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

TY - JOUR

T1 - Exploiting FcRn engagement of an albumin-CpG oligonucleotide covalent conjugate for potent TLR9 immune induction

AU - Pilati, Diego

AU - Agyei, Eugene Kusi

AU - Elkhashab, Marwa

AU - Fuchs, Elisabeth

AU - Nielsen, Ian Helstrup

AU - Bjerg, Tobias Wang

AU - Anthi, Aina Karen

AU - Jiménez-Reinoso, Anaïs

AU - Iversen, Marie Beck

AU - Pohl, Layla

AU - Narita, Ryo

AU - Frago, Susana

AU - Jakobsen, Martin R

AU - Andersen, Jan Terje

AU - Degn, Søren E

AU - Paludan, Søren R

AU - Alvarez-Vallina, Luis

AU - Howard, Kenneth A

PY - 2025/6

Y1 - 2025/6

N2 - 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.

AB - 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.

KW - CpG ODN

KW - FcRn

KW - TLR9

KW - albumin

KW - immune adjuvant

KW - innate immunity

KW - lymph nodes

UR - http://www.scopus.com/inward/record.url?scp=105005210701&partnerID=8YFLogxK

U2 - 10.1016/j.jbc.2025.108508

DO - 10.1016/j.jbc.2025.108508

M3 - Journal article

C2 - 40222546

SN - 0021-9258

VL - 301

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 6

M1 - 108508

ER -