Self-Assembly of Toll-Like Receptor (TLR2/6) Agonist Lipidated Amino Acid or Peptide Conjugates: Distinct Morphologies and Bioactivities

Toll-like receptor (TLR) agonists are of interest in immunotherapy and cancer vaccines. The most common agonists of TLR2 are based on Pam₂Cys or Pam₃Cys. In the former, two palmitoyl (Pam) fatty acids are linked to a glycerylcysteine motif by ester linkages. Pam₃Cys is analogous but contains an extra Pam group on the α-amine. Here, we compare the self-assembly in aqueous solution of the parent Pam₂CysOH and Pam₃Cys amino acid conjugates to that of Pam₂CysSK₄ and Pam₃CysSK₄ which are potent TLR2 agonists bearing the CysSK₄ peptide sequence. All four conjugates exhibit a critical aggregation concentration above which self-assembled structures are formed. We find through a combination of small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), and confocal fluorescence microscopy remarkable differences in self-assembled nanostructures. Pam₂CysOH and Pam₃CysOH both form unilamellar vesicles, although these are larger for the latter compound, an effect ascribed to enhanced membrane rigidity. This is in contrast to previously reported morphologies for Pam₂CysSK₄ and Pam₃CysSK4, which are spherical micelles or predominantly wormlike micelles, respectively [Hamley, I. W.; et al. <i>Toll-like Receptor Agonist Lipopeptides Self-Assemble into Distinct Nanostructures</i>. Chem. Comm. 2014, 50, 15948-15951]. We also examine the effect of introduction in the bulky <i>N</i>-terminal Fmoc [fluorenylmethoxycarbonyl] group on the self-assembly of Fmoc-Pam₂CysOH. This compound also forms vesicles (above a critical aggregation concentration, determined from dye probe fluorescence experiments) in aqueous solution, larger than those for Pam₂CysOH and with a population of perforated/compound vesicles. The carboxyl-coated (and amino-coated for Pam₂CysOH) vesicles demonstrated here represent a promising system for future development toward bionanotechnology applications such as immune therapies. Conjugates Pam₂CysOH, Pam₂CysSK₄, and Pam₃CysSK₄ show good cytocompatibility at low concentrations, and in fact, the cell compatibility extends over a wider concentration range for Pam₂CysOH. The TLR2/6 agonist activity was assessed using an assay that probes secreted alkaline phosphatase (SEAP) in NF-κB-SEAP reporter HEK293 cells expressing human TLR2 and TLR6, and Pam₂CySOH shows significant activity, although not to the extent of Pam₂CysSK4 or Pam₃CysSK₄. Thus, Pam₂CysOH in particular is of interest as a vesicle-forming TLR2/6 agonist and stimulator of immune response.