Probing Immune Signatures of Conjugated Pattern Recognition Receptor Ligands Identifies Chimeras with Potent Adjuvant and Antitumor Activities.

Pattern recognition receptor (PRR) ligands represent a promising class of immunostimulants. Here, we demonstrate that the covalent conjugation of PRR ligands enables coordinated receptor engagement and amplifies immune responses beyond those achievable with unlinked mixtures. We synthesized a focused panel of chimeric PRR ligands comprising defined pairwise agonist combinations targeting selected extracellular and intracellular PRRs. Using phenotypic screening in human peripheral blood mononuclear cells, we identified conjugates that induced distinct cytokine signatures and enhanced cytotoxic immune activity. Among these, chimeric TLR4/TLR7 and TLR7/RIG-I ligands elicited broad innate immune activation in vitro and enhanced antigen-specific immune responses in murine vaccination models. Notably, intratumoral administration of the conjugated TLR4/TLR7 ligand resulted in significant antitumor activity in a syngeneic B16F10 melanoma model. Collectively, these findings establish covalent PRR ligand conjugation as a powerful chemical strategy for modulating innate immune signaling and support the development of conjugated PRR ligands as next-generation vaccine adjuvants and immunotherapeutics.