virus‐encoded receptor with specificity to type I interferons, at 0.5 μg/mL final concentration (open bars). Cytokine levels in the supernatants were determined by a bead‐based multiplex immunoassay or ELISA. Cells were stained with antibodies against lineage‐ and activation markers and analyzed by flow cytometry as described in Materials and Methods section. Frequencies or MFI of activation markers within the cell population are shown. Means and SEM resulting from four individual experiments are displayed.
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Related In: Results  -  Collection

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iid3126-fig-0003: Dependency of dSLIM® and CpG‐ODN effects on type I interferon. PBMC were treated with the TLR9 agonists dSLIM®, ProMune®, or class A CpG‐ODN (ODN2216) at final concentrations of 3 μM or medium alone for 48 h. To block type I interferons, cell cultures were co‐incubated with B18R, a vaccinia virus‐encoded receptor with specificity to type I interferons, at 0.5 μg/mL final concentration (open bars). Cytokine levels in the supernatants were determined by a bead‐based multiplex immunoassay or ELISA. Cells were stained with antibodies against lineage‐ and activation markers and analyzed by flow cytometry as described in Materials and Methods section. Frequencies or MFI of activation markers within the cell population are shown. Means and SEM resulting from four individual experiments are displayed.
Mentions: The CG‐dependent activation of TLR9 negative cells within PBMC by dSLIM® is probably mediated by IFN‐α. Co‐incubation of dSLIM‐activated PBMC with the vaccinia virus protein B18R, that is known to complex type I interferon 36, abolishes the secretion of IP‐10 as well as the activation of monocytes, NK cells, and NKT cells (Fig. 3). Blocking of type I interferon also impairs the stimulation of PBMC by ODN2216 (see Fig. 3 and others 19), a class A CpG‐ODN with partial PTO‐protection at the termini, known for strong induction of IFN‐α, whereas the ProMune® effects depicted in Figure 3 are hardly affected.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: DNA‐based TLR9 agonists are potent activators of the immune system. ProMune® and dSLIM® belong to different families of TLR9 agonists and both have been established as cancer immunotherapeutics in clinical proof‐of‐concept studies. Unfortunately, ProMune® failed in pivotal oncological trials. dSLIM®, the active ingredient of Lefitolimod (MGN1703), successfully finished a double‐blinded, placebo‐controlled phase II study in patients with advanced colorectal cancer, exhibiting improved progression‐free survival and durable disease control.

Methods: To explain the different systemic efficacies of dSLIM® and ProMune®, both TLR9 agonists and chimeric molecules thereof are analyzed side‐by‐side in a panel of in vitro assays for immune activation.

Results and conclusions: Indeed, dSLIM® exposure results in an IFN‐α dependent broad activation of immune cells whereas ProMune® strongly stimulates B cells. Moreover, all functional effects of dSLIM® strictly depend on the presence of CG‐motifs within its dumbbell‐shaped, covalently closed structural context. Conversely, several immunological effects of ProMune® like IL‐8 secretion are independent of CG‐motifs and could be ascribed to the phosphorothioate‐modifications of its DNA backbone, which may have caused the side effects of ProMune® in clinical trials. Finally, we showed that the implementation of ProMune® (ODN2006) base sequence into the characteristic dSLIM® dumbbell form resulted in dSLIM2006 with all beneficial effects for immunostimulation combined from both TLR9 classes without any CG‐independent effects.

No MeSH data available.


Related in: MedlinePlus