Engineered HMGB1 construct with tandem Box B domains promotes tissue regeneration without potential for inflammation

Fully reduced high-mobility group box 1 (HMGB1) binds CXCL12 and signals via CXCR4 when released into the extracellular space. It acts as a chemokine and transitions stem cells from quiescent G₀ to a primed G_Alert state. Cells in G_Alert rapidly enter G₁ in response to activating factors released by tissue injury to promote tissue repair. However, oxidative conversion of FR-HMGB1 into the disulfide form activates proinflammatory pathways via TLR-2, TLR-4 and RAGE. Peptide mapping and nuclear magnetic resonance (NMR) spectroscopy identified a conserved CXCL12-binding motif within each Box and adjacent flanking regions. We decoupled the regenerative and inflammatory functions using an engineered construct (dBB12L), comprising tandem Box B domains with a flexible linker. dBB12L exhibited CXCL12 binding and accelerated repair equivalent to FR-HMGB1. Importantly, dBB12L lacked detectable RAGE binding and did not signal via TLR-2 and TLR-4, establishing it as a potential therapeutic to promote tissue repair without deleterious inflammation.