Transcriptional profiling of liver from mice treated with Toll-like receptor 5 agonists

Entolimod, a pharmacological derivative of the natural Toll-like receptor 5 (TLR5) agonist flagellin, has strong therapeutic potential for several indications including radioprotection and cancer immunotherapy. However, in Phase 1 studies, entolimod induced a rapid neutralizing immune response, presumably due to flagellin-reactive immune memory derived from life-long exposure of adult humans to flagellated enterobacteria. Since applications requiring repeated dosing could be precluded by entolimod’s neutralizing antigenicity and immunogenicity, we used structure-guided reengineering to develop a new, substantially deimmunized entolimod variant, GP532. We used RNAseq-based analysis of global gene expression profiles in C57BL/6 mice following systemic administration of entolimod vs GP532 to evaluate the biocomparability of the two drugs. In this experiment, we used wild type (WT) and TLR5-/- mice (on the same genetic background) to address the TLR5-dependence/specificity of responses and focused on the liver since it was previously defined as the major organ responsive to TLR5 systemic administration in mice . Analysis of livers collected 30 minutes after injection of entolimod or GP532 was aimed at assessing direct primary TLR5 responses, while a 24-hour time point was included (for wild type mice) to define long-term overall (direct and indirect) effects of TLR5 activation. Overall, our RNAseq-based analysis of the effects of entolimod and GP532 on transcription confirmed our expectations of their mechanism of action and overall biocomparability. These results establish GP532 as a highly optimized TLR5 agonist suitable for multi-dose therapeutic regimens and for patients with high titers of preexisting flagellin-neutralizing antibodies. Gene expression profiles were generated by deep sequencing for livers of WT and TLR5-/- C57BL/6 mice treated with vehicle, entolimod or GP532. Livers were collected at 30min and 24h for WT mice and at 30 min only for TLR5-/-. All experiments were performed in triplicates with the exception of TLR5-/- liver treated with entolimod at 30 minutes after administration which was performed in duplicate.