Dual-targeting immunoadsorbent employing a bispecific nanobody for the simultaneous removal of IL-6 and TNF-α from serum

The dysregulated overproduction of pro-inflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) is a hallmark of severe inflammatory diseases. Extracorporeal blood purification offers a promising therapeutic strategy, but current adsorbents often lack the specificity required for precise intervention. This study reports the development of a bispecific nanobody (bsNb)-based immunoadsorbent for the simultaneous and specific removal of IL-6 and TNF-α. The functional bsNb, produced in a prokaryotic system via tandem fusion, was covalently immobilized on Sepharose CL-6B matrix to prepare the affinity adsorbent. Comprehensive characterization confirmed its high saturation adsorption capacity (6.9 mg/mL for IL-6 and 2.2 mg/mL for TNF-α) and rapid binding kinetics. In a simulated hemoperfusion system using human serum, the adsorbent achieved removal efficiencies exceeding 85% for both cytokines across a range of pathophysiological concentrations. Crucially, the bsNb adsorbent effectively captured both targets simultaneously from a complex serum matrix. This work highlights the significant potential of bispecific nanobodies as robust and selective ligands for preparing targeted immunoadsorbents. The developed platform represents a superior alternative to non-specific adsorbents, paving the way for more precise and effective extracorporeal therapies against cytokine-driven inflammatory disorders.