Probing immune signatures of conjugated pattern recognition receptor ligands identifies chimeras with adjuvant and antitumor activity

Pattern recognition receptor (PRR) ligands hold great promise as adjuvants and immunotherapeutics. Here, we demonstrate that chemical conjugation of PRR ligands results in synergistic immune response amplification inaccessible to unlinked agonist mixtures. To identify potent immune agonists, we synthesized conjugated PRR ligands incorporating distinct agonist pairings, each targeting two carefully selected PRRs. We used a phenotypic screen using human peripheral blood mononuclear cells (PBMCs) to single out chimeric PRR ligands capable of inducing robust immune response both in terms of cytokine response and cytotoxicity against cancer cells. Chimeric TLR4/TLR7 and TLR7/RIG-I ligands showed broad immune activation in vitro as well as enhancement of antigen-specific cellular and humoral responses in mice. Intratumoral delivery of chimeric TLR4/TLR7 ligand induced robust antitumor response in a syngeneic mouse B16F10 tumor model. These results demonstrate the profound effects that conjugation can have on immune response and support the use of conjugated PRR ligands as adjuvants/immunotherapeutics.