Exploiting FcRn engagement of an albumin-CpG oligonucleotide covalent conjugate for potent TLR9 immune induction.

CpG-oligodeoxynucleotide (CpG ODN)-based Toll-like receptor (TLR) agonists are promising immunostimulatory adjuvants; however, low entry into TLR-rich cellular endosomal compartments and poor lymphatic accumulation limit clinical translation. In this work, we introduce a recombinant human serum albumin (rHA)-CpG ODN covalent conjugate (rHA-CpG) designed to exploit the neonatal Fc receptor (FcRn)-driven albumin cellular sorting pathway to maximize CpG delivery into TLR9-rich endosomes and accumulate in lymph nodes. Site-selective conjugation of CpG to albumin cysteine 34, distant from its main FcRn-binding interface, resulted in a retained pH-dependent human FcRn binding, and receptor-driven endosomal trafficking in a cellular recycling assay. Induction of tumor necrosis factor (TNF) secretion in THP-1 cells and interferon alpha (IFN-α) in human hematopoietic stem and progenitor cell (HSPC)-derived plasmacytoid dendritic cells (pDCs), in contrast, to a myeloid differentiation primary response 88 (MyD88) and TLR9 knockout cells, respectively, support TLR9-engagement. The rHA-CpG construct induced greater TNF-α than free CpG ODN in mouse RAW 264.7 cells, and in human peripheral blood mononuclear cells (PBMCs) and expansion of classical (CD14(+)CD16(-)) monocytes. Furthermore, greater accumulation of Cy5.5-labelled CpG in the inguinal (>3-fold) and axillary (>18-fold) lymph nodes was observed when conjugated to rHA compared to an unconjugated rHA/CpG mix following subcutaneous injection in mice. Moreover, increased LN accumulation of an rHA variant engineered with high FcRn-binding affinity supports an FcRn-driven mechanism. Demonstration of FcRn-mediated albumin targeting at intra- and extracellular sites provides the mechanistic basis for the potent immune induction observed using the novel rHA-CpG conjugate design class introduced in this work.