Potent and Prolonged Innate Immune Activation by Enzyme-Responsive Imidazoquinoline TLR7/8 Agonist Prodrug Vesicles

Abstract

Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent activators of antigen-presenting cells (APCs), however, they also induce severe side effects due to leakage from the site of injection into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of an imidazoquinoline TLR7/8 agonist that in aqueous medium forms vesicular structures of 200 nm. The conjugate contains an endosomal enzyme-responsive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native form after endocytosis, which results in high in vitro TLR agonist activity. In a mouse model, locally administered vesicles provoke significantly more potent and long-lasting immune stimulation in terms of interferon expression at the injection site and in draining lymphoid tissue compared to a nonamphiphilic control and the native TLR agonist. Moreover, the vesicles induce robust activation of dendritic cells in the draining lymph node in vivo.

Figure 1

Self-assembly of PEG-modified imidazoquinoline (IMDQ) amphiphile prodrug into vesicular nanoparticles. An optimized amphiphile prodrug bearing two benzyl repeating units in the linker and PEG5k self-assembles into vesicles. Upon endocytosis by antigen presenting cells, the vesicles degrade and native IMDQ is released, thereby binding to TLR7/8 receptors and triggering immune-activation.

Figure 2

(A) TEM image of self-assembled vesicles based on PEG5k-GL2-IMDQ (B) DLS plot of the PEG5k-GL2-IMDQ vesicles with a mean diameter of 195 nm and low dispersity (0.2). (C) Stability of the PEG5k-GL2-IMDQ vesicles after dilution in PBS 7.4 and incubated at 37 °C. (n=3) (D) Enzymatic degradation scheme of PEG5k-GL2-IMDQ. (E) In vitro release of IMDQ from PEG5k-GL2-IMDQ vesicles in the presence of an esterase and β-GUS at pH 5.0 and 37 °C (n=3).

Figure 3

In vitro RAW Blue assay andin vivo immune activation. (A1) Time dependent activation curves. (A2) EC50 values of the compounds. (n=6: Student t-test; ****:p<0.0001, **:p<0.01) (B1) IVIS imaging of IFNβ+/Δβ-luc reporter mice upon subcutaneous injection of PEG5k-GL2-IMDQ vesicles and soluble PEG-IMDQ into the footpad. (B2) Fluorescence signal detected at the local injection site and draining LN of the IFNβ+/Δβ-luc reporter mice. (n=3)

Figure 4

In vivo recruitment and activation of dendritic cells (DCs) in draining lymphoid tissue. (A) Flow cytometry analysis of the DC population in the draining lymph node in response to subcutaneous injection of PEG5k-GL2-IMDQ vesicles. (A1) contour plots and (A2) relative numbers of DCs. (n=3: Student t-test; *:p<0.05) (B) Flow cytometry analysis of the expression of maturation markers on DCs in the draining lymph node in response to subcutaneous injection of PEG5k-GL2-IMDQ vesicles. (B1) histograms and (B2) percentages of DCs expressing a specific maturation marker. (n=3 Student t-test; ***:p<0.001, **:p<0.05)

Scheme 1

Synthesis route for PEG-GLn-IMDQ (n=1-3).